# Patent

## Title
Method for identifying a compound that modulates telomerase activity

## Abstract

The present invention embraces methods for identifying compounds that modulate the activity of telomerase. Compounds of the invention are identified by designing or screening for a compound which binds to at least one amino acid residue of the TRBD, “thumb,” “finger,” and/or “palm” domain; or FP-pocket, PT-pocket or Th-pocket of telomerase and testing the compound for its ability to modulate the activity of telomerase.

## Background
INTRODUCTION
This application is a continuation-in-part of PCT/US2008/080604, filed Oct. 21, 2008, which claims benefit of priority to U.S. Provisional Patent Application Ser. No. 61/090,726, filed Aug. 21, 2008, and Ser. No. 60/981,548, filed Oct. 22, 2007, the contents of which are incorporated herein by reference in their entireties.


BACKGROUND OF THE INVENTION
Any organism with linear chromosomes faces a substantial obstacle in maintaining the terminal sequence of its DNA often referred to as the “end replication problem” (Blackburn (1984) Annu. Rev. Biochem. 53:163-194; Cavalier-Smith (1974) Nature 250:467-470; Cech & Lingner (1997) Ciba Found. Symp. 211:20-34; Lingner, et al. (1995) Science 269:1533-1534; Lundblad (1997) Nat. Med. 3:1198-1199; Ohki, et al. (2001) Mol. Cell. Biol. 21:5753-5766). Eukaryotic cells overcome this problem through the use of a specialized DNA polymerase, called telomerase. Telomerase adds tandem, G-rich, DNA repeats (telomeres) to the 3′-end of linear chromosomes that serve to protect chromosomes from loss of genetic information, chromosome end-to-end fusion, genomic instability and senescence (Autexier & Lue (2006) Annu. Rev. Biochem. 75:493-517; Blackburn & Gall (1978) J. Mol. Biol. 120:33-53; Chatziantoniou (2001) Pathol. Oncol. Res. 7:161-170; Collins (1996) Curr. Opin. Cell Biol. 8:374-380; Dong, et al. (2005) Crit. Rev. Oncol. Hematol. 54:85-93).
The core telomerase holoenzyme is an RNA-dependent DNA polymerase (TERT) paired with an RNA molecule (TER) that serves as a template for the addition of telomeric sequences (Blackburn (2000) Nat. Struct. Biol. 7:847-850; Lamond (1989) Trends Biochem. Sci. 14:202-204; Miller & Collins (2002) Proc. Natl. Acad. Sci. USA 99:6585-6590; Miller, et al. (2000) EMBO J. 19:4412-4422; Shippen-Lentz & Blackburn (1990) Science 247:546-552). TERT is composed of four functional domains one of which shares similarities with the HIV reverse transcriptase (RT) in that it contains key signature motifs that are hallmarks of this family of proteins (Autexier & Lue (2006) supra; Bryan, et al. (1998) Proc. Natl. Acad. Sci. USA 95:8479-8484; Lee, et al. (2003) J. Biol. Chem. 278:52531-52536; Peng, et al. (2001) Mol. Cell. 7:1201-1211). The RT domain, which contains the active site of telomerase is thought to be involved in loose associations with the RNA template (Collins & Gandhi (1998) Proc. Natl. Acad. Sci. USA 95:8485-8490; Jacobs, et al. (2005) Protein Sci. 14:2051-2058). TERT however is unique, when compared to other reverse transcriptases in that it contains two domains N-terminal to the RT domain that are essential for function. These include the far N-terminal domain (TEN), which is the least conserved among phylogenetic groups, but is required for appropriate human, yeast and ciliated protozoa telomerase activity in vitro and telomere maintenance in vivo (Friedman & Cech (1999) Genes Dev. 13:2863-2874; Friedman, et al. (2003) Mol. Biol. Cell 14:1-13). The TEN domain has both DNA- and RNA-binding properties. DNA-binding facilitates loading of telomerase to the chromosomes while RNA-binding is non-specific and the role of this interaction is unclear (Hammond, et al. (1997) Mol. Cell. Biol. 17:296-308; Jacobs, et al. (2006) Nat. Struct. Mol. Biol. 13:218-225; Wyatt, et al. (2007) Mol. Cell. Biol. 27:3226-3240). A third domain, the telomerase RNA binding domain (TRBD), is located between the TEN and RT domains, and unlike the TEN-domain is highly conserved among phylogenetic groups and is essential for telomerase function both in vitro and in vivo (Lai, et al. (2001) Mol. Cell. Biol. 21:990-1000). The TRBD contains key signature motifs (CP- and T-motifs) implicated in RNA recognition and binding and makes extensive contacts with stem I and the TBE of TER, both of which are located upstream of the template (Bryan, et al. (2000) Mol. Cell. 6:493-499; Cunningham & Collins (2005) Mol. Cell. Biol. 25:4442-4454; Lai, et al. (2002) Genes Dev. 16:415-420; Lai, et al. (2001) supra; Miller, et al. (2000) supra; O'Connor, et al. (2005) J. Biol. Chem. 280:17533-17539). The TRBD-TER interaction is required for the proper assembly and enzymatic activity of the holoenzyme both in vitro and in vivo, and is thought to play an important role (although indirect) in the faithful addition of multiple, identical telomeric repeats at the ends of chromosomes (Lai, et al. (2002) supra; Lai, et al. (2003) Mol. Cell. 11:1673-1683; Lai, et al. (2001) supra).
Unlike TERT, TER varies considerably in size between species. For example, in Tetrahymena thermophila TER is only 159 nucleotides long (Greider & Blackburn (1989) Nature 337:331-337), while yeast harbors an unusually long TER of 1167 nucleotides (Zappulla & Cech (2004) Proc. Natl. Acad. Sci. USA 101:0024-10029). Despite the large differences in size and structure, the core structural elements of TER are conserved among phylogenetic groups, suggesting a common mechanism of telomere replication among organisms (Chen, et al. (2000) Cell 100:503-514; Chen & Greider (2003) Genes Dev. 17:2747-2752; Chen & Greider (2004) Trends Biochem. Sci. 29:183-192; Ly, et al. (2003) Mol. Cell. Biol. 23:6849-6856; Theimer & Feigon (2006) Curr. Opin. Struct. Biol. 16:307-318). These include the template, which associates loosely with the RT domain, and provides the code for telomere synthesis, and the TBE, which partly regulates telomerase's repeat addition processivity. In Tetrahymena thermophila, the TBE is formed by stem II and the flanking single stranded regions, and is located upstream and in close proximity to the template (Lai, et al. (2002) supra; Lai, et al. (2003) supra; Licht & Collins (1999) Genes Dev. 13:1116-1125). Low-affinity TERT-binding sites are also found in helix IV and the template recognition element (TRE) of Tetrahymena thermophila TER.
TERT function is regulated by a number of proteins, some of which act by direct association with the TERT/TER complex, while others act by regulating access of telomerase to the chromosome end through their association with the telomeric DNA (Aigner, et al. (2002) Curr. Opin. Genet. Dev. 12:80-85; Cong, et al. (2002) Microbiol. Mol. Biol. Rev. 66:407-425; Dong, et al. (2005) supra; Loayza & de Lange (2004)Cell 117:279-280; Smogorzewska & de Lange (2004) Annu. Rev. Biochem. 73:177-208; Smogorzewska, et al. (2000) Mol. Cell. Biol. 20:1659-1668; Witkin & Collins (2004) Genes Dev. 18:1107-1118; Witkin, et al. (2007) Mol. Cell. Biol. 27:2074-2083). For example, p65 in the ciliated protozoan Tetrahymena thermophila or its homologue p43 in Euplotes aediculatus, are integral components of the telomerase holoenzyme (Aigner & Cech (2004) RNA 10:1108-1118; Aigner, et al. (2003) Biochemistry 42:5736-5747; O'Connor & Collins (2006) Mol. Cell. Biol. 26:2029-2036; Prathapam, et al. (2005) Nat. Struct. Mol. Biol. 12:252-257; Witkin & Collins (2004) supra; Witkin, et al. (2007) supra). Both p65 and p43 are thought to bind and fold TER, a process required for the proper assembly and full activity of the holoenzyme. In yeast, recruitment and subsequent up regulation of telomerase activity requires the telomerase-associated protein Est1 (Evans & Lundblad (2002) Genetics 162:1101-1115; Hughes, et al. (1997) Ciba Found. Symp. 211:41-52; Lundblad (2003) Curr. Biol. 13:R439-441; Lundblad & Blackburn (1990) Cell 60:529-530; Reichenbach, et al. (2003) Curr. Biol. 13:568-574; Snow, et al. (2003) Curr. Biol. 13:698-704). Est1 binds the RNA component of telomerase, an interaction that facilitates recruitment of the holoenzyme to the eukaryotic chromosome ends via its interaction with the telomere binding protein Cdc13 (Chandra, et al. (2001) Genes Dev. 15:404-414; Evans & Lundblad (1999) Science 286:117-120; Lustig (2001) Nat. Struct. Biol. 8:297-299; Pennock, et al. (2001) Cell 104:387-396).
How telomerase and associated regulatory factors physically interact and function with each other to maintain appropriate telomere length is under investigation. Structural and biochemical characterization of these factors, both in isolation and complexed with one another, can be used to determine how the interaction of the TRBD domain with stem I and the TBE of TER facilitate the proper assembly and promote the repeat addition processivity of the holenzyme.
While in vitro and in vivo screening assays have been developed to identify agents which modulate telomerase activity or telomere binding, focus has not been placed on identifying agents with a degree of specificity for particular domains or substrate pockets. See, U.S. Pat. Nos. 7,067,283; 6,906,237; 6,787,133; 6,623,930; 6,517,834; 6,368,789; 6,358,687; 6,342,358; 5,856,096; 5,804,380; and 5,645,986 and US 2006/0040307.
SUMMARY OF THE INVENTION
The present invention features methods for identifying a compound which modulates the activity of telomerase. The methods of this invention involve, (a) designing or screening for a compound which binds to at least one amino acid residue of the TRBD domain, “thumb” domain, “palm” domain, “finger” domain, Motif 2, Motif B′, Thumb loop or Thumb helix domains; and (b) testing the compound designed or screened for in (a) for its ability to modulate the activity of telomerase, thereby identifying a compound that modulates the activity of telomerase. In one embodiment, the TRBD domain of telomerase contains the amino acid residues set forth in Table 1. In another embodiment, the “thumb,” “palm” and/or “finger” domain contains the amino acid residues set forth in Table 2. In other embodiments, step (a) is carried out in silico. Compounds identified by this method are also embraced by the present invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the structure of telomerase (TERT). FIG. 1A shows the primary of human, yeast and Tetrahymena thermophila TERT showing the functional domains and conserved motifs. FIG. 1B is the primary structure and conserved motifs of the Tribolium castaneum TERT. FIG. 1C shows TERT domain organization with the RNA-binding domain (TRBD), the reverse transcriptase domain composed of the “fingers” and “palm” subdomains, and the “thumb” domain depicted.
FIGS. 2A and 2B show a sequence alignment and schematic of secondary structure of Tetrahymena thermophila TRBDs (TETTH; SEQ ID NO:1) compared with the TRBDs from ciliated protozoa such as, Euplotes aediculatus (EUPAE; SEQ ID NO:2) and Oxytricha trifallax (OXYTR; SEQ ID NO:3); mammals such as human (SEQ ID NO:4) and mouse (SEQ ID NO:5); fungi such as Schizosaccharomyces pombe (SCHPO; SEQ ID NO:6) and Saccharomyces cerevisiae (YEAST; SEQ ID NO:7); and plants such as Arabidopsis thaliana (ARATH; SEQ ID NO:8) produced by ALSCRIPT Barton (1993) Protein Eng. 6:37-40). Conserved residues in key signature motifs are indicated and mutated residues that affect RNA-binding and telomerase function are also indicated. The solid triangles define the boundaries of the TRBD construct used in the studies herein.
FIGS. 3A-3C show the sequence alignment and surface conservation of Tribolium castaneum TERT (TRICA; SEQ ID NO:9) compared with TERTs from various phylogenetic groups including mammals such as mouse (SEQ ID NO:10) and human (SEQ ID NO:11); plants such as Arabidopsis thaliana (ARATH; SEQ ID NO:12); fungi such as Saccharomyces cerevisiae (YEAST; SEQ ID NO:13) and Schizosaccharomyces pombe (SCHPO; SEQ ID NO:14); and protozoa such as Tetrahymena thermophila (TETTH; SEQ ID NO:15) and Euplotes aediculatus (EUPAE; SEQ ID NO:16) produced by ClustalW2 (Larkin et al. (2007) Bioinformatics 23:2947-2948). Conserved residues in key signature motifs are indicated. K210 of helix α10 and polar residues (K406, K416, K418, N423) of the “thumb” domain implicated in direct contacts with the backbone of the DNA substrate are also shown.
FIG. 4 is a schematic of the primary structure of the RNA component (TER) of telomerase from Tetrahymena thermophila. Stem I, TBE and the template are indicated.
FIG. 5 depicts TERT RNA template associations. The nucleotide located at the 5′-end of the RNA template (rC1) is coordinated by Ile196 and Val197 of motif 2 and Gly309 of motif B′. rU2 interacts with Pro311 of motif B′ and rG3 coordinates the backbone of helix α15 via a water molecule (Wat18).
FIG. 6 depicts TERT telomeric DNA associations. Interactions between the thumb loop and the DNA are mostly backbone and solvent mediated. Also, the side chains of Lys416 and Asn423 that form part of the thumb loop extend toward the center of the ring and coordinate the DNA backbone.
FIG. 7 depicts DNA interactions with the primer grip region and the active site. Shown is a stereo view of the DNA interactions with motif E and the active site residues. The tip of the primer grip region (loop shown on left), formed by the backbone of residues Cys390 and Gly391 abuts the ribose group of C22 and this interaction guides the 3′-end of the DNA at the active site of the enzyme for nucleotide addition. The active site bound magnesium ion (sphere) coordinates the DNA backbone formed by the last two nucleotides. The nucleotide binding pocket of TERT, which is partially occupied by the last DNA nucleotide, is in part formed by the highly conserved residue Val342 and the invariant Tyr256 and Gly308.



DETAILED DESCRIPTION OF THE INVENTION
Telomerase, a ribonucleoprotein complex, replicates the linear ends of eukaryotic chromosomes, thus taking care of the “end of replication problem”. TERT contains an essential and universally conserved domain (TRBD; FIG. 1) that makes extensive contacts with the RNA (TER) component of the holoenzyme and this interaction facilitates TERT/TER assembly and repeat addition processivity. The TRBD domain is highly conserved among phylogenetic groups and is essential for the function of telomerase. Extensive biochemical and mutagenesis studies have localized TRBD binding to stem I and the TEB, interactions that are thought to be important for the proper assembly and stabilization of the TERT/TER complex as well as the repeat addition processivity of the holoenzyme. The atomic structure of the TRBD domain has now been identified, thereby providing information about TERT/TER binding. The RNA-binding site of TRBD is an extended groove on the surface of the protein that is partly hydrophilic and partly hydrophobic in nature and is formed by the previously identified T- and CP-motifs shown to be important for telomerase function. The size, organization and chemical nature of this groove indicates that the TRBD domain interacts with both double- and single-stranded nucleic acid, possibly stem I or II and the ssRNA that connects them.
In addition to the structure of the TRBD domain, it has now been shown that three highly conserved domains, TRBD, the reverse transcriptase (RT) domain, and the C-terminal extension thought to represent the putative “thumb” domain of TERT, are organized into a ring-like structure (FIG. 1C) that shares common features with retroviral reverse transcriptases, viral RNA polymerases and B-family DNA polymerases. Domain organization places motifs implicated in substrate binding and catalysis in the interior of the ring, which can accommodate seven-to-eight bases of double stranded nucleic acid. Modeling of an RNA/DNA heteroduplex in the interior of this ring reveals a perfect fit between the protein and the nucleic acid substrate and positions the 3′-end of the DNA primer at the active site of the enzyme providing evidence for the formation of an active telomerase elongation complex.
Indeed, as the high-resolution structure of the telomerase catalytic subunit, TERT, in complex with its RNA-templating region, part of the complementary DNA sequence, nucleotides and magnesium ions shows, the RNA-DNA hybrid adopts an A-form helical structure and is docked in the interior cavity of the TERT ring. Protein-nucleic acid associations induce global TERT conformational changes that lead to a decrease in the diameter of the interior cavity of the ring facilitating the formation of a tight telomerase elongation complex. The enzyme, which uses a two-metal binding mechanism for nucleotide association and catalysis, contains two nucleotides at its active site. One of the nucleotides is in position for attack by the 3′-hydroxyl of the incoming DNA primer while the second site appears to hold the nucleotide transiently, allowing for RNA template-dependent selectivity and telomerase processivity.
The TRBD domain, as well as RT and “thumb” domains, are highly conserved domains among phylogenetic groups. As such, these domains serve as ideal candidates for telomerase inhibitors. In this regard, telomerase is an ideal target for treating human diseases or conditions relating to cellular proliferation and senescence.
Accordingly, the present invention relates to the use of the high-resolution structure of Tetrahymena thermophila and Tribolium castaneum telomerases for the identification of effector molecules that modulate the activity of telomerase. The term “effector” refers to any agonist, antagonist, ligand or other agent that affects the activity of telomerase. Effectors include, but are not limited to, peptides, carbohydrates, nucleic acids, lipids, fatty acids, hormones, organic compounds, and inorganic compounds. The information obtained from the crystal structures herein reveal detailed information which is useful in the design, isolation, screening and determination of potential compounds which modulate the activity of telomerase. Compounds that bind the TRBD domain and, e.g., sterically block TER binding or block RNP assembly act as effective telomerase-specific inhibitors, whereas compounds that mimic or facilitate TER binding or RNP assembly act as effective telomerase-specific activators. Compounds that bind and block the active site or nucleotide binding site can also modulate telomerase activity. Similarly, compounds that interact with one or more amino acid residues of telomerase in direct contact with DNA can block DNA binding and act as effective telomerase-specific inhibitors, whereas compounds that mimic DNA act as effective telomerase-specific activators.
The effector molecules of the invention have a wide variety of uses. For example, it is contemplated that telomerase modulators will be effective therapeutic agents for treatment of human diseases or conditions. Screening for agonists provides for compositions that increase telomerase activity in a cell (including a telomere-dependent replicative capacity, or a partial telomerase activity). Such agonistic compositions provide for methods of immortalizing otherwise normal untransformed cells, including cells which can express useful proteins. Such agonists can also provide for methods of controlling cellular senescence. Conversely, screening for antagonist activity provides for compositions that decrease telomere-dependent replicative capacity, thereby mortalizing otherwise immortal cells, such as cancer cells. Screening for antagonistic activity provides for compositions that decrease telomerase activity, thereby preventing unlimited cell division of cells exhibiting unregulated cell growth, such as cancer cells. In general, the effector molecules of the invention can be used whenever it is desired to increase or decrease telomerase activity in a cell or organism.
Broadly, the methods of the invention involve designing or screening for a test compound which binds to at least one amino acid residue of an essential telomerase domain or pocket disclosed herein; and testing the compound designed or screened for its ability to modulate the activity of telomerase. In certain embodiments, the method of the present invention is carried out using various in silico, in vitro and/or in vivo assays based on detecting interactions between one or more domains or domain residues; or pockets or pocket residues of telomerase and a test compound.
In the context of the present invention, telomerase refers to a family of enzymes which maintain telomere ends by addition of the telomere repeat TTAGGG. Telomerases are described, e.g., by Nakamura, et al. (1997) Science 277(5328):955-9 and O'Reilly, et al. (1999)Curr. Opin. Struct. Biol. 9(1):56-65. Exemplary telomerase enzymes of use in accordance with the present invention are set forth herein in SEQ ID NOs:1-16 (FIGS. 2A-2B and FIGS. 3A-3C) and full-length sequences for telomerase enzymes are known in the art under GENBANK Accession Nos. AAC39140 (Tetrahymena thermophila), NP—197187 (Arabidopsis thaliana), NP—937983 (Homo sapiens), CAA18391 (Schizosaccharomyces pombe), NP—033380 (Mus musculus), NP—013422 (Saccharomyces cerevisiae), AAC39163 (Oxytricha trifallax), CAE75641 (Euplotes aediculatus) and NP—001035796 (Tribolium castaneum). For the purposes of the present invention, reference to telomerase refers to allelic and synthetic variants of telomerase, as well as fragments of telomerase. Synthetic variants include those which have at least 80%, preferably at least 90%, homology to a telomerase disclosed herein. More preferably, such variants correspond to the sequence of a telomerase provided herein, but have one or more, e.g., from 1 to 10, or from 1 to 5 substitutions, deletions or insertions of amino acids. Fragments of telomerase and variants thereof are preferably between 20 and 500 amino acid residues in length or between 50 and 300 amino acids in length. An exemplary fragment includes the approximately 250 amino acid residues encompassing the TRBD domain of telomerase. Other fragments include the “thumb” domain and the reverse transcriptase domain and its subdomains, i.e., the “finger” and “palm” subdomains. As depicted in FIG. 1A and FIGS. 2A and 2B, the TRBD domain encompasses amino acid residues at or about 254-519 of T. thermophila telomerase. As depicted in FIG. 1B and FIGS. 3A-3C, the reverse transcriptase domain encompasses amino acid residues at or about 160-403 of T. castaneum telomerase, and the “thumb” domain encompasses amino acid residues at or about 404-596 of T. castaneum telomerase. Based upon the amino acid sequence comparisons depicted in FIGS. 2A, 2B, and 3A-3C, suitable domains and fragments of telomerases from other species can be readily obtained based upon the location of equivalent amino acid residues in a telomerase from other species.
The nearly all-helical structure of TRBD provides a nucleic acid binding fold suitable for TER binding. An extended pocket on the surface of the protein, formed by two conserved motifs (CP- and T-motifs) provides TRBD's RNA-binding pocket. The width and the chemical nature of this pocket indicate that it binds both single- and double-stranded RNA, likely stem I and the template boundary element (TBE). As disclosed herein, the structure of T. thermophila telomerase identified key amino acid residues involved in RNP assembly of telomerase and the interaction between telomerase TRBD and TER. Accordingly, the present invention embraces a compound, which binds to at least one amino acid residue of the TRBD domain. Essential amino acid residues of this domain are provided in Table 1. The location of these residues in telomerases from other organisms is also listed in Table 1. In particular embodiments, a compound of the invention binds to one or more of the amino acid residues listed in Table 1, thereby modulating the activity of telomerase.








TABLE 1







Essential



TRBD
Location in telomerases of other organisms
















Residues*
Tt#
Tc†
At#
Sp#
Hs#
Mm#
Sc#
Ot#
Ea#





T-motif*











F476
F233
Y134
F265
F185
F246
F231
F186
F202
F206


Y477
Y234
Y135
Y266
Y186
Y247
Y232
Y187
Y203
Y207


T479
T236
P137
T268
T188
T249
T234
T189
T205
T209


E480
E237
I138
E269
E189
E250
E235
E190
E206
E210


Y491
Y248
I149
Y280
F200
Y261
Y246
F200
Y217
Y221


R492
R249
R150
R281
R201
R262
R247
R201
R218
R222


K493
K250
K151
K282
K202
K263
K248
H202
K219
L223


W496
W253
Y154
W285
W205
W266
W251
W205
W222
W226


CP-motif*


F323
F80
L36
L95
L55
Y90
Y90
Y58
F55
F59


L327
L84
K39
L99
Y59
L94
L94
L62
L59
L63


K328
K85
H40
D100
N60
K95
R95
N63
S60
T64


K329
K86
K41
K101
H61
T96
S96
S64
K61
K65


C331
C88
K43
C103
C63
C98
C98
C66
C63
C67


L333
L90
P45
L108
—
L100
—
—
L65
L69


P334
P91
V46
Q109
—
R101
—
—
P66
P70


QFP-motif*


Q375
Q132
Q47
Q158
Q83
Q145
Q130
R87
Q103
C107


I376
I133
I48
V159
V84
V146
V131
V88
I104
V108


L380
L137
L52
I163
L88
V150
L135
I92
L108
I112


I383
I140
I55
I166
I91
C153
C138
I95
F111
F115


I384
I141
I56
C167
L92
L154
L139
L96
V112
F116


C387
C144
—
I170
V95
L157
V142
L99
V115
I119


V388
V145
—
V171
F96
V158
V143
L100
F116
L120


P389
P146
P57
P172
P97
P159
S144
P101
P117
P121


L392
L149
Y60
L175
I100
L162
L147
M104
F120
F124


L393
L150
F61
L176
W101
W163
W148
F105
L121
L125


N397
N154
N66
Q181
I106
N168
N153
N110
N125
N129


L405
L162
V74
I189
L114
T176
L161
L118
M133
V137


F408
F165
I77
F192
F117
F179
F164
L121
F136
Y140


Y422
Y179
L91
F206
L131
L193
L178
L135
L150
L154


I423
I180
H92
L207
M132
T194
M179
L136
L151
L155


M426
M183
Y95
V210
I135
M197
M182
L139
F154
I158


W433
W190
W102
F217
W142
W204
W189
W146
W161
W165


F434
F191
L103
F218
L143
L205
L190
L147
L162
M166





Tt, Tetrahymena thermophila;


At, Arabidopsis thaliana;


Hs, Homo sapiens;


Sp, Schizosaccharomyces pombe;


Mm, Mus musculus;


Sc, Saccharomyces cerevisiae;


Tc, Tribolium castaneum;


Ot, Oxytricha trifallax and


Ea, Euplotes aediculatus.


*Location is with reference to the full-length T. thermophila telomerase.


#Location is with reference to the telomerase sequences depicted in FIGS. 2A and 2B, i.e., SEQ ID NOs: 1-8.


†location is with reference to the telomerase sequence depicted in FIGS. 3A-3C.






As further disclosed herein, the structure of T. castaneum telomerase identified key amino acid residues of the reverse transcriptase and “thumb” domains. In particular, key amino acid residues of the nucleotide binding pocket were identified as well as amino acid residues which appear to make direct contacts with the backbone of the DNA substrate. Accordingly, the present invention also embraces a compound, which binds to at least one amino acid residue of the nucleotide binding pocket of telomerase or residues which make direct contact with DNA. These residues are found in the “palm” and “finger” subdomains of the reverse transcriptase domain and the “thumb” domain of T. castaneum telomerase. Essential amino acid residues of these domains are provided in Table 2. The location of these amino acid residues in various species is also listed in Table 2.








TABLE 2







Domain
Location in telomerases of other organisms*














Residues*
Tt
At
Sp
Hs
Mm
Sc
Ea





“Palm”









K210
R573
K644
K547
N666
N656
E483
T558


V250
L617
A690
I589
V711
A704
F529
M602


D251
D618
V691
D590
D712
D705
D530
D603


I252
I619
D692
I591
V713
V706
V531
I604


A255
C622
A695
C594
A716
A706
C534
C607


Y256
Y623
F696
Y595
Y717
Y707
Y535
Y608


G257
D624
D697
D596
D718
D708
D536
D609


G305
G770
G801
G703
G830
G823
G629
G734


L306
I771
I802
I704
I831
I824
L630
I735


L307
P772
P803
P705
P832
P825
F631
P736


Q308
Q773
Q804
Q706
Q833
Q826
Q632
Q737


G309
G774
H805
G707
G834
G827
G633
G738


V342
T814
I859
V741
V867
V860
A669
T780


D343
D815
D860
D742
D868
D861
D670
D781


D344
D816
D861
D743
D869
D862
D671
D782


Y345
Y817
Y862
F744
F870
F863
L672
Y783


F346
L818
L863
L745
L871
L864
F673
L784


F347
F819
F864
F746
L872
L865
I674
L785


C348
I820
V865
I747
V873
V866
I675
I786


S349
S821
S866
T748
T874
T867
S676
T787


N369
N846
N891
S773
N899
N892
N701
N812


K372
K849
K894
K776
K902
K895
K704
K815


T373
I850
F895
T777
T903
T896
I705
L816


“Finger”


L184
L533
F612
I502
L621
L611
M438
L514


N185
R534
R613
R503
R622
R612
P439
R515


I186
I535
F614
L504
F623
F613
I440
L516


I187
I536
L615
L505
I624
I614
I441
I517


P188
P537
P616
P506
P625
P615
P442
P518


K189
K538
K617
K507
K626
K616
K443
K519


F193
F542
V621
F511
L630
L620
N447
F523


R194
R543
R622
R512
R631
R621
E448
R524


A195
P544
M623
L513
P632
P622
F449
P525


I196
I545
V624
I514
I633
I623
R450
I526


V197
M546
L625
T515
V634
V624
I451
M527


“Thumb”


K406
Q888
T937
P815
S943
S936
S729
N860


K416
T898
T947
T825
S953
S946
K739
T869


K418
N900
S949
S826
T955
T948
S741
N871


N423
K906
K955
H832
K961
K954
R746
K877





Tt, Tetrahymena thermophila;


At, Arabidopsis thaliana;


Hs, Homo sapiens;


Sp, Schizosaccharomyces pombe;


Mm, Mus musculus;


Sc, Saccharomyces cerevisiae;; and


Ea, Euplotes aediculatus.


*Location is with reference to the Tribolium castaneum telomerase sequence depicted in FIGS. 3A-3C.






Compounds designed or screened for in accordance with the present invention can interact or bind with at least one of the amino acid residues of one or more domains disclosed herein via various heterogeneous interactions including, but not limited to van der Waals contacts, hydrogen bonding, ionic interactions, polar contacts, or combinations thereof. In this regard, the terms “bind,” “binding,” “interact,” or “interacting” are used interchangeably herein to describe the interactions between telomerase and modulators thereof. In general, it is desirable that the compound interacts with 2, 3, 4, 5, 6 or more of the amino acid residues of a domain disclosed herein to enhance the specificity of the compound for one or more telomerase proteins. Accordingly, it is desirable that the compound interacts with between 2 and 20, 2 and 10, or 2 and 5 of one or more of the domains or pockets disclosed herein.
In one embodiment, the compound interacts with one or more essential amino acids of the QFP-motif, T-motif or CP-motif. In another embodiment, the compound interacts with one or more essential amino acids of the T-motif and CP-motif. In a further embodiment, the compound interacts with one or more essential amino acids as set forth in Table 1. In particular embodiments, the compound interacts with between 2 and 33 amino acid residues of the TRBD domain. In a particular embodiment, the compound interacts with one or more essential amino acid residues set forth in Table 1, which have not been previously identified by mutation to affect RNA-binding and telomerase activity.
In another embodiment of the invention, the compound interacts with one or more essential amino acids of the nucleotide binding pocket. In a further embodiment, the compound interacts with one or more essential amino acids of telomerase in direct contact with DNA. In yet a further embodiment, the compound interacts with one or more essential amino acids as set forth in Table 2. In a particular embodiment, the compound interacts with one or more essential amino acid residues set forth in Table 2, which have not been previously identified by mutation to affect nucleotide binding, DNA binding or telomerase activity. In particular embodiments, the compound interacts with between 2 and 34 amino acid residues of the finger and palm domains. In specific embodiments, the compound binds to one or more of the amino acid residues selected from the group of K189, R194, Y256, Q308, V342, and K372 of T. castaneum telomerase or their equivalents in other species. In other embodiments, the compound interacts with between 2 and 15 amino acid residues of the palm and thumb domains. In specific embodiments, the compound binds to one or more of the amino acid residues selected from the group of K210, K406, K416, K418, or N423 of T. castaneum telomerase or their equivalents in other species.
As further disclosed herein, the structure of T. castaneum TERT in complex with its RNA-templating region, part of the complementary DNA, nucleotides and magnesium ions identified key amino acid residues of various domains which coordinate to place substrates in proximity with the active site (D251, D343 and D344) of telomerase. These key amino acid residues are located in Motif 2, Motif B′, Thumb Loop, and Thumb Helix regions of telomerase (FIG. 3). Accordingly, the present invention further embraces a compound, which binds to at least one amino acid residue of the Motif 2, Motif B′, Thumb Loop, or Thumb Helix of telomerase. Amino acid residues of Motif 2 and Motif B′ are selected from the group of Ile196, Val197, Gly309 and Pro311 of T. castaneum telomerase or their equivalents in other species. Amino acid residues of the Thumb Loop and Thumb Helix are selected from the group of Tyr256, Gln308, Val342, Cys390, Gly391, Lys416, and Asn423 of T. castaneum telomerase or equivalent amino acid residues thereof in a telomerase from another species.
In accordance with the present invention, molecular design techniques can be employed to design, identify and synthesize chemical entities and compounds, including inhibitory and stimulatory compounds, capable of binding to one or more amino acids of telomerase. The structure of the domains of telomerase can be used in conjunction with computer modeling using a docking program such as GRAM, DOCK, HOOK or AUTODOCK (Dunbrack, et al. (1997) Folding& Design 2:27-42) to identify potential modulators of telomerase proteins. This procedure can include computer fitting of compounds to domains or pockets disclosed herein to, e.g., ascertain how well the shape and the chemical structure of the compound will complement the TRBD domain; or to compare the compound with the binding of TER in the TRBD; or compare the compound with the binding of a DNA molecule to the “thumb” domain; compare the compound with binding of a nucleotide substrate to the nucleotide binding pocket; or compare the compound to binding of TERT substrates in the Motif 2, Motif B′, Thumb Loop, and Thumb Helix. Computer programs can also be employed to estimate the attraction, repulsion and stearic hindrance of the telomerase protein and effector compound. Generally, the tighter the fit, the lower the stearic hindrances, the greater the attractive forces, and the greater the specificity, which are important features for a specific effector compound which is more likely to interact with the telomerase protein rather than other classes of proteins. In so far as the present invention has identified the amino acid residues specifically involved in substrate binding, the present invention offers specificity not heretofore possible with conventional screening assays.
Alternatively, a chemical-probe approach can be employed in the design of telomerase modulators or effectors. For example, Goodford ((1985) J. Med. Chem. 28:849) describes several commercial software packages, such as GRID (Molecular Discovery Ltd., Oxford, UK), which can be used to probe the telomerase domains with different chemical probes, e.g., water, a methyl group, an amine nitrogen, a carboxyl oxygen, and a hydroxyl. Favored sites for interaction between these regions or sites of the telomerase domains and each probe are thus determined, and from the resulting three-dimensional pattern of such regions or sites a putative complementary molecule can be generated.
The compounds of the present invention can also be designed by visually inspecting the three-dimensional structure of the telomerase domains to determine more effective inhibitors or activators. This type of modeling is generally referred to as “manual” drug design. Manual drug design can employ visual inspection and analysis using a graphics visualization program such as “O” (Jones, et al. (1991) Acta Crystallographica Section A A47:110-119).
Initially effector compounds are selected by manual drug design. The structural analog thus designed can then be modified by computer modeling programs to better define the most likely effective candidates. Reduction of the number of potential candidates is useful as it may not be possible to synthesize and screen a countless number of compound variations that may have some similarity to known inhibitory molecules. Such analysis has been shown effective in the development of HIV protease inhibitors (Lam, et al. (1994) Science 263:380-384; Wlodawer, et al. (1993) Ann. Rev. Biochem. 62:543-585; Appelt (1993) Perspectives in Drug Discovery and Design 1:23-48; Erickson (1993) Perspectives in Drug Discovery and Design 1:109-128). Alternatively, random screening of a small molecule library could lead to modulators whose activity may then be analyzed by computer modeling as described above to better determine their effectiveness as inhibitors or activators.
Programs suitable for searching three-dimensional databases include MACCS-3D and ISIS/3D (Molecular Design Ltd, San Leandro, Calif.), ChemDBS-3D (Chemical Design Ltd., Oxford, UK), and Sybyl/3 DB Unity (Tripos Associates, St Louis, Mo.). Programs suitable for compound selection and design include, e.g., DISCO (Abbott Laboratories, Abbott Park, Ill.), Catalyst (Bio-CAD Corp., Mountain View, Calif.), and ChemDBS-3D (Chemical Design Ltd., Oxford, UK).
The compounds designed using the information of the present invention can bind to all or a portion of the TRBD domain, nucleotide binding domain, “thumb” domain, Motif 2, Motif B′, Thumb Loop, and/or Thumb Helix of telomerase and may be more potent, more specific, less toxic and more effective than known inhibitors of telomerase. The designed compounds can also be less potent but have a longer half-life in vivo and/or in vitro and therefore be more effective at modulating telomerase activity in vivo and/or in vitro for prolonged periods of time. Such designed modulators are useful to inhibit or activate telomerase activity to, e.g., alter lifespan or proliferative capacity of a cell.
The present invention also provides the use of molecular design techniques to computationally screen small molecule databases for chemical entities or compounds that can bind to telomerase in a manner analogous to its natural substrates. Such computational screening can identify various groups which interact with one or more amino acid residues of a domain disclosed herein and can be employed to synthesize modulators of the present invention.
In vitro (i.e., in solution) screening assays are also embraced by the present invention. For example, such assays include combining telomerase, the telomerase TRBD domain (e.g., as disclosed herein), or other fragments of telomerase (e.g., motifs or pockets disclosed herein) with or without substrates or cofactors (e.g., TER, complementary DNA, nucleotides, or Mg) in solution and determining whether a test compound can block or enhance telomerase activity. In this respect, in vitro screening assays can be carried out to monitor nucleotide or DNA binding in the presence or absence of a test compound.
Compounds screened in accordance with the methods of the present invention are generally derived from libraries of agents or compounds. Such libraries can contain either collections of pure agents or collections of agent mixtures. Examples of pure agents include, but are not limited to, proteins, polypeptides, peptides, nucleic acids, oligonucleotides, carbohydrates, lipids, synthetic or semi-synthetic chemicals, and purified natural products. Examples of agent mixtures include, but are not limited to, extracts of prokaryotic or eukaryotic cells and tissues, as well as fermentation broths and cell or tissue culture supernates. Databases of chemical structures are also available from a number of sources including Cambridge Crystallographic Data Centre (Cambridge, UK) and Chemical Abstracts Service (Columbus, Ohio). De novo design programs include Ludi (Biosym Technologies Inc., San Diego, Calif.), Sybyl (Tripos Associates) and Aladdin (Daylight Chemical Information Systems, Irvine, Calif.).
Library screening can be performed using any conventional method and can be performed in any format that allows rapid preparation and processing of multiple reactions. For in vitro screening assays, stock solutions of the test compounds as well as assay components can be prepared manually and all subsequent pipeting, diluting, mixing, washing, incubating, sample readout and data collecting carried out using commercially available robotic pipeting equipment, automated work stations, and analytical instruments for detecting the signal generated by the assay. Examples of such detectors include, but are not limited to, luminometers, spectrophotometers, and fluorimeters, and devices that measure the decay of radioisotopes.
After designing or screening for a compound which binds to at least one amino acid residue of a domain or pocket disclosed herein, the compound is subsequently tested for its ability to modulate the activity of telomerase. Such activities of telomerase include telomerase catalytic activity (which may be either processive or non-processive activity); telomerase processivity; conventional reverse transcriptase activity; nucleolytic activity; primer or substrate (telomere or synthetic telomerase substrate or primer) binding activity; dNTP binding activity; RNA (i.e., TER) binding activity; and protein binding activity (e.g., binding to telomerase-associated proteins, telomere-binding proteins, or to a protein-telomeric DNA complex). See, e.g., assays disclosed in U.S. Pat. No. 7,262,288.
Telomerase catalytic activity is intended to encompass the ability of telomerase to extend a DNA primer that functions as a telomerase substrate by adding a partial, one, or more than one repeat of a sequence (e.g., TTAGGG) encoded by a template nucleic acid (e.g., TER). This activity may be processive or non-processive. Processive activity occurs when a telomerase RNP adds multiple repeats to a primer or telomerase before the DNA is released by the enzyme complex. Non-processive activity occurs when telomerase adds a partial, or only one, repeat to a primer and is then released. In vivo, however, a non-processive reaction could add multiple repeats by successive rounds of association, extension, and dissociation. This can occur in vitro as well, but it is not typically observed in standard assays due to the vastly large molar excess of primer over telomerase in standard assay conditions. Conventional assays for determining telomerase catalytic activity are disclosed, for example, in Morin (1989) Cell 59:521; Morin (1997) Eur. J. Cancer 33:750; U.S. Pat. No. 5,629,154; WO 97/15687; WO 95/13381; Krupp, et al. (1997) Nucleic Acids Res. 25:919; Wright, et al. (1995) Nuc. Acids Res. 23:3794; Tatematsu, et al. (1996) Oncogene 13:2265.
Telomerase conventional reverse transcriptase activity is described in, e.g., Morin (1997) supra, and Spence, et al. (1995) Science 267:988. Because telomerase contains conserved amino acid motifs that are required for reverse transcriptase catalytic activity, telomerase has the ability to transcribe certain exogenous (e.g., non-TER) RNAs. A conventional RT assay measures the ability of the enzyme to transcribe an RNA template by extending an annealed DNA primer. Reverse transcriptase activity can be measured in numerous ways known in the art, for example, by monitoring the size increase of a labeled nucleic acid primer (e.g., RNA or DNA), or incorporation of a labeled dNTP. See, e.g., Ausubel, et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
Because telomerase specifically associates with TER, the DNA primer/RNA template for a conventional RT assay can be modified to have characteristics related to TER and/or a telomeric DNA primer. For example, the RNA can have the sequence (CCCTAA)n, where n is at least 1, or at least 3, or at least 10 or more. In one embodiment, the (CCCTAA)n region is at or near the 5′ terminus of the RNA (similar to the 5′ locations of template regions in telomerase RNAs). Similarly, the DNA primer may have a 3′ terminus that contains portions of the TTAGGG telomere sequence, for example XnTTAG, XnAGGG, etc., where X is a non-telomeric sequence and n is 6-30. In another embodiment, the DNA primer has a 5′ terminus that is non-complementary to the RNA template, such that when the primer is annealed to the RNA, the 5′ terminus of the primer remains unbound. Additional modifications of standard reverse transcription assays that may be applied to the methods of the invention are known in the art.
Telomerase nucleolytic activity is described in, e.g., Morin (1997) supra and Collins & Grieder (1993) Genes Dev. 7:1364. Telomerase preferentially removes nucleotides, usually only one, from the 3′ end of an oligonucleotide when the 3′ end of the DNA is positioned at the 5′ boundary of the DNA template sequence, in humans and Tetrahymena, this nucleotide is the first G of the telomeric repeat (TTAGG in humans). Telomerase preferentially removes G residues but has nucleolytic activity against other nucleotides. This activity can be monitored using conventional methods known in the art.
Telomerase primer (telomere) binding activity is described in, e.g., Morin (1997) supra; Collins, et al. (1995) Cell 81:677; Harrington, et al. (1995) J. Biol. Chem. 270:8893. There are several ways of assaying primer binding activity; however, a step common to most methods is incubation of a labeled DNA primer with telomerase or telomerase/TER under appropriate binding conditions. Also, most methods employ a means of separating unbound DNA from protein-bound DNA. Such methods can include, e.g., gel-shift assays or matrix binding assays. The DNA primer can be any DNA with an affinity for telomerase, such as, for example, a telomeric DNA primer like (TTAGGG)n, where n could be 1-10 and is typically 3-5. The 3′ and 5′ termini can end in any location of the repeat sequence. The primer can also have 5′ or 3′ extensions of non-telomeric DNA that could facilitate labeling or detection. The primer can also be derivatized, e.g., to facilitate detection or isolation.
Telomerase dNTP binding activity is described in, e.g., Morin (1997) supra and Spence, et al. (1995) supra. Telomerase requires dNTPs to synthesize DNA. The telomerase protein has a nucleotide binding activity and can be assayed for dNTP binding in a manner similar to other nucleotide binding proteins (Kantrowitz, et al. (1980) Trends Biochem. Sci. 5:124). Typically, binding of a labeled dNTP or dNTP analog can be monitored as is known in the art for non-telomerase RT proteins.
Telomerase RNA (i.e., TER) binding activity is described in, e.g., Morin (1997) supra; Harrington, et al. (1997) Science 275:973; Collins, et al. (1995) Cell 81:677. The RNA binding activity of a telomerase protein of the invention may be assayed in a manner similar to the DNA primer binding assay described supra, using a labeled RNA probe. Methods for separating bound and unbound RNA and for detecting RNA are well known in the art and can be applied to the activity assays of the invention in a manner similar to that described for the DNA primer binding assay. The RNA can be full length TER, fragments of TER or other RNAs demonstrated to have an affinity for telomerase or TRBD. See U.S. Pat. No. 5,583,016 and WO 96/40868.
In addition to determining either RNA or DNA binding, assays monitoring binding of the RNA-templating region and the complementary telomeric DNA sequence can also be carried out. By way of illustration, Example 5 describes the use of fluorescence polarization to determine binding of compounds to TERT protein.
To further evaluate the efficacy of a compound identified using the method of the invention, one of skill will appreciate that a model system of any particular disease or disorder involving telomerase can be utilized to evaluate the adsorption, distribution, metabolism and excretion of a compound as well as its potential toxicity in acute, sub-chronic and chronic studies. For example, the effector or modulatory compound can be tested in an assay for replicative lifespan in Saccharomyces cerevisiae (Jarolim, et al. (2004) FEMS Yeast Res. 5(2):169-77). See also, McChesney, et al. (2005) Zebrafish 1(4):349-355 and Nasir, et al. (2001) Neoplasia 3(4):351-359, which describe marine mammal and dog tissue model systems for analyzing telomerase activity.
Compounds which bind to at least one amino acid residue of one or more of the telomerase domains or pockets disclosed herein can be used in a method for modulating (i.e., blocking or inhibiting, or enhancing or activating) a telomerase. Such a method involves contacting a telomerase either in vitro or in vivo with an effective amount of a compound that interacts with at least one amino acid residue of a domain or pocket of the invention so that the activity of telomerase is modulated. An effective amount of an effector or modulatory compound is an amount which reduces or increases the activity of the telomerase by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to telomerase not contacted with the compound. Such activity can be monitored by enzymatic assays detecting activity of the telomerase or by monitoring the expression or activity of proteins which are known to be associated with or regulated by telomerase.
One of skill in the art can appreciate that modulating the activity of telomerase can be useful in selectively analyzing telomerase signaling events in model systems as well as in preventing or treating diseases, conditions, and disorders involving telomerase. The selection of the compound for use in preventing or treating a particular disease or disorder will be dependent upon the particular disease or disorder. For example, human telomerase is involved in cancer and therefore a compound which inhibits telomerase will be useful in the prevention or treatment of cancer including solid tumors (e.g., adenocarcinoma of the breast, prostate, and colon; melanoma; non-small cell lung; glioma; as well as bone, breast, digestive system, colorectal, liver, pancreatic, pituitary, testicular, orbital, head and neck, central nervous system, acoustic, pelvic, respiratory tract, and urogenital neoplasms) and leukemias (e.g., B-cell, mixed-cell, null-cell, T-cell, T-cell chronic, lymphocytic acute, lymphocytic chronic, mast-cell, and myeloid). Cancer cells (e.g., malignant tumor cells) that express telomerase activity (telomerase-positive cells) can be mortalized by decreasing or inhibiting the endogenous telomerase activity. Moreover, because telomerase levels correlate with disease characteristics such as metastatic potential (e.g., U.S. Pat. Nos. 5,639,613; 5,648,215; 5,489,508; Pandita, et al. (1996) Proc. Am. Ass. Cancer Res. 37:559), any reduction in telomerase activity could reduce the aggressive nature of a cancer to a more manageable disease state (increasing the efficacy of traditional interventions).
By way of illustration, Example 6 describes cell-based assays and animal model systems which are useful for assessing the inhibition of tumor cell growth by one or more compounds of the invention. Another useful method for assessing anticancer activities of compounds of the invention involves the multiple-human cancer cell line screening assays run by the National Cancer Institute (see, e.g., Boyd (1989) in Cancer: Principles and Practice of Oncology Updates, DeVita et al., eds, pp. 1-12). This screening panel, which contains approximately 60 different human cancer cell lines, is a useful indicator of in vivo antitumor activity for a broad variety of tumor types (Greyer, et al. (1992) Seminars Oncol. 19:622; Monks, et al. (1991) Natl. Cancer Inst. 83:757-766), such as leukemia, non-small cell lung, colon, melanoma, ovarian, renal, prostate, and breast cancers. Antitumor activities can be expressed in terms of ED50 (or GI50), where ED50 is the molar concentration of compound effective to reduce cell growth by 50%. Compounds with lower ED50 values tend to have greater anticancer activities than compounds with higher ED50 values.
Upon the confirmation of a compound's potential activity in one or more in vitro assays, further evaluation is typically conducted in vivo in laboratory animals, for example, measuring reduction of lung nodule metastases in mice with B16 melanoma (e.g., Schuchter, et al. (1991) Cancer Res. 51:682-687). The efficacy of a compound of the invention either alone or as a drug combination chemotherapy can also be evaluated, for example, using the human B-CLL xenograft model in mice (e.g., Mohammad, et al. (1996) Leukemia 10:130-137). Such assays typically involve injecting primary tumor cells or a tumor cell line into immune compromised mice (e.g., a SCID mouse or other suitable animal) and allowing the tumor to grow. Mice carrying the tumors are then treated with the compound of interest and tumor size is measured to follow the effect of the treatment. Alternatively, the compound of interest is administered prior to injection of tumor cells to evaluate tumor prevention. Ultimately, the safety and efficacy of compounds of the invention are evaluated in human clinical trials.
Compounds that activate or stimulate telomerase activity can be used in methods for treating or preventing a disease or condition induced or exacerbated by cellular senescence in a subject; methods for decreasing the rate of senescence of a subject, e.g., after onset of senescence; methods for extending the lifespan of a subject; methods for treating or preventing a disease or condition relating to lifespan; methods for treating or preventing a disease or condition relating to the proliferative capacity of cells; and methods for treating or preventing a disease or condition resulting from cell damage or death. Certain diseases of aging are characterized by cell senescence-associated changes due to reduced telomere length (compared to younger cells), resulting from the absence (or much lower levels) of telomerase activity in the cell. Telomerase activity and telomere length can be increased by, for example, increasing the activity of telomerase in the cell. A partial listing of conditions associated with cellular senescence in which increased telomerase activity can be therapeutic includes Alzheimer's disease, Parkinson's disease, Huntington's disease, and stroke; age-related diseases of the integument such as dermal atrophy, elastolysis and skin wrinkling, graying of hair and hair loss, chronic skin ulcers, and age-related impairment of wound healing; degenerative joint disease; osteoporosis; age-related immune system impairment (e.g., involving cells such as B and T lymphocytes, monocytes, neutrophils, eosinophils, basophils, NK cells and their respective progenitors); age-related diseases of the vascular system; diabetes; and age-related macular degeneration. Moreover, telomerase activators can be used to increase the proliferative capacity of a cell or in cell immortalization, e.g., to produce new cell lines (e.g., most human somatic cells).
Prevention or treatment typically involves administering to a subject in need of treatment a pharmaceutical composition containing an effective of a compound identified via screening methods of the invention. In most cases this will be a human being, but treatment of agricultural animals, e.g., livestock and poultry, and companion animals, e.g., dogs, cats and horses, is expressly covered herein. The selection of the dosage or effective amount of a compound is that which has the desired outcome of preventing, reducing or reversing at least one sign or symptom of the disease or disorder being treated. Methods for treating cancer and other telomerase-related diseases in humans are described in U.S. Pat. Nos. 5,489,508, 5,639,613, and 5,645,986. By way of illustration, a subject with cancer (including, e.g., carcinomas, melanomas, sarcomas, lymphomas and leukaemias) can experience unexplained weight loss, fatigue, fever, pain, skin changes, sores that do not heal, thickening or lump in breast or other parts of the body, or a nagging cough or hoarseness, wherein treatment with a compound of the invention can prevent, reduce, or reverse one or more of these symptoms.
Pharmaceutical compositions can be in the form of pharmaceutically acceptable salts and complexes and can be provided in a pharmaceutically acceptable carrier and at an appropriate dose. Such pharmaceutical compositions can be prepared by methods and contain carriers which are well-known in the art. A generally recognized compendium of such methods and ingredients is Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, Pa., 2000. A pharmaceutically-acceptable carrier, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, is involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated.
Examples of materials which can serve as pharmaceutically acceptable carriers include sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
The compositions of the present invention can be administered parenterally (for example, by intravenous, intraperitoneal, subcutaneous or intramuscular injection), topically (including buccal and sublingual), orally, intranasally, intravaginally, or rectally according to standard medical practices.
The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of a compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. This is considered to be within the skill of the artisan and one can review the existing literature on a specific compound or similar compounds to determine optimal dosing.
The invention is described in greater detail by the following non-limiting examples.
Example 1
Structure of Tetrahymena thermophila TERT
Protein Expression and Purification. The T. thermophila TERT residues 254-519 was identified by limited proteolysis and cloned into a modified version of the pET28b vector containing a cleavable hexa-histidine tag at its N-terminus. The protein was over-expressed in E. coli BL21 (pLysS) at 20° C. for 4 hours. The cells were lysed by sonication in 50 mM Tris-HCl, 10% glycerol, 0.5 M KCl, 5 mM β-mercaptoethanol, and 1 mM PMSF, pH 7.5 on ice. The protein was first purified over a Ni-NTA column followed by TEV cleavage of the hexa-histidine tag overnight at 4° C. The TRBD/TEV mix was diluted so that the concentration of imidazole was at 15 mM and the protein mix was passed over a Ni-NTA column to remove the TEV, the cleaved tag and any tagged protein. The Ni-NTA flow through was concentrated to 1 ml and diluted to a salt concentration of 0.15 M. The diluted TRBD sample was then passed over a POROS-HS column (PerSeptive Biosystems, Framingham, Mass.). At this stage, the protein was more than 99% pure. The protein was finally passed over a SEPHADEX-S200 sizing column pre-equilibrated with 50 mM Tris-HCl, 10% glycerol, 0.5 M KCl, and 2 mM DTT, pH 7.5 to remove any TRBD aggregates. The pure, monodisperse protein as indicated by SDS-page and dynamic light scattering, respectively, was concentrated to 8 mg/ml using an AMICON 10K cutoff (MILLIPORE, Billerica, Mass.) and the protein was stored at 4° C. for subsequent studies. Stock protein was dialyzed in 5 mM Tris-HCl, 500 mM KCl, 1 mM TCEP, pH 7.5 prior to crystallization trials.
Protein Crystallization and Data Collection. Initial plate-like clusters of TRBD that diffracted poorly ( ̃4 Å resolution) were grown at 4° C. using the sitting drop method by mixing on volume of dialyzed protein with one volume of reservoir solution containing 20% PEG 3350, 0.2 M NaNO3. Single, well diffracting crystals were grown in microbatch trays under paraffin oil by mixing one volume of dialyzed protein with an equivalent volume of 50 mM HEPES (pH 7.0), 44% PEG 400, 0.4 M NaNO3, 0.4 M NaBr and 1 mM TCEP at 4° C. Crystals were harvested into cryoprotectant solution that contained 25 mM HEPES (pH 7.0), 25% PEG 400, 0.2 M NaNO3, 0.2 M NaBr and 1 mM TCEP and were flash frozen in liquid nitrogen. Data were collected at the NSLS, beam line X6A and processed with HKL-2000 (Minor (1997) Meth. Enzymol. Macromole. Crystallogr. Part A 276:307-326) (Table 3). The crystals belong to the monoclinic space group P21 with one monomer in the asymmetric unit.










TABLE 3









Native
Holmium-Derivative










TRBD(254-519)
λ
Ho-λ1
Ho-λ2













Wavelength (Å)
0.9795
1.5347
1.5595


Space group
P21
P21
P21


Cell
39.4 67.2
39.2 68.2
39.2 68.2


dimensions (Å)
51.5 90.7
50.1 91.6
50.1 91.6


Resolution (Å)
  20-1.71
  50-2.59
  50-2.63



 (1.77-1.71)*
(2.69-2.59)
(3.02-2.63)


Redundancy
3.7 (3.0)
1.7 (1.8)
1.7 (1.8)


Completeness
99.3 (93.3)
92.5 (88.1)
92.9 (88.7)


(%)


Rsym (%)
 4.7 (48.1)
 7.3 (23.8)
 7.0 (21.5)


I/σ (I)
27.3 (2.6) 
  9 (3.4)
9.4 (3.7)


Phasing Analysis


Resolution (Å)
 50-2.7


Number of sites
2


Mean figure of merit
0.43


(FOM)





*Values in parentheses correspond to the highest resolution shell.






Structure Determination and Refinement. Initial phases were obtained from a two-wavelength MAD holmium (Ho) derivative that was prepared by co-crystallizing the protein with 5 mM HoCl3. Heavy atom sites were located using SOLVE (Terwilliger (2003) Methods Enzymol. 374:22-37) and the sites were refined and new phases calculated with MLPHARE (CCP4 (1994) Acta Crystallogr. D 50:760-763) as implemented in ELVES (Holton & Alber (2004) Proc. Natl. Acad. Sci. USA 101:1537-1542) (Table 3). Initial maps showed well-defined density only for the larger half of the molecule. The density for the smaller half of the molecule was weak mostly due to its intrinsic mobility with respect to larger half of the molecule. The problem associated with building the model into the density was exacerbated by the lack of information regarding the location of specific side chains such as selenomethionines. Key factors in building a complete model were successive rounds of PRIME and SWITCH in RESOLVE (Terwilliger (2002) Acta Crystallogr. D Biol. Crystallogr. 58:1937-1940) followed by manual building in COOT (Emsley & Cowtan (2004) Acta Crystallogr. D Biol. Crystallogr. 60:2126-2132). The model was refined using both CNS-SOLVE (Brunger, et al. (1998) Acta Crystallogr. D Biol. Crystallogr. 54:905-921) and REFMAC5 (Murshudov, et al. (1997) Acta Crystallogr. D Biol. Crystallogr. 53:240-255). The last cycles of refinement were carried out with TLS restraints as implemented in REFMAC5 (Table 4). Figures were prepared in PYMOL (DeLano (2002)) and electrostatic surfaces in APBS (Baker, et al. (2001) Proc. Natl. Acad. Sci. USA 98:10037-10041).







TABLE 4





TRBD(254-519)


















Refinement Statistics




Resolution (Å)
20-1.71



Rwork/Rfree (%)
20.0/23.9



RMSD bonds (Å)
0.008



RMSD angles (°)
0.831



Number of atoms



Protein
2145



Bromine
7



Water
213



Average B (Å2)



Protein
27.41



Bromine
42.63



Water
31.22



Ramachandran % (no res.)



Most favored
91.6



Allowed
8.4










TRBD Structure. To explore the role of the essential RNA-binding domain of telomerase (TRBD), a construct identified by limited proteolysis, containing residues 254-519 from T. thermophila (FIG. 1A) was purified to homogeneity. This protein construct was monomeric in solution as indicated by both gel filtration and dynamic light scattering. Crystals of this construct grew readily and diffracted to 1.71 Å resolution (Table 4). The protein was phased to 2.7 Å resolution by the multiwavelength anomalous dispersion method (MAD) using a holmium derivative and the phases were extended with the native dataset to 1.71 Å resolution (Table 4). In the refined structure there was clear density for residues 257-266 and 277-519.
The structure contains twelve α-helices linked together by several long loops and two short β-strands. The helices are organized so that the molecule is divided into two asymmetric halves linked together by three extended loops. The larger half is composed of nine α-helices, one of which (α11) runs along the middle of the domain and spans its entire length making contacts with all other eight helices. The smaller half of the molecule is composed of three helices (α4, α5 and α12), all of which are arranged at a  ̃120° angle to the plane of the larger half of the protein. The smaller half of the protein is somewhat more flexible than the larger half as suggested by its high B factors reflecting the intrinsic mobility of this region and may result from the absence of observable contacts with the RNA substrate. An interesting feature of the structure is a β-hairpin formed by the 15-residues that connect helices α11 and α12 of the larger and the smaller halves, respectively. The β-hairpin protrudes from the base of the crevice formed by the two halves of the protein and stands at a 45° angle to the plane of the smaller half of the molecule. The positioning and the fact that this hairpin is well-defined in the density could be attributed to helix α7 and the loop that connects it to helix α8. Both of these elements are conveniently positioned at the back of this hairpin holding it in place. A search in the protein structure database using the Dali server (Holm & Sander (1996) Science 273:595-603) produced no structural homologues, indicating that the TRBD domain of telomerase is a novel nucleic acid binding fold. The overall organization of the two halves of the protein has significant implications for nucleic acid recognition and binding.
The TRBD RNA-Binding Motifs. The ability of the TRBD domain to interact with TER has been attributed to two conserved motifs known as the CP-, and T-motifs, while a third motif known as the QFP-motif is thought to be important for RNP assembly (FIGS. 2A and 2B) (Bosoy, et al. (2003) J. Biol. Chem. 278:3882-3890; Bryan, et al. (2000) supra; Jacobs, et al. (2005) supra; Xia, et al. (2000) Mol. Cell. Biol. 20:5196-5207). The TRBD structure shows that the QFP-motif is formed by several mostly hydrophobic residues, which are located on the larger half of the molecule and are buried within the core of the domain making extensive hydrophobic contacts with the surrounding residues aiding in the fold of the protein. These residues included Gln375, Ile376, Leu380, Ile383, Ile384, Cys387, Val388, Pro389, Leu392, Leu393, Asn397, Leu405, Phe408, Tyr422, Ile423, Met426, Trp433, and Phe434. The location and the contacts of the QFP-residues indicate that they are not directly involved in nucleic acid binding.
The T-motif is located at the center of the molecule where the two halves of the protein meet and it is composed of residues that form both part of the β-hairpin and helix α12. Together these structural elements form a narrow ( ̃10 Å), well-defined pocket (T-pocket) that is lined by several solvent exposed and highly conserved residues (Phe476, Tyr477, Thr479, Glu480, Tyr491, Arg492, Lys493, and Trp496). Of particular note are the side chains of the invariant residues Tyr477 and Trp496, which are part of the β-hairpin and helix α12, respectively. Together these residues form a “hydrophobic pincer” that could sandwich the purine/pirimidine moiety of an interacting RNA nucleotide. In this structure, the side chains of Tyr477 and Trp496 are only 4 Å apart, which is not sufficient to accommodate a nucleotide base. Insertion of a base between the two side chains would require structural rearrangement of the T-pocket, possibly splaying of the two halves of the molecules apart. In addition to its hydrophobic part, the T-pocket also contains several hydrophilic residues such as Arg492 and Lys493 both of which are solvent exposed and are located at the interface of the T- and CP-pocket connecting the two together.
The CP-motif is formed by helix α3 and the following loop. In contrast to the T-motif, which is a narrow well-defined pocket, the CP-motif is composed a shallow, wide ( ̃20 Å), highly positively charged cavity located adjacent and beneath the entry of the T-pocket. Several of the conserved residues that form the CP-motif include Phe323, Leu327, Lys328, Lys329, Cys331, Leu333, and Pro334. These residues are buried in the core of the larger half or the region that connects the two halves of the molecule and are contributing to the protein fold. Of particular interest are residues Leu327, Cys331, Leu333 and Pro334 all of which are buried and make direct contacts with structural elements of the T-motif thus aiding in the formation of both the CP- and the T-pockets. For example, Leu327 and Cys331 are within Van der Waal contacts of the large hydrophobic side chain of the invariant Phe476 and the aliphatic part of the side chain of the conserved Arg492 both of which form part of the β-hairpin. Interestingly, Arg492 is located at the base of helix α12 and its contact with Leu327, Cys331, and Leu333 partially helps position this helix αt a 45° angle of the plane that runs parallel with the larger half of the molecule thus further facilitating the formation of the T-pocket. Moreover, the interaction of Arg492 with Leu327, Cys331, and Leu333 helps position the guanidine moiety, the only solvent-exposed part of this residue, at the interface formed by the CP- and T-pockets. The CP-pocket also contains several surface-exposed, conserved residues that are mainly hydrophilic in nature. These include Lys328 and Lys329 both of which are located beneath the T-pocket and in close proximity of Arg492 and Lys493 together forming a single large, positively charged surface area that almost spans the entire side of the molecule.
TRBD Structure and Existing Mutants. Several mutants of TERT that affect RNA-binding and telomerase activity have been isolated. Several of these mutants are found in the TRBD domain and specifically within the T- and CP-motifs. Single- and double- as well as stretches of 4-10 amino acid alanine substitutions within these two motifs showed moderate to severe loss (20-100%) of RNA-binding affinity and polymerase activity when compared to the wild type enzyme (Bryan, et al. (2000) supra; Lai, et al. (2002) supra; Miller, et al. (2000) supra).
One set of mutants, Phe476Ala, Tyr477Ala, Thr479Ala, Glu480Ala, Arg492Ala and Trp496Ala, showed severe loss (80-100%) of RNA-binding affinity and telomerase activity suggesting that these residues mediate direct contacts with the RNA substrate (Bryan, et al. (2000) supra; Lai, et al. (2002) supra). All five residues are part of the T-motif and, with the exception of Phe476, all of their side chains are solvent exposed. In the structure, both Tyr477 and Trp496 are located at the base of the T-pocket and their side chains form a “hydrophobic pincer”. Assuming that the solvent-exposed side chains of these residues are involved in stacking interactions with the ssRNA, mutating them to small alanines would likely compromise substrate binding which explains the dramatic loss of RNA-binding affinity and telomerase function. In contrast to Tyr477 and Trp496, Phe476 is buried and is not accessible for interactions with the nucleic acid substrate. Instead, Phe476 is located at the base of the β-hairpin and contributes significantly to the formation of the T-pocket. Mutating the large hydrophobic side chain of this residue to the small alanine would likely lead to conformational rearrangements of this pocket and loss of RNA-binding affinity and telomerase activity.
A second set of alanine mutants, Leu327Ala, Lys329Ala, Cys331Ala, and Pro334Ala, which showed moderate loss of RNA-binding affinity and telomerase activity has also been isolated (Bryan, et al. (2000) supra; Miller, et al. (2000) supra). Both Leu327 and Cys331 make direct contacts with Phe476 and the aliphatic part of the side chain of Arg492, both of which are located at the base of the T-motif. Mutation to the smaller alanine residue could result in the rearrangement of the T-pocket potentially leading to loss of interactions with the nucleic acid substrate and loss of function. Likewise, Pro334 is located at the back of helix α12 and makes direct contacts with residues of this structural element. Helix α12 contains the invariant Trp496 and the conserved Lys493, both of which form part of the T-pocket. Mutating Pro334 into an alanine could lead to the displacement of helix α10 and reorganization of the T-pocket leading to loss of function. Lys329 is also located on helix α3 and unlike Leu327Ala, Cys331Ala, and Pro334Ala, is solvent exposed possibly making direct contacts with the nucleic acid substrate. Mutating it to an alanine would lead to lose of RNA interactions and loss of RNA-binding affinity and telomerase activity.
TRBD Domain-Mediated Formation of Stable RNP Complex and Repeat Addition Processivity. In vivo, telomerase exists as a stable ribonucleoprotein complex and contacts between the protein (TERT) and the RNA components (TER) are mediated by the TEN, TRBD and the RT domains. Extensive biochemical and mutagenesis studies have shown that the TRBD is involved in extensive, specific interactions with stem I and the TBE of TER (Lai, et al. (2001) supra; O'Connor, et al. (2005) supra) (FIG. 4). Contacts between the TRBD and TER are thought to facilitate the proper assembly and stabilization of the RNP complex and promote repeat addition processivity (Lai, et al. (2003) supra). In ciliates, in addition to the TRBD, a conserved motif (CP2) located N-terminally to the TRBD domain is thought to be required for TERT-TER assembly and template boundary definition (Lai, et al. (2002) supra; Miller, et al. (2000) supra). However, until now it has been unclear as to how the telomerase TRBD carries out this process. The present analysis indicates that the TRBD domain is divided into two asymmetric halves connected by several long loops that are shaped like a boomerang, an arrangement that has significant implications for RNA recognition and binding. The overall organization of the two lobes of the molecule results in the formation of two well-defined cavities (CP- and T-pockets) on the surface of the protein that consist of several solvent-exposed, invariant/conserved residues. The T-pocket is a narrow, deep cavity located at the junction of the two halves of the molecule with part of it being hydrophobic in nature while the part that is located in proximity of the CP-pocket is positively charged. Interestingly, the hydrophobic side chains of Tyr477 and Trp496 are solvent-exposed and are stacked against each other forming a narrow “hydrophobic pincer” that in this structure could not accommodate a nucleotide base. It is, however, worth noting that helix α12, which contains Trp496, is somewhat flexible with respect to the β-hairpin that contains Tyr477. The ability of helix α12 and therefore Trp496 to move could splay the two side chains apart thus allowing for the space required for the accommodation of a nucleotide base between them. Another possibility is that the polar moiety of Tyr477 and Trp496 could act together as a nucleotide base that would allow for the formation of pseudo Watson Crick interactions with an incoming nucleotide base. Pseudo Watson Crick interactions have been previously observed for a number of protein nucleic acid complexes including the Rho transcription termination factor (Bogden, et al. (1999) Mol. Cell. 3:487-493) and the signal recognition particle (Wild, et al. (2001) Science 294:598-601). The width and the organization of the hydrophobic part of the T-pocket indicate that it binds ssRNA, most likely the TBE, possibly mediated by a network of stacking interactions.
In contrast to the T-pocket, the CP-pocket is a positively charged, shallow cavity located on the side of the molecule and forms an extension of the T-pocket. Together the hydrophilic part of the T-pocket and the CP-pocket are lined with several lysines and arginines the side chains of which are solvent exposed and could be involved in direct contacts with the backbone of double-stranded RNA. The width and the chemical nature of this pocket indicate that it binds double-stranded RNA, most likely stem I or stem II (FIG. 4). The nature and the extent of the protein/nucleic acid interactions mediated by the TRBD binding pockets provides the stability required for the proper assembly of a functional ribonucleoprotein enzyme and guide TERT to a TER binding site (between stem I and II) that has significant implications for telomerase function.
Telomerase is unique in its ability to add multiple short oligonucleotide repeats at the end of linear chromosomes. The enzyme's ability to do so has been partly attributed to the interactions of the TRBD domain with the TBE and in ciliates both the TRBD and the CP2 motif (Lai, et al. (2002) supra; Lai, et al. (2003) supra; Miller, et al. (2000) supra). The TBE is composed of stem II and the flanking ssRNA regions and is located downstream of stem I and only a few nucleotides upstream of the RNA template (FIG. 4). The TRBD structure indicates that the T-pocket, a narrow, hydrophobic cavity located on the surface of the protein that can only accommodate ssRNA, may play an important role in this process. Assuming that the T-pocket binds the ssRNA that connects stem I and stem II, this interaction likely forces stem II to act as a steric block, which in turn forces the TRBD domain to stay within the boundaries of stem I and stem II. The stem I- and II-locked TRBD domain then may act as an anchor that constrains the distance the RT domain can travel and prevents it from moving beyond the boundaries of the RNA template thus promoting telomerase addition processivity. In ciliates however, the TRBD domain alone is not sufficient for template boundary definition and it requires the action of the CP2 motif (Lai, et al. (2002) supra; Miller, et al. (2000) supra). It is contemplated that CP2 binding to TER promotes template boundary definition either via contributing to the stabilization of the RNP complex or, like the TRBD, it may act as an anchor that prevents slippage of the active site of the RT domain beyond the RNA template.
Example 2
Structure of Tribolium castaneum TERT in Complex with DNA Substrate
Protein Expression and Purification. The synthetic gene of T. castaneum full-length TERT was cloned into a modified version of the pET28b vector containing a cleavable hexahistidine tag at its N-terminus. The protein was over-expressed in E. coli BL21 (pLysS) at 30° C. for 4 hours. The cells were lysed by sonication in 50 mM Tris-HCl, 10% glycerol, 0.5 M KCl, 5 mM β-mercaproethanol, and 1 mM PMSF, pH 7.5 on ice. The protein was first purified over a Ni-NTA column followed by TEV cleavage of the hexahistidine tag overnight at 4° C. The TERT/TEV mixture was dialyzed to remove the excess imidazole and the protein was further purified over a second Ni-NTA column that was used to remove all his-tagged products. The Ni-NTA flow through was then passed over a POROS-HS column (Perseptive Biosystems) to remove any trace amounts of protein contaminants. At this stage the protein was more than 99% pure. The protein was finally purified over a SEPHEDEX-S200 sizing column pre-equilibrated with 50 mM Tris-HCl, 10% glycerol, 0.5 M KCl, and 1 mM Tris(2-Carboxyethyl) phosphine (TCEP), pH 7.5 to remove any TERT aggregates and the protein was concentrated to 10 mg/ml using an AMICON 30K cutoff (MILLIPORE) and stored at 4° C. for subsequent studies. Stock protein was dialyzed in 10 mM Tris-HCl, 200 mM KCl, 1 mM TCEP, pH 7.5 prior to crystallization trials.
Protein Crystallization and Data Collection. Initial crystal trials of the protein alone did not produce crystals. Co-crystallization of the protein with single stranded telomeric DNA ((TCAGG)3) produced two rod-like crystal forms one of which belongs to the orthorhombic space group P212121 and diffracted to 2.71 Å and the other to the hexagonal space group P61 and diffracted to 3.25 Å resolution. The protein nucleic acid mix was prepared prior to setting crystal trials by mixing one volume of dialyzed protein with 1.2-fold excess of the DNA substrate. Both crystal forms where grown by the vapor diffusion, sitting drop method by mixing on volume of the protein-DNA mix with one volume of reservoir solution. Orthorhombic crystals where grown in the presence of 50 mM HEPES, (pH 7.0) and 1.5 M NaNO3 while hexagonal crystals grew in the presence of 100 mM Tris (pH 8.0) and 2 M (NH4)2SO4 and both at room temperature. Orthorhombic crystals were harvested into cryoprotectant solution that contained 50 mM HEPES (pH 7.0), 25% glycerol, 1.7 M NaNO3, 0.2 M KCl and 1 mM TCEP and were flash frozen in liquid nitrogen. Hexagonal crystals were harvested into cryoprotectant solution that contained 100 mM Tris (pH 8.0), 25% glycerol, 2 M (NH4)2SO4, 0.2 M KCl and 1 mM TCEP and were also flash frozen in liquid nitrogen. Data were collected at the NSLS, beam line X6A and processed with HKL-2000 (Minor (1997) Methods in Enzymology: Macromolecular Crystallography, part A 276:307-326) (Table 5). Both crystal forms contain a dimer in the asymmetric unit.












TABLE 5







Native 1
Native 2
Hg1
Hg2




















Data collection






Space group
P212121
P61
P212121
P212121


Cell dimensions


a, b, c (Å)
85.0420,
200.0670,
86.7165,
86.9260,



122.6570,
200.0670,
123.3500,
123.4100,



212.4060
96.4100
211.4530
211.4160


Resolution (Å)
  40-2.70
  40-3.25
 40-3.5
 40-3.5



 (2.78-2.71)*
(3.32-3.25)
(3.69-3.5) 
(3.69-3.5) 


Rsym or Rmerge
10.7 (48.1)
14.9 (42.6)
14.5 (41.7)
16.1 (43.7)


I/σI
9.3 (1.7)
6.4 (2.4)
7.0 (3.5)
7.3 (3.6)


Completeness (%)
96.97 (95.84)
98.85 (98.1) 
85.7 (83.1)
93.8 (94.2)


Redundancy
4.2 (4.2)
2.8 (2.5)
4.7 (4.8)
5.3 (5.3)


Refinement


Resolution (Å)
  20-2.71
  20-3.25


No. reflections
56173
32773


Rwork/Rfree
23.8/27.7
24.3/29.6


No. atoms


Protein
4982
4982


Water
358
77


B-factors


Protein
52.5
37.8


Water
41.3
26.5


R.m.s deviations


Bond lengths (Å)
0.007
0.006


Bond angles (°)
0.848
0.735


Ramachandran plot


(%)


Most favored
83.3
86.4


Allowed
15.2
11.5


Generously allowed
1.4
1.7


Disallowed
0.2
0.4





*Highest resolution shell is shown in parenthesis.






Structure Determination and Refinement. Initial phases for the orthorhombic crystals were obtained using the method of single isomorphous replacement with anomalous signal (SIRAS) using two datasets collected from two different mercury (CH3HgCl) derivatized crystals at two different wavelengths (Hg1-1.00850 Å; Hg2-1.00800 Å) (Table 5). The derivatives were prepared by soaking the crystals with 5 mM methyl mercury chloride (CH3HgCl) for 15 minutes. Initially, twelve heavy atom sites were located using SOLVE (Terwilliger (2003) Methods Enzymol. 374:22-37) and refined and new phases calculated with MLPHARE (Collaborative Computational Project 4 (1994) Acta Crystallogr. D 50:760-763). MLPHARE improved phases were used to identify the remaining heavy atom sites (twenty two in total) by calculating an anomalous difference map to 3.5 Å resolution. MLPHARE phases obtained using all the heavy atom sites where then used in DM with two-fold NCS and phase extension using the high-resolution (2.71 Å) dataset collected, at 1.00800 Å wavelength, to calculate starting experimental maps. These maps were sufficiently good for model building which was carried out in COOT (Emsley & Cowtan (2004) Acta Crystallogr D Biol Crystallogr 60:2126-32). The electron density map revealed clear density for all 596 residues of the protein. However, density for the nucleic acid substrate in the structure was not observed. The model was refined using both CNS-SOLVE (Brunger, et al. (1998) Acta Crystallogr D Biol Crystallogr 54:905-21) and REFMAC5 (Murshudov, et al. (1997) Acta Crystallogr D Biol Crystallogr 53:240-55). The last cycles of refinement were carried out with TLS restraints as implemented in REFMAC5 (Table 5). The P212121 refined model was used to solve the structure of the TERT crystallized in the P61 crystal form (data collected at 0.97980 Å wavelength) by molecular replacement with PHASER (Potterton, et al. (2003) Acta Crystallogr D Biol Crystallogr 59:1131-7).
Architecture of the TERT Structure. The structure of the full-length catalytic subunit of the T. castaneum active telomerase, TERT, was determined to 2.71 Å resolution. As indicated, there was a dimer in the asymmetric unit (AU), however the protein alone was clearly monomeric in solution as indicated by gel filtration and dynamic light scattering, indicating that the dimer observed in the crystal was the result of crystal packing.
This was further supported by the fact that a different crystal form (Table 5) of the same protein also contained a dimer in the AU of different configuration. It is worth noting that the TERT from this organism does not contain a TEN domain, a low conservation region of telomerase (FIG. 1B).
The TERT structure is composed of three distinct domains, a TER-binding domain (TRBD), the reverse transcriptase (RT) domain, and the C-terminal extension thought to represent the putative “thumb” domain of TERT (FIGS. 1A and 1B). As indicated herein, the TRBD is mostly helical and contains an indentation on its surface formed by two conserved motifs (CP and T) which bind double- and single-stranded RNA, respectively, and has been defined as the template boundary element of the RNA substrate of telomerase, TER. Structural comparison of the TRBD domain from T. castaneum with that of the structure from T. thermophila shows that the two structures are similar (RMSD 2.7 Å), indicating a high degree of structural conservation between these domains across organisms of diverse phylogenetic groups.
The RT domain is a mix of α-helices and β-strands organized into two subdomains that are most similar to the “fingers” and “palm” subdomains of retroviral reverse transcriptases such as HIV reverse transcriptase (PDB code ID 1N5Y; Sarafianos, et al. (2002) EMBO J. 21:6614-24), viral RNA polymerases such as hepatitis C viral polymerase (Code ID 2BRL Di Marco, et al. (2005) J. Biol. Chem. 280:29765-70) and B-family DNA polymerases such as RB69 (PDB Code ID 1WAF; Wang, et al. (1997) Cell 89:1087-99), and contain key signature motifs that are hallmarks of these families of proteins (Lingner, et al. (1997) Science 276:561-7) (FIGS. 3A-3C). Structural comparison of TERT with the HIV RTs, shows that the “fingers” subdomain of TERT (i.e., motifs 1 and 2) are arranged in the open configuration with respect to the “palm” subdomain (i.e., motifs A, B′, C, D, and E), which is in good agreement with the conformation adopted by HIV RTs in the absence of bound nucleotide and nucleic acid substrates (Ding, et al. (1998) J. Mol. Biol. 284:1095-111). One striking difference between the putative “palm” domain of TERT and that HIV reverse transcriptases is a long insertion between motifs A and B′ of TERT referred to as the IFD motif that is required for telomerase processivity (Lue, et al. (2003) Mol. Cell. Biol. 23:8440-9). In the TERT structure, the IFD insertion is composed of two anti-parallel α-helices (α13 and α14) located on the outside periphery of the ring and at the interface of the “fingers” and the “palm” subdomains. These two helices are almost in parallel position with the central axis of the plane of the ring and make extensive contacts with helices α10 and α15 and play an important role in the structural organization of this part of the RT domain. A similar structural arrangement is also present in viral polymerases, and the equivalent of helix α10 in these structures is involved in direct contacts with the nucleic acid substrate (Ferrer-Orta, et al. (2004) J. Biol. Chem. 279:47212-21).
In contrast to the RT domain, the C-terminal extension is an elongated helical bundle that contains several surface exposed, long loops. A search in the protein structure database using the software SSM (Krissinel & Henrick (2004) Acta Cryst. D60:2256-2268; Krissinel (2007) Bioinformatics 23:717-723) produced no structural homologues suggesting that the CTE domain of telomerase adopts a novel fold. Structural comparison of TERT with the HIV RT, the viral RNA polymerases and B-family DNA polymerases places the “thumb” domain of these enzymes and the CTE domain of TERT in the same spatial position with respect to the “fingers” and “palm” subdomains, indicating that the CTE domain of telomerase is the “thumb” domain of the enzyme, a finding that is in good agreement with previous biochemical studies (Hossain, et al. (2002) J. Biol. Chem. 277:36174-80).
TERT domain organization brings the TRBD and “thumb” domains, which constitute the terminal domains of the molecule, together, an arrangement that leads to the formation of a ring-like structure that is reminiscent of the shape of a donut (FIG. 10). Several lines of evidence indicate that the domain organization of the TERT structure presented herein is biologically relevant. First, the domains of four TERT monomers observed in two different crystal forms (two in each asymmetric unit) are organized the same (average RMSD=0.76 Å between all four monomers). Second, contacts between the N- and C-terminal domains of TERT are extensive (1677 Å2) and largely hydrophobic in nature involving amino acid residues Tyr4, Lys76, Thr79, Glu84, Ser81, His87, Asn142, His144, Glu145, Tyr411, His415, Phe417, Trp420, Phe422, Ile426, Phe434, Thr487, Ser488, Phe489, and Arg592. This observation is in agreement with previous biochemical studies (Arai, et al. (2002) J. Biol. Chem. 277:8538-44). Third, TERT domain organization is similar to that of the polymerase domain (p66 minus the RNase H domain) of its closest homologue, HIV reverse transcriptase (Sarafianos, et al. (2002) supra), the viral RNA polymerases (Di Marco, et al. (2005) supra) and the B-family DNA polymerases and in particular RB69 (Wang, et al. (1997) supra). The arrangement of the TERT domains creates a hole in the interior of the particle that is  ̃26 Å wide and  ̃21 Å deep, sufficient to accommodate double-stranded nucleic acids approximately seven to eight bases long, which is in good agreement with existing biochemical data (Forstemann & Lingner (2005) EMBO Rep. 6:361-6; Hammond & Cech (1998) Biochemistry 37:5162-72).
The TERT Ring Binds Double-Stranded Nucleic Acid. To understand how the TERT ring associates with RNA/DNA to form a functional elongation complex, a double-stranded nucleic acid was modeled into the interior using the HIV reverse transcriptase—DNA complex (Sarafianos, et al. (2002) supra), TERT's closest structural homologue. The TERT-RNA/DNA model immediately showed some striking features that supported the model of TERT-nucleic acid associations. The hole of the TERT ring and where the nucleic acid heteroduplex was projected to bind was lined with several key signature motifs that are hallmarks of this family of polymerases and have been implicated in nucleic acid association, nucleotide binding and DNA synthesis. Moreover, the organization of these motifs resulted in the formation of a spiral in the interior of the ring that resembled the geometry of the backbone of double-stranded nucleic acid. Several of the motifs, identified as contact points with the DNA substrate, were formed partly by positively charged residues, the side chains of which extended toward the center of the ring and were poised for direct contact with the backbone of the DNA substrate. For example, the side chain, of the highly conserved K210 that forms part of helix α10, is within coordinating distance of the backbone of the modeled DNA thus providing the stability required for a functional telomerase enzyme. Helix α10 lies in the upper segment of the RT domain and faces the interior of the ring. The location and stabilization of this helix is heavily influenced by its extensive contacts with the IFD motif implicated in telomerase processivity (Lue, et al. (2003) Mol. Cell. Biol. 23:8440-9). Disruption of the IFD contacts with helix α10 through deletion or mutations of this motif would lead to displacement of helix α10 from its current location, which would in turn effect DNA-binding and telomerase function.
Structural elements of the “thumb” domain that localized to the interior of the ring also made several contacts with the modeled DNA substrate. In particular, the loop (“thumb” loop) that connects the “palm” to the “thumb” domain and constitutes an extension of the E motif also known as the “primer grip” region of telomerase, preserves to a remarkable degree, the geometry of the backbone of double stranded nucleic acid. The side chains of several lysines (e.g., Lys406, Lys416, Lys418) and asparagines (e.g., Asn423) that formed part of this loop extended toward the center of the TERT molecule and were within coordinating distance of the backbone of modeled double-stranded nucleic acid. Of particular interest was Lys406. This lysine was located in proximity of motif E and its side chain extended toward the nucleic acid heteroduplex and was poised for direct contacts with the backbone of the nucleotides located at the 3′ end of the incoming DNA primer. It is therefore possible that the side chain of this lysine together with motif E help facilitate placement of the 3′-end of the incoming DNA substrate at the active site of the enzyme during telomere elongation. Sequence alignments of the “thumb” domain of TERTs from a wide spectrum of phylogenetic groups showed that the residues predicted to contact the DNA substrate are always polar (FIGS. 3A-3C). Another feature of the “thumb” domain that supported double-stranded nucleic acid binding was helix α19, a 310 helix (“thumb” 310 helix) that extended into the interior of the ring and appeared to dock itself into the minor groove of the modeled double-stranded nucleic acid thus facilitating RNA/DNA hybrid binding and stabilization. Deletion or mutation of the corresponding residues in both yeast and human TERT results in sever loss of TERT processivity clearly indicating the important role of this motif in TERT function (Hossain, et al. (2002) J. Biol. Chem. 277:36174-80; Huard, et al. (2003) Nucleic Acids Res. 31:4059-70; Banik, et al. Mol. Cell. Biol. 22:6234-46).
The Active Site of TERT and Nucleotide Binding. The T. castaneum TERT structure presented herein was crystallized in the absence of nucleotide substrates and magnesium, however, the location and organization of TERT's active site and nucleotide binding pocket was determined on the basis of existing biochemical data (Lingner, et al. (1997) supra) and structural comparison with the polymerase domain of its closest homologue, the HIV reverse transcriptase (Das, et al. (2007) J. Mol. Biol. 365:77-89). The TERT active site is composed of three invariant aspartic acids (Asp251, Asp343 and Asp344) that form part of motifs A and C, two short loops located on the “palm” subdomain, and adjacent to the “fingers” of TERT. Structural comparison of TERT with HIV reverse transcriptases, as well as RNA and DNA polymerases showed a high degree of similarity between the active sites of these families of proteins indicating that telomerase also employs a two-metal mechanism for catalysis. Alanine mutants of these TERT aspartic acids resulted in complete loss of TERT activity indicating the essential role of these residues in telomerase function (Lingner, et al. (1997) supra).
The telomerase nucleotide binding pocket is located at the interface of the “fingers” and “palm” subdomains of TERT and is composed of conserved residues that form motifs 1, 2, A, C, B′ and D implicated in template and nucleotide binding (Bosoy & Lue (2001) J. Biol. Chem. 276:46305-12; Haering, et al. (2000) Proc. Natl. Acad. Sci. USA 97:6367-72). Structural comparisons of TERT with viral HIV reverse transcriptases bound to ATP (Das, et al. (2007) supra) supports nucleotide substrate in this location. Two highly conserved, surface-exposed residues Tyr256 and Val342 of motifs A and C, respectively, form a hydrophobic pocket adjacent to and above the three catalytic aspartates and could accommodate the base of the nucleotide substrate. Binding of the nucleotide in this oily pocket places the triphospate moiety in proximity of the active site of the enzyme for coordination with one of the Mg2+ ions while it positions the ribose group within coordinating distance of an invariant glutamine (Gln308) that forms part of motif B′ thought to be an important determinant of substrate specificity (Smith, et al. (2006) J. Virol. 80:7169-78). Protein contacts with the triphospate moiety of the nucleotide are mediated by motif D, a long loop, located beneath the active site of the enzyme. In particular, the side chain of the invariant Lys372 is within coordinating distance of the γ-phosphate of the nucleotide an interaction that most likely helps position and stabilize the triphosphate group during catalysis. The side chains of the highly conserved Lys189 and Arg194 of motifs 1 and 2, which together form a long β-hairpin that forms part of the “fingers” subdomain, are also within coordinating distance of the both the sugar and triphosphate moieties of the modeled nucleotide. Contacts with either or both the sugar moiety and the triphosphate of the nucleotide substrate would facilitate nucleotide binding and positioning for coordination to the 3′-end of the incoming DNA primer.
TRBD Facilitates Template Positioning at the Active Site of TERT. As with most DNA and RNA polymerases, nucleic acid synthesis by telomerase requires pairing of the templating region (usually seven to eight bases or more) of TER with the incoming DNA primer (Lee & Blackburn (1993) Mol. Cell. Biol. 13:6586-99). TRBD-RT domain organization forms a deep cavity on the surface of the protein that spans the entire width of the wall of the molecule, forming a gap that allows entry into the hole of the ring from its side. The arrangement of this cavity with respect to the central hole of the ring provides an elegant mechanism for placement of the RNA template, upon TERT-TER assembly, in the interior of the ring and where the enzyme's active site is located. Of particular significance is the arrangement of the β-hairpin that forms part of the T-motif. This hairpin extends from the RNA-binding pocket and makes extensive contacts with the “thumb” loop and motifs 1 and 2. Contacts between this hairpin and both the “fingers” and the “thumb” domains place the opening of the TRBD pocket that faces the interior of the ring in proximity to the active site of the enzyme. It is therefore likely that this β-hairpin acts as an allosteric effector switch that couples RNA-binding in the interior of the ring and placement of the RNA template at the active site of the enzyme. Placement of the template into the interior of the molecule would facilitate its pairing with the incoming DNA substrate, which together would form the RNA/DNA hybrid required for telomere elongation. RNA/DNA pairing is a prerequisite of telomere synthesis in that it brings the 3′-end of the incoming DNA primer in proximity to the active site of the enzyme for nucleotide addition while the RNA component of the heteroduplex provides the template for the faithful addition of identical repeats of DNA at the ends of chromosomes. Strikingly, modeling of the RNA/DNA heteroduplex in the interior of the TERT ring places the 5′-end of the RNA substrate at the entry of the RNA-binding pocket and where TERT is expected to associate with TER while it places the 3′-end of the incoming DNA primer at the active site of TERT providing a snapshot of the organization of a functional telomerase elongation complex.
Example 3
Structure of T. castaneum Telomerase Catalytic Subunit TERT Binding to RNA Template and Telomeric DNA
Methods for Expressing and Purifying Proteins. Wild-type and mutant (Asp251Ala) T. castaneum, full length TERT proteins were overexpressed and purified as described herein with subtle modifications that increased protein yield. Specifically, the proteins were over-expressed in E. coli Rosetta (DE3) pLysS (Novagen) at 30° C. for 5 hours with slow shaking (shaker/incubator setting, 120 rpm). Stock protein (10 mg/ml) was dialyzed in 10 mM Tris-HCl, 100 mM KCl, 1 mM TCEP, pH 7.5 prior to crystallization trials.
Methods for Preparing T. castaneum Extracts and Isolating RNA. Twenty T. castaneum larvae or pupae were ground in liquid N2, homogenized with 200 μl of extraction buffer (25 mM Tris-HCl, 5 mM β-mercaptoethanol (β-ME), 1 mM EGTA, 0.1 mM benzamidine, 200 mM KCl, 10% (w/v) glycerol, 10 mM imidazole and RNasin (PROMEGA, Madison, Wis.), pH 7.5) and placed on ice for 30 minutes. After the homogenate was centrifuged at 12,000 g at 4° C. for 20 minutes, the supernatant was collected, flush frozen in liquid nitrogen and stored at −80° C. before use. The total RNA was then extracted from the T. castaneum homogenate using the RNEASY Protect Mini Kit (QIAGEN, Valencia, Calif.).
Methods for In Vitro Reconstitution of T. castaneum Telomerase. The telomerase RNA of T. castaneum had not been previously identified. Therefore, total RNA was isolated from beetle larvae and used with the recombinant TERT to assemble the telomerase complex in vitro. Twenty μg of the his-tagged TERT (25 mM Tris, 200 mM KCl, 10% (w/v) glycerol, 5 mM β-ME and 10 mM imidazole, pH 7.5) was mixed with 50 μl of T. castaneum larvae total RNA and the two were incubated in T. castaneum lysate for two hours at room temperature in the presence of RNasin. The telomerase complex was then purified over a Ni-NTA column and tested for activity using a modified TRAP assay (Sasaki & Fujiwara (2000) Eur. J. Biochem. 267:3025-3031) as described herein.
Telomerase Repeat Amplification Protocol (TRAP) Assays. The activity of the in vitro reconstituted T. castaneum telomerase was tested using the following TRAP assay. The telomerase elongation step was carried out in a 50 μl reaction mixture composed of 20 mM Tris-HCl (pH 8.3), 7.5 mM MgCl2, 63 mM KCl, 0.05% (w/v) TWEEN 20, 1 mM EGTA, 0.01% (w/v) BSA, 0.5 mM of each dNTP, 1 μM DNA primer (5′-aag ccg tcg agc aga gtc-3′; SEQ ID NO:17) (Tcas-TS)) and 4 μg of Ni-NTA purified, T. castaneum telomerase. After incubation at 30° C. for 60 minutes, each reaction mixture was phenol-chloroform extracted and precipitated with ethanol. Each sample was re-suspended in 50 μl of PCR reaction buffer (10 mM Tris/HCl (pH 8.0), 50 mM KCl, 2 mM MgCl2, 100 μM dNTPs (dATP dTTP dGTP), 10 μM [32P]dCTP (80 Ci per mmol), 1 μM Tcas-CX primer (5′-gtg tga cct gac ctg acc-3′; SEQ ID NO:18) and HOTSTARTAQ DNA polymerase (QIAGEN). The PCR products were resolved on Tris-borate-EDTA (TBE)-polyacrylamide gels. The presence of multiple telomeric repeats (TCAGG)n was also confirmed by subcloning and sequencing the TRAP products.
RNA-DNA Hairpins. The RNA-DNA hairpins tested for TERT binding, activity and subsequently for structural studies are shown in Table 6. All three hairpins contain the putative RNA-templating region (5′-rCrUrGrArCrCrU-3′) (one and a half times the telomeric repeat of T. castaneum, TCAGG) and the complementary telomeric DNA sequence (5′-GTCAGGT-3′). The RNA-templating region and the DNA complement were connected for stability with an RNA-DNA linker (loop). The hairpins were designed to contain a 5′-RNA overhang so that the telomerase complex could be trapped in its replication state upon co-crystallization with the complementary non-hydrolysable nucleotide(s). The only difference between the three hairpins is the length of the linker.









TABLE 6





Hairpin
Sequence (5′->3′)
SEQ ID NO:







21 mer
rCrUrGrArCrCrUrGrArCTTCGGTCAGGT
19


 


17 mer
rCrUrGrArCrCrUrGTTCGCAGGT
20


 


15 mer
rCrUrGrArCrCrUTTCGAGGT
21





*Underlined sequences represent linker sequences. Bold sequences form the loop of the hairpin.






TERT, Reverse Transcriptase (RT) Assays. Standard reverse transcriptase assays were carried out using the recombinant T. castaneum TERT and the RNA-DNA hairpin (Table 6) to test TERT's ability to replicate the end of the DNA substrate that includes part of the RNA-DNA hairpin. RT assays were carried out in telomerase buffer (50 mM Tris-HCl, 100 mM KCl, 1.25 mM MgCl2, 5 mM DTT, 5% (w/v) glycerol, pH 8 at room temperature), 100 μM dNTPs (dATP dTTP dGTP), 10 μM [32P]dCTP (80 Ci per mmol), 5 μM RNA-DNA hairpin and 1 μM recombinant TERT. The samples were incubated for two hours at room temperature, then phenol/chloroform extracted and ethanol precipitated. The DNA pellet was re-suspended in a solution composed of 90% (w/v) formamide and 10% (w/v) glycerol and the sample was run on a 12% (w/v) polyacrylamide-7 M urea gel in 1×TBE at 220V for 70 minutes at 4° C.
Protein Crystallization and Data Collection. The binary complex was prepared by adding to the dialyzed protein 1.2 molar excess nucleic acid (RNA-DNA hairpin purchased from Integrated DNA Technologies), 5 mM dNTPaS (Jena Biosciences GmbH) and 5 mM MgCl2. Crystals of the monoclinic space group P21 that diffracted to 2.7 Å resolution appeared in three days and grew to final size in two weeks. Crystals were grown by the vapor diffusion, sitting drop method by mixing one volume of the ternary complex with one volume of reservoir solution containing 0.1 M HEPES, (pH 7.5) and 12% 1,6-hexanediol or PEG 4K and 0.2 M KCl. Crystals were transferred into cryoprotectant solution that contained 0.1 M HEPES (pH 7.5), 15% (w/v) 1,6-hexanediol or PEG 4K, 15% (w/v) glycerol, 0.2 M KCl and 1 mM TCEP and were harvested by flash freezing in liquid nitrogen. Data were collected at the NSLS, beam line X25 and processed with MOSFILM as implemented in WEDGER-ELVES (Holton & Alber (2004) Proc. Natl. Acad. Sci. USA 101:1537-1542) (Table 7). There is one monomer in the asymmetric unit.







TABLE 7





TERT Complex


















Data Collection




Space group
P21



Cell dimensions



a, b, c (Å)
77.2 52.8 101.6



α, β, γ (°)
90 101.9 90



Resolution (Å)
   20-2.7 (2.85-2.70)*



Rsym or Rmerge
11.4 (45.8)



I/σI
7.9 (2.3)



Completeness (%)
93.7 (96.1)



Redundancy
2.8 (2.5)



Refinement



Resolution (Å)
 20-2.7



No. Reflections
19815



Rwork/Rfree
24.2/28.7



Number of atoms
4982



Protein
4982



Ligand/Ion
496/1 



Water
54



B-factors



Protein
45



Ligand/Ion
37



Water
21



R.m.s. Deviations



Bond lengths (Å)
0.006



Bond angles (°)
0.887







*Number of crystals used - one.



*Values in parentheses are for highest-resolution shell.






Structure Determination and Refinement. Phases where calculated by molecular replacement (MR) using PHASER (Potterton, et al. (2003) Acta Crystallogr D Biol Crystallogr 59:1131-1137) as implemented in CCP4 suit of programs using the substrate-free TERT structure described herein as a search model. Maps calculated after one cycle of refinement by CNS-SOLVE revealed clear 2fo-fc density for all 596 residues of TERT and fo-fc density for the nucleic acid substrate at 3.0 σ contour level. Model building was carried out in COOT (Emsley & Cowtan (2004) Acta Crystallogr D Biol Crystallogr 60:2126-2132) and the model was refined using both CNS-SOLVE (Brunger, et al. (1998) Acta Crystallogr. D Biol. Crystallogr. 54:905-921) and REFMAC5 (Murshudov, et al. (1997) Acta Crystallogr. D Biol. Crystallogr. 53:240-255). The last cycles of refinement were carried out with TLS restraints as implemented in REFMAC5. The structure was refined to good stereochemistry with 84.3, 14.3 and 1.4 of the residues in the most favorable, additional allowed and generously allowed of the Ramachandran plot, respectively.
TERT Structure Overview. The full length (FIG. 1B), active, T. castaneum, TERT was co-crystallized with an RNA-DNA hairpin containing the putative RNA templating region (5′-rCrUrGrArCrCrU-3′) and the complementary telomeric DNA (5′-GTCAGGT-3′) joined together with a short RNA-DNA linker (Table 6). It is worth noting that the T. castaneum TERT lacks the TEN domain, required for activity and processivity in several eukaryotic telomerase genes, including human (Wyatt, et al. (2009) PLoS One 4:e7176) and Tetrahymena thermophila (Zaug, et al. (2008) Nat. Struct. Mol. Biol. 15:870-872; Finger & Bryan (2008) Nucleic Acids Res. 36:1260-1272). The absence of the TEN domain from T. castaneum could explain the reduced activity observed for this enzyme when compared to those containing this domain. The RNA component of T. castaneum telomerase has not been previously identified. Therefore, the following information was used to predict its templating region: The RNA templating region of telomerase is usually one and a half telomeric repeats (Lee & Blackburn (1993) Mol. Cell. Biol. 13: 6586-6599; Lingner, et al. (1994) Genes Dev. 8: 1984-1998; Shippen-Lentz & Blackburn (1990) Science 247: 546-552) and this sequence is known for many organisms (telomerase database). For example the mammalian telomerase templating region is “CUAACCCU” and the telomeric repeat is “TTAGGG”. The telomeric repeat for T. castaneum is “TCAGG”(Osanai, et al. (2006) Gene 376:281-89; Richards, et al. (2008) Nature 452:949-955). The RNA-DNA hairpin was designed to contain a three-nucleotide overhang at the 5′-end of the RNA template, so that the enzyme could be trapped in its catalytic state upon co-crystallization of the protein-nucleic acid assembly with Mg++ ions and non-hydrolysable nucleotides. In an effort to identify a hairpin suitable for crystallographic studies, a number of RNA-DNA hairpins were screened (15 mer, 18 mer and 21 mer; Table 6) where the templating region and the complementary DNA sequence were kept the same but the length of the linker was changed. Although, all three hairpins tested had the same binding affinity for TERT and reverse transcriptase activity, only the 21 mer was amenable to crystallographic studies. It was noted that in the structure, the hairpin linker extends out of the TERT ring and is only involved in crystal contacts with adjacent molecules. The crystals were also grown in the presence of the slowly hydrolysable nucleotide analogues dNTPaS and Mg++ ions.
The structure was solved at 2.7 Å resolution using the method of molecular replacement with the substrate-free TERT described herein as a search model. All of the TERT molecule and the RNA-DNA hybrid were interpretable in electron density maps. Unexpectedly, there was extra density for three nucleotides at the 3′-end of the telomeric DNA, indicating that TERT had extended the 3′-end of the DNA substrate in the crystallization drop. There was no evidence for nucleotide at the active site of the enzyme, which was partially occupied by the nucleotide located at the 3′-end of the DNA substrate. Several lines of evidence indicate that the association of the RNA templating region and the DNA substrate with TERT in the structure presented here are biologically relevant. First, TERT is an active polymerase in the presence of the nucleic acid substrate used in this study both by standard reverse transcriptase assays and in the crystallization drop. Second, the RNA template makes contacts with conserved motifs that are hallmarks of this family of enzymes. Third, contacts between TERT and the RNA template position the solvent accessible bases adjacent to and above the active site of the enzyme for nucleotide binding thus facilitating selectivity. Moreover, TERT-RNA associations position the 5′-end of the RNA-template at the entry of the TRBD RNA-binding pocket and where the template boundary element located upstream of the RNA-template in many organisms is thought to bind. Fourth, interactions between the DNA substrate and the primer grip region (a characteristic shared among telomerase and HIV RTs) place the 3′-end of the DNA substrate at the active site of the enzyme where it is accessible for nucleotide addition. Fifth, TERT-nucleic acid associations are strikingly similar to those observed of HIV reverse transcriptase, TERT's closest structural homologue.
The four major TERT domains: the RNA-binding domain (TRBD); the fingers domain, implicated in nucleotide binding and processivity (Bosoy & Lue (2001) J. Biol. Chem. 276:46305-46312); the palm domain, which contains the active site of the enzyme; and the thumb domain, implicated in DNA binding and processivity (Hossain, et al. (2002) J. Biol. Chem. 277:36174-80), were organized into a ring configuration similar to that observed for the substrate-free enzyme described herein (FIG. 1C). The arrangement of the TERT domains created a highly positively charged cavity in the interior of the TERT ring, which was 22 Å wide and 21 Å deep and could accommodate seven bases of double stranded nucleic acid. Within this cavity binds one molecule of the RNA-DNA hybrid, which assembles together, via Watson-Crick base pairing into a helical structure similar to both the DNA-DNA and RNA-DNA structure bound to HIV reverse transcriptase (Huang, et al. (1998) Science 282:1669-75; Sarafianos, et al. (2001) EMBO J. 20:1449-61).
TERT Nucleic Acid Associations. Structure analysis indicated that interactions between the protein and the RNA templating region were mediated by the fingers, the palm and thumb domains. The 5′-end RNA cytosine (rC1) and uracil (rU2) were located at the interface of the fingers and palm domains and were involved in a network of interactions with conserved residues (FIG. 3) of motifs 2 and B′ both of which are located in proximity of the active site of the enzyme. In particular, the 2′-OH and the base carbonyl of rC1 is within hydrogen bonding distance of the backbone carbonyls of Val197 of motif 2 and Gly309 of motif B′ while the pyrimidine base sits over the otherwise solvent exposed, hydrophobic side chain of the conserved Ile196 that also forms part of motif 2 (FIG. 5). Contacts between rU2 and the protein are mediated by the short aliphatic side chain of Pro311 and the ribose group (FIG. 5). Interactions between rC1 and rU2 with motifs 2 and B′ place the cytosine base in proximity of the active site of the enzyme, where it is well-positioned for Watson-Crick base-pairing with the incoming nucleotide substrate.
Stabilization and placement of the 5′-end bases of the templating region above the active site of the enzyme was in large part mediated by the interactions of the remaining five ribonucleotides with the incoming DNA primer. Limited contacts between this part of the RNA and the protein were mediated via a water molecule (Wat18) which coordinates the 2′-OH of guanosine (rG3) with the backbone of helix α15 (FIG. 5). Notably, the structural organization of helix α15 was influenced by the IFD motif, a long insertion, composed of two helices (α13 and α14), between motifs A and B′, which explains why mutations in this motif lead to loss of telomerase function (Lue, et al. (2003) Mol. Cell. Biol. 23:8440-49).
Contacts between TERT and the DNA substrate were mediated in large part via backbone interactions with the thumb loop and helix. The thumb helix, as described herein, sits in the minor groove of the RNA-DNA heteroduplex, making extensive contacts with the phosphodiester backbone and the ribose groups of the RNA-DNA hybrid. The mode of action of the thumb helix of telomerase appears to be similar to that proposed for the equivalent helix (helix H) of retroviral reverse transcriptases (Jacobo-Molina, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6320-24; Kohlstaedt, et al. (1992) Science 256:1783-1790). Another conserved element of the thumb domain, known as the thumb loop, runs almost parallel to the curvature of the DNA primer and the two are involved in a network of backbone and solvent mediated interactions (FIG. 6). Interactions between the DNA and the thumb loop included the side chains of Lys416 and Asn423, both of which extended toward the center of the ring and were within hydrogen bonding distance of the DNA backbone. Contacts between the thumb domain and the DNA position the nucleotides located at its 3′-end within coordinating distance of the primer grip region (motif E), a short, rigid loop located at the interface of the palm and thumb domains, and in proximity of the active site of the enzyme (FIG. 7). The backbone of the tip of this loop formed by the conserved residues Cys390 and Gly391 abuts the ribose group of C22 and this interaction guides the 3′-end DNA nucleotides toward the active site of the enzyme (FIG. 7). The active site of the enzyme, and where the incoming nucleotide is projected to bind, is partially occupied by the nucleotide (G24) located at the 3′-end of the DNA (FIG. 7). The ribose group, and to a certain extend the guanosine base of G24, which makes Watson-Crick pairing interactions with the rC1 located at the 5′-end of the RNA template, sit in a well-defined hydrophobic pocket formed by the side chains of the invariant Tyr256, Gln308 and conserved Val342 of motifs A, B′ and C respectively, while the α-phosphate is coordinated by the magnesium ion occupying the active site aspartates (FIG. 7). The important role of Val342 in telomerase selectivity has been shown for the human telomerase holoenzyme (Drosopoulos & Prasad (2007) Nucleic Acids Res. 35:1155-1168).
TERT Domain Rearrangements Upon Nucleic Acid Binding. Comparisons of the nucleic acid bound and substrate-free TERT structures indicate that TERT nucleic acid associations induce small rigid-body changes in orientation between subunits of the enzyme that lead to a 3.5 Å decrease of the diameter of the interior cavity of the ring. The decrease in the diameter of the central cavity arises from a 6° inward rotation together with a 3.5 Å translation, of the thumb domain with respect to the fingers and palm domains. Translation of the thumb domain toward the center of the ring is accompanied by the TRBD, which is also shifted 3.5 Å toward the finger domain creating a more narrow RNA-binding pocket than the substrate-free enzyme. The role of this subtle structural rearrangement may have implications for TERT association with the full-length RNA, TER.
Common Aspects of Substrate Binding Between TERT and HIV RTs. It has been postulated that telomerase uses a mechanism of DNA replication that resembles that of other retroviral reverse transcriptases, a suggestion supported by the structure of the TERT-nucleic acid complex presented here. Structural comparison of the RNA-DNA bound TERT and HIV RT (PDB ID: 1HYS36) shows a striking similarity in the overall domain organization and nucleic acid binding between the two structures. Like HIV RTs, telomerase-dependent telomere replication requires pairing of the templating region with the incoming DNA primer and placement of the 3′-end of the DNA into the enzyme's active site for nucleotide addition. Moreover, TERT- or HIV RT-nucleic acid associations are accompanied by domain rearrangements that facilitate the formation of a tight, catalytic, protein nucleic acid assembly and positioning of the DNA 3′-end at the active site of the enzyme for catalysis (Kohlstaedt, et al. (1992) supra; Rodgers, et al. (1995) Proc. Natl. Acad. Sci. USA 92:1222-1226; Steitz (1997) Harvey Lect. 93:75-93). Contacts between the protein and the RNA templating region are specific and involve telomerase key signature motifs (motif 2 and B′ of the fingers and palm domains, respectively) that are hallmarks of these families of enzymes and are required for positioning of the solvent accessible bases of the RNA template in proximity of the active site for nucleotide binding and selectivity. Contacts between the protein and the DNA substrate are mediated by the thumb domain and despite the lack of sequence homology in this region between the two families of enzymes, the mode of action of the thumb helix of telomerase is similar to that proposed for helix H of HIV RTs (Jacobo-Molina, et al. (1993) supra; Kohlstaedt, et al. (1992) supra; Beese, et al. (1993) Science 260: 352-355). Placement of the DNA 3′-end at the active site of the enzyme is further facilitated by the primer grip region another highly conserved motif between TERT and HIV RTs44 (FIG. 7).
Although the enzyme was not trapped in its catalytic state, the partially occupied active site of the enzyme, formed by a number of invariant (Asp251, Tyr256, Gln308, Asp343, Asp344) or highly conserved residues (Val342), by the nucleotide G24 located at the 3′-end of the DNA (FIG. 7) suggests the mechanism of nucleotide binding and selectivity of telomerase during the replication process. In a similar manner, the invariant Tyr256 and G1n308 are also present in HIV RTs where, like in TERT, they are involved in nucleotide binding and selectivity through positioning for interactions with the templating region (Huang, et al. (1998) supra; Cases-Gonzalez, et al. (2000) J. Biol. Chem. 275:19759-19767), further supporting common mechanistic aspects of DNA replication between these families of enzymes.
TERT Rigid Conformational Changes and Function. Domain re-organization, upon nucleic acid binding, are a common feature of RNA-, DNA-polymerases and retroviral reverse transcriptases, and are geared toward the formation of a tight, catalytic protein nucleic acid assembly and positioning of the DNA 3′-end at the active site of the enzyme for catalysis (Kohlstaedt, et al. (1992) supra; Rodgers, et al. (1995) supra; Steitz (1997) supra). Unlike HIV RTs, telomerase appears to exist, at least in the absence of the full-length integral RNA component, in a closed ring configuration, an arrangement mediated by extensive contacts between the TRBD and the thumb domains. Comparison of the nucleic acid bound and substrate-free TERT structures indicate that TERT nucleic acid associations induce subtle, rigid-body changes in orientation between subunits of the enzyme that lead to a 3.5 Å decrease of the diameter of the interior cavity of the ring. These observations were unexpected because in most polymerases, including the HIV RT, the fingers and thumb domains undergo significant conformational changes required for substrate binding and function (Steitz (1997) supra; Ding, et al. (1998) J. Mol. Biol. 284:1095-1111; Steitz (1999) J. Biol. Chem. 274:17395-98). For example, the fingers domain, which is known to bind and position the nucleotide at the active site of the enzyme, undergoes significant conformational changes referred to as the open and closed states (Ding, et al. (1998) supra). It is therefore possible that the interactions between the TRBD and the thumb domains lock the fingers domain in place, thus preventing the conformational rearrangements observed in other polymerases, which would indicate the possibility of a preformed active site. A preformed active site has been observed for the Hepatitis C viral RNA polymerase (NS5B)(Bressanelli, et al. (2002) J. Virol. 76:3482-92), a close structural homologue of TERT but also the Y-family DNA polymerases (Ling, et al. (2001) Cell 107:91-102). Another possibility is that the substrate-free TERT enzyme was trapped in the closed fingers conformation. Assuming the later is true, significant movement of the fingers domain of TERT would most likely require that the TRBD and the thumb domains are splayed apart. Contacts between the TRBD and the thumb domain are extensive and would require significant energy to force them apart. This could be accomplished by accessory proteins that possibly act in a similar manner to that of the sliding clamp loader of DNA polymerases (Jeruzalmi, et al. (2002) Curr. Opin. Struct. Biol. 12:217-224).
Repeat Addition Processivity. Telomerase, unlike most polymerases, has the ability to add multiple identical repeats of DNA to the ends of chromosomes, known as repeat addition processivity. This unique characteristic of telomerase has been attributed in part to the association of the N-terminal portion of TERT with TER and the telomeric overhang as well as the IFD motif (Lue, et al. (2003) supra). The TEN domain, present in several organisms, and its weak interaction with the DNA substrate is thought to be a determinant of repeat addition processivity (Wyatt, et al. (2007) Mol. Cell. Biol. 27:3226-40; Moriarty, et al. (2004) Mol. Cell. Biol. 24:3720-33), while most recently the TRBD and its stable association with TER has also been shown to be involved in this process (see data presented herein). In the complex presented here, the RNA does not directly engage the RNA-binding pocket of TRBD. The structure shows that TERT-RNA contacts position the 5′-end of the templating region at the entry of the RNA-binding pocket of TRBD. This arrangement would place the template boundary element (Chen &7 Greider (2003) Genes Dev. 17:2747-2752; Lai, et al. (2002) Genes Dev. 16:415-20; Tzfati, et al. (2000) Science 288:863-867) present in most organisms or the short oligonucleotide overhang of rodent TER51 within the RNA binding pocket of TRBD. The stable association of TER with the TRBD would force the enzyme to stall when reaching the nucleotide located at the 5′-end of the RNA template thus preventing replication beyond this point. Stalling of the enzyme for extended periods would lead to destabilization and dissociation of the RNA-DNA heteroduplex and initiation of another round of telomere replication.
Collectively, the data presented here supports common mechanistic aspects of substrate binding and DNA replication between telomerase and HIV reverse transcriptases, indicating an evolutionary link between these families of enzymes. It also provides novel insights into the basic mechanisms of telomere replication and length homeostasis by telomerase. Moreover, the structure presented here provides a detailed picture of the physical contacts between TERT and its nucleic acid substrates, information of use in the design of small molecule inhibitors of telomerase having therapeutic value in the treatment of cancer and other diseases associated with cellular aging.
Example 4
Modulators of Telomerase Activity
The X-ray crystal structure (complex of TERT with RNA-DNA, nucleotides and magnesium ions) showed that the organization of the fingers and palm domains between the substrate-free and substrate-bound TERT molecules is highly similar, indicating that like the Hepatitis C viral RNA polymerase (NS5B), telomerase has a preformed active site. This observation is surprising since in most polymerases, including the HIV reverse transcriptase, the fingers domain undergoes significant conformational changes referred to as the open and closed states. In HIV RTs, conformational rearrangements of the fingers domain with respect to the palm domain are essential for nucleotide binding and positioning at the active site of the enzyme and their absence from telomerase suggests possible mechanistic differences in nucleotide binding and selectivity between these families of enzymes.
The organization of the fingers and palm domains of TERT creates a deep, well-defined narrow mostly hydrophobic Fingers-Palm pocket (FP-pocket; residues Y170, F193, A195, D254, W302, H304, L307, Q308) that opens into the interior cavity of the TERT ring and is solvent accessible. Moreover, the entry of this pocket is located in close proximity of the active site (D251, D343 and D344) of the enzyme, which is absolutely essential for telomerase function. Alanine mutants of any of the above three invariant aspartates that form the active site of the enzyme completely abolishes telomerase activity.
Based on the fact that the FP-pocket is a stable, well-defined, solvent accessible cavity and is located in close proximity of the active site of the enzyme indicates that it is likely a useful target for the identification of small molecule inhibitors-activators that modulate telomerase activity. Based on the structure of the FP-pocket, it is expected that inhibitors will occlude the active site of telomerase thus interfering with nucleotide association and therefore telomere replication. On the other hand, activators are expected to be in close proximity to the active site of the enzyme without occluding the active site of the enzyme. These compounds could therefore be involved in favorable interactions with the incoming nucleotide substrate producing a tight and stable catalytic active complex thus increasing telomerase activity.
Example 5
Fluorescence Polarization Binding Assay
The TERT protein was expressed and purified as described herein. Protein was concentrated to 4.72 mg ml-1 using an AMICON 30K cutoff filter (Millipore) and stored at −80° C. for binding assays.
For the binding assay, TERT protein was diluted to a 500 nM concentration in a solution of 50 mM Tris-HCl, pH 7.5, 200 mM KCl, and 20% glycerol. A 1.2-fold molar excess of the Chim21m RNA-DNA hybrid (Integrated DNA Technologies), containing the template for telomere extension (5′-rCrUrG rArCrC rUrGrA rCTT CGG TCA GGT-3′; SEQ ID NO:19), was then added to these samples and kept on ice. The cyanine 5-dCTP (Perkin Elmer) for the binding study was made up to a 20 nM concentration in sterile, RNase, DNase free water with 40 nM MgCl2.
Compound screens were carried out as 30.5 μl reactions in a 384-well, round bottom plate (Thermo). Fifteen μl of TERT-Chim21m complex was first added to each well. Then 0.5 μl of library compound (ChemBridge) was added to this solution. Finally, 15 μl of the nucleotide-MgCl2 sample was added, and the solution mixed to provide final concentrations of 250 nM protein-nucleic acid, 10 nM cyanine 5-dCTP, and 20 nM MgCl2. Final compound concentrations were 50 μM and 5 μM. All additions were completed using a Janus 96/384 Modular Dispensing Tool (Perkin Elmer), performed in triplicate, and equilibrated for 40 minutes. Fluorescence polarization was measured at λex=620 nm and λem=688 nm on an EnVision Xcite Multilabel plate reader (Perkin Elmer). Polarization was calculated using the standard equation: P=(V−H)/(V+H), where P denotes polarization, V denotes vertical emission intensity, and H denotes horizontal emission intensity.
Example 6
Efficacy of Telomerase Inhibitors
Novel telomerase inhibitors of the instant invention can be analyzed in a variety of systems. The compounds can be assessed in defined well-known model systems used to assess cellular permeability, toxicity, and pharmacodynamic effects. These assays include both cell-based and animal based assays.
Cell-Based Assay. Cells from a P388 cell line (CellGate, Inc., Sunnyvale, Calif.) or human malignant melanoma cell line SK-MEL-2 are grown in RPMI 1640 cell medium containing fetal calf serum (10%), L-glutamine, penicillin, streptomycin and are split twice weekly. All compounds are first diluted with DMSO. Later serial dilutions are done with a phosphate-buffered saline solution. All dilutions are done in glass vials and the final DMSO concentration is generally below 0.5% by volume. Final two-fold dilutions are done in a 96-well plate using cell media so that each well contains 50 μL. All compounds are assayed over multiple concentrations. Cell concentration is measured using a hemacytometer and the final cell concentration is adjusted to about 1×104 cells/mL with cell medium. The resulting solution of cells (50 μL) is then added to each well and the plates are incubated for 5 days in a 37° C., 5% CO2, humidified incubator. MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, 10 μL) is then added to each well and the plates are re-incubated under identical conditions for 2 hours. To each well is then added acidified isopropanol (150 μL of i-PrOH solution containing 0.05 N HCl) and mixed thoroughly. The plates are then scanned at 595 nm and the absorbances are read (Wallac Victor 1420 Multilabel Counter). The resulting data is then analyzed to determine an ED50 value. Compounds that kill cancer cells, but fail to kill normal cells, find application in the prevention or treatment of cancer.
Mouse Ovarian Carcinoma Zenograft Model. Compounds of the invention are evaluated in an ovarian carcinoma xenograft model of cancer, based on that described by Davis, et al. ((1993) Cancer Research 53:2087-2091). This model, in brief, involves inoculating female nu/nu mice with 1×109 OVCAR3-icr cells into the peritoneal cavity. One or more test compounds are administered, e.g., prior to or after tumor cell injection, by the oral route as a suspension in 1% methyl cellulose or intraperitoneally as a suspension in phosphate-buffered saline in 0.01% TWEEN-20.
At the conclusion of the experiment (4-5 weeks) the number of peritoneal cells are counted and any solid tumor deposits weighed. In some experiments tumor development is monitored by measurement of tumor specific antigens.
Rat Mammary Carcinoma Model. Compounds of the invention are evaluated in a HOSP.1 rat mammary carcinoma model of cancer (Eccles, et al. (1995) Cancer Res. 56:2815-2822). This model involves the intravenous inoculation of 2×104 tumor cells into the jugular vein of female CBH/cbi rats. One or more test compounds are administered, e.g., prior to or after tumor cell injection, by the oral route as a suspension in 1% methyl cellulose or intraperitoneally as a suspension in phosphate-buffered saline and 0.01% TWEEN-20. At the conclusion of the experiment (4-5 weeks) the animals are killed, the lungs are removed and individual tumors counted after 20 hours fixation in Methacarn.
Mouse B16 Melanoma Model. The anti-metastatic potential of compounds of the invention is evaluated in a B16 melanoma model in C57BL/6. Mice are injected intravenously with 2×105 B16/F10 murine tumor cells harvested from in vitro cultures. Inhibitors are administered by the oral route as a suspension in 1% methyl cellulose or intraperitoneally as a suspension in phosphate-buffered saline pH 7.2 and 0.01% TWEEN-20. Mice are killed 14 days after cell inoculation and the lungs removed and weighed prior to fixing in Bouin's solution. The number of colonies present on the surface of each set of lungs is then counted.

## Claims

1. A method for identifying a compound which modulates the activity of telomerase comprising
(a) using a computer program to generate a three-dimensional structure of the Fingers-Palm pocket of a telomerase protein of SEQ ID NO:9 in complex with an RNA template and complementary telomeric DNA, wherein said Fingers-Palm pocket consists of amino acid residues 170, 193, 195, 254, 302, 304, 307 and 308 of SEQ ID NO:9 and said three-dimensional structure has a space group of P21 and unit cell parameters of a=77.2 Å, b=52.8 Å, c ̃101.6 Å, α=90 Å, β=101.9 Å, and γ=90 Å at a resolution of 2.7 Å;
(b) designing or screening for a compound that binds to said pocket; and
(c) testing the compound designed or screened for in (b) by in vitro or in vivo assay for its ability to modulate the activity of telomerase, thereby identifying a compound that modulates the activity of telomerase.