Patent Application: US-99324501-A

Abstract:
this invention relates to crystalline structures of the topoisomerase i and their use in designing new anti - cancer agents anti - viral agents and anti - microbial agents .

Description:
in order that the invention described herein may be more fully understood , the following detailed description is set forth . topoisomerase i ( topo i ) is an essential eukaryotic enzyme that acts to relax torsional stress in supercoiled dna generated during transcription and replication . champoux , j . j ., ann . rev . biochem ., 70 , pp . 369 - 413 ( 2001 ). topo i mediates dna relaxation by creating a transient single strand break , allowing the broken strand to rotate around its intact complement . this nicking results from the transesterification of an active - site tyrosine at a dna phosphodiester bond forming a 3 ′- phosphotyrosine covalent enzyme - dna complex . after dna relaxation , the covalent intermediate is reversed when the released 5 ′- oh of the broken strand re - attacks the phosphotyrosine intermediate in a second transesterification reaction champoux , j . j ., ann . rev . biochem ., 70 , pp . 369 - 413 ( 2001 ). topo i is the sole molecular target of a family of anti - cancer compounds called camptothecins , wall , m . e ., et al ., j . am . chem . soc ., 88 , pp . 3888 - 3890 ( 1966 ), hsiang , y . h ., et al ., j . biol . chem ., 260 , pp . 14873 - 14878 ( 1985 ), nitiss , j . l . and wang , j . c ., proc . natl . acad . sci . u . s . a ., 85 , pp . 7501 - 7505 ( 1988 ) ( cpts ). it is generally believed that cpts act as uncompetitive inhibitors by binding to the covalent topo i - dna intermediate and blocking the second transesterification reaction , hertzberg , r . p ., et al ., biochem ., 28 , pp . 4629 - 4638 ( 1989 ), thus converting the enzyme into a molecular poison . chen , a . y . and liu , l . f ., rev . pharmacol . toxicol ., 34 , pp . 191 - 218 ( 1994 ). several other families of compounds exist which are known to inhibit topoisomerase i and are believed to bind at the same site as the camptothecin family of compounds . these compounds includes indolacarbazoles , such as the anti - microbial marcellomycin ; indenoisoquinolines , such as the experimental anti - cancer compound mj - ii - 38 ; silatecan derivatives which are camptothecin compounds with silicon derivitizations , such as ag260 . additionally , other compounds have been shown to inhibit topoisomerase i , but it is not known if they bind at the same site as the camptothecin compounds . these compounds include minor groove binding compounds such as hoecsht - 33342 . we have shown that these compounds do not bind at the same site as camptothecin . to determine the structural basis for the mechanism of inhibitory activity , we have solved several new crystal structures of a fully active version of human topo i covalently joined to duplex dna in the absence ( form7 ) and presence of topotecan , a camptothecin derivative ( form 9 - ttc ); ag260 , a sialyl - tecan compound ( form 9 - ag260 ); mj - ii - 38 , an indenoisoquinoline compound ( form 10 ); and hoechst - 33342 , a dna minor groove binding compound ( form 11 ). examination of the form9 - ttc , form9 - ag260 , and form 10 , structures reveals that theses compounds intercalate at the site of dna cleavage , forming base - stacking interactions with both the − 1 ( upstream ) and + 1 ( downstream ) base pairs . a detailed examination of the topotecan structure follows . the planar five - membered ring system of topotecan mimics a base pair in the dna duplex , and occupies the same space as the + 1 base pair in the structure without drug bound ( fig1 ). approximately 61 % of the topotecan surface is covered by base stacking interactions , and 27 % is covered by protein contacts . the intercalation pocket is stabilized by several protein - dna interactions . the hydroxyl of thr718 makes a hydrogen bond contact with the non - bridging phosphodiester oxygen of guanosine at position + 1 of the cleaved strand , and arg364 makes a hydrogen bond contact with n3 of adenosine at position − 1 of the uncleaved strand . consistent with this binding mode , mutation at position 364 is expected to destabilize the binding site and results in camptothecin resistance . li , x . g ., et al ., biochem . pharmacol ., 53 , pp . 1019 - 1027 ( 1997 ). the intercalation also results in a 3 . 4 å shift of the downstream duplex that displaces the reactive 5 ′- oh of the cleaved strand 10 å away from the phosphotyrosine . in order for a religation event to occur , the topotecan molecule must be released from the nicked dna and diffuse out of the complex . the e - ring of camptothecin is known to be in equilibrium between a closed lactone form and a hydrolyzed open carboxylate form . wall , m . e ., et al ., j . am . chem . soc ., 88 , pp . 3888 - 3890 ( 1966 ) ( fig1 ). it is widely believed that the closed lactone e - ring is essential for inhibition of topo i . kehrer , d . f . s ., et al ., anti - cancer drugs , 12 , pp . 89 - 105 ( 2001 ). however , there is experimental evidence for e - ring opening upon formation of the ternary protein - dna - drug complex . chourpa , i ., r10u , j . f ., millot , j ., pommier , y ., manfait , m ., biochem ., 37 , pp . 7284 - 7291 ( 1998 ). in addition , despite a general belief that the carboxylate form is inactive , it has been shown that the sodium carboxylate form of camptothecin does have topo i inhibitory activity in vitro hsiang , y . h ., et al ., cancer res , 49 , pp . 4385 - 9 ( 1989 ) and in in vivo cell killing assays . giovanella , b . c ., et al ., science , 246 , pp . 1046 - 8 ( 1989 ). close inspection of the topotecan electron density allowed positioning of both the open and closed e - ring conformers ( fig1 b ). an unrestrained full matrix refinement of occupancy factors sheldrick , g . m ., pp . ( 1997 ) ( with all positional and thermal parameters fixed ) for the closed lactone and open carboxylate versions of topotecan converged to an occupancy of 63 % ( standard uncertainty 7 %) closed lactone and 37 % ( standard uncertainty 7 %) open carboxylate forms of topotecan . as another test , each conformer of topotecan was then placed into the structure and refined independently . analysis of the difference fourier maps demonstrates the presence of both the lactone and carboxylate forms of topotecan ( fig1 c and 15 d ). these results are typical of crystallographic structures in which multiple conformations of an amino acid side chain are present in a protein structure smith , j . l ., hendrickson , w . a ., honzatko , r . b ., sheriff , s ., biochem ., 25 , pp . 5018 - 5027 ( 1986 ). surprisingly , there is only one protein - drug interaction stabilizing the lactone ( e - ring closed ) form of topotecan ( fig1 ). asp533 hydrogen bonds to the 20 ( s ) hydroxyl of topotecan . in turn , asp533 is coordinated by arg364 , which is positioned only 4 å from the b - ring nitrogen . additionally , there are two water - mediated hydrogen bonds that assist in coordinating the topotecan into the cleaved dna intermediate . the oxygen of the d - ring pyridone makes a water mediated contact to asn722 , and the c - 21 oxygen of the e - ring is bridged by a water molecule to the phosphotyrosine and catalytic residues arg488 , arg590 and his632 champoux , j . j ., ann . rev . biochem ., 70 , pp . 369 - 413 ( 2001 ). consistent with the structural model , mutations at residues asp533 , arg364 and asn722 would be expected to destabilize the bound drug and are known to result in camptothecin resistance li , x . g ., et al ., biochem . pharmacol ., 53 , pp . 1019 - 1027 ( 1997 ), tamura , h ., et al ., nucleic acids res ., 19 , pp . 69 - 75 ( 1991 ), fertala , j ., et al ., j . biol . chem , 275 , pp . 15246 - 15253 ( 2000 ). it is not possible to determine the relative affinities of open ( carboxylate ) vs . closed ( lactone ) forms of topotecan based on the crystal structures , however the carboxylate form of topotecan would be expected to have a slower rate of dissociation since three additional direct hydrogen bonds are possible between the open e - ring and the protein - dna complex ( fig1 ). in the carboxylate model , the 22 - hydroxyl is 2 . 7 å from the r - group of asn722 . the 21 - carboxylate oxygen is 2 . 8 å from lys532 , a known catalytic residue , krogh , b . o ., shuman , s ., mol . cell , 5 , pp . 1034 - 1041 ( 2000 ). the 20 ( s )- hydroxyl still coordinates asp533 , and makes an additional hydrogen bond contact ( 3 . 1 å ) to the 1 - nitrogen of arg364 , a residue known to be involved in camptothecin sensitivity , li , x . g ., et al ., biochem . pharmacol ., 53 , pp . 1019 - 1027 ( 1997 ). finally , it is also important to note that in the carboxylate structure , one of the 21 - carboxylate oxygens is 2 . 7 å from the o2 of the − 1 thymidine of the cleaved strand and the second carboxylate oxygen makes a water mediated contact with the phosphotyrosine phosphodiester . topotecan therefore appears to inhibit religation by displacing the reactive 5 ′- oh and by simultaneously coordinating several active - site functional groups . in addition to preventing dna religation , topo i poisons such as camptothecin have been shown to inhibit the rotation / relaxation process in vitro champoux , j . j ., ann . n . y . acad . sci ., 922 , pp . 56 - 64 ( 2000 ). it has been a mystery why camptothecins stabilize the nicked complex but prevent dna relaxation — nicked dna should be able to rotate and allow dna relaxation champoux , j . j ., ann . n . y . acad . sci ., 922 , pp . 56 - 64 ( 2000 ). topoisomerase i has been proposed to relax dna via a mechanism of “ controlled rotation ,” in which the dna duplex located downstream of the cleavage site rotates around the − 1 /+ 1 phosphodiester linkage of the intact strand , effectively passing the unbroken strand through the single strand break with each complete rotation event stewart , l ., et al ., science , 729 , pp . 1534 - 1541 ( 1998 ). a comparison of the unbound and topotecan - bound structures shows that topotecan displaces the critical − 1 /+ 1 phosphodiester linkage of the non - scissile strand into a binding pocket , producing several interactions that are predicted to inhibit rotation ( fig1 ). one non - bridging oxygen of the − 1 /+ 1 phosphodiester is hydrogen bonded to the main chain nitrogen atoms of arg362 and gly363 . the other non - bridging oxygen forms a hydrogen bond to the terminal nitrogen of lys374 . the hydrogen bond contact to lys374 is also present in the non - drug bound structure , indicating that this side chain can move to accommodate a shift in position of the − 1 /+ 1 phosphodiester . the shifted − 1 /+ 1 phosphodiester is also positioned close to the phe361 side chain which would provide and additional steric block to rotation . the tight positioning of the − 1 /+ 1 intact phosphodiester against the peptide backbone , together with support from phe361 and a molecular clamping of the upstream duplex by topo i redinbo , m . r ., stewart , l ., kuhn , p ., champoux , j . j ., hol , w . g . j ., science , 279 , pp . 1504 - 1513 ( 1998 ), effectively restrains 3 ( α , β , γ ) saenger , w ., springer advanced texts in chemistry , pp . 556 ( 1984 ) of the 5 potentially rotatable backbone bonds . this tight packing arrangement is expected to interfere with the conformational changes in the dna required to complete a 360 degree rotation of the downstream dna about the − 1 /+ 1 intact phosphodiester in what we propose is a “ hinge - lock ” mechanism . this model provides a rationale for understanding how camptothecins can inhibit dna relaxation through an intercalative binding mode , and is consistent with the observations that phe361 , gly363 , and arg364 are required for sensitivity to camptothecin li , x . g ., et al ., biochem . pharmacol ., 53 , pp . 1019 - 1027 ( 1997 ), rubin , e ., et al ., j biol chem , 269 , pp . 2433 - 2439 ( 1994 ), fiorani , p ., et al ., mol pharmacol , 56 , pp . 1105 - 1115 ( 1999 ). the hinge - lock mechanism would not eliminate all possible dna rotation . for example , rotation could still occur at the + 2 ( or + 3 , etc .) phosphodiester . however , additional base - pair hydrogen bond interactions would have to be broken to allow this rotation . alternatively , rotation could still occur at + 1 since two rotatable bonds are not hindered . however in both cases , the trajectory of the rotating dna would be significantly altered and this would require conformational flexibility that is not likely to be present in the protein . the protein encircles the dna , and both the linker and nose cone domains of topo i contain a positively charged residues that are likely to contact the dna during rotation stewart , l ., et al ., science , 729 , pp . 1534 - 1541 ( 1998 ). this may at least partially explain why reconstituted “ linker - less ” human topo i is resistant to the relaxation - inhibition effect of topotecan stewart , l ., et al ., j . biol . chem ., 274 , pp . 32950 - 32960 ( 1999 ), as well as the camptothecin resistant phenotype of an ala653pro mutation which destabilizes the linker domain , fiorani , p ., et al ., mol pharmacol , 56 , pp . 1105 - 1115 ( 1999 ). the coding sequences for wild type human topo70 ( residues 175 to 765 of the natural protein plus an n - terminal initiating methionine ) were derived from plasmid pgst - topo70 wt biochemical and biophysical analyses of recombinant forms of human topoisomerase i described in , stewart , l ., et al ., j . biol . chem ., 271 , pp . 7593 - 7601 ( 1996 ). a bamhi - ecor1 restriction fragment from pgst - topo70 wt was transferred into linear pfastbac baculovirus transfer vector ( life technologies , inc .) that was prepared by cleavage with bamhi and ecor1 . the resulting plasmid called “ pfastbac - topo70 wt ” was used , according to standard protocol ( life technologies , inc . ), to generate recombinant baculovirus stock that expresses the recombinant topo70 . recombinant baculoviruses were used to produce topo70 in insect cells and the protein was purified according to known procedures for purification of baculovirus expressed human dna topoisomerase i . in protocols for dna topoisomerases : i . dna topology and enzyme purification , stewart , l ., et al ., j . biol . chem ., 271 , pp . 7593 - 7601 ( 1996 ). the topo58 / 6 . 3 protein was prepared as described previously with minor modification . stewart , l ., et al ., j . mol . biol ., 269 , pp . 355 - 372 ( 1997 ). the purification of oligonucleotides and the hybridization of complementary oligonucleotides to generate duplex oligonucleotide substrates was described . stewart , l ., et al ., science , 729 , pp . 1534 - 1541 ( 1998 ). three - dimensional structures of reconstituted human topoisomerase i in covalent and non - covalent complex with dna is described in redinbo , m . r ., stewart , l ., kuhn , p ., champoux , j . j ., hol , w . g . j ., science , 279 , pp . 1504 - 1513 ( 1998 ). the synthesis of suicide substrates that contain a 5 ′- bridging phosphorothiolate at the site of topo i cleavage wherein the base immediately downstream of the cleavage site is a thymidine as described , burgin jr ., a . b ., huizenga , b . n ., nash , h . a ., nucleic acids res ., 23 , pp . 2973 - 2979 ( 1995 ). these synthetic routes have been used to produce oligonucleotides containing a 5 ′- bridging phosphorothiolate at the site of topo i cleavage immediately preceding a thymidine , adenine , guanine , or cytocine . fig9 illustrates the process . the synthetic routes of 5 ′- bridging phosphorthiolate at the site of topo i cleavage wherein the base immediately downstream of the cleavage site is a adenine , guanine or cytosine are described in u . s . patent application ser . no . 09 / 882 , 274 ( burgin , sdsu patent application ). while a 22 mer duplex dna is preferred , those skilled in the art will recognize that larger dna sequences having 15 - 40 bp are operative and the dna sequence can very as long as the dna is linked to the topo i cleavage site . topotecan is a trade name for the structure shown in fig1 . related compounds are described in u . s . pat . no . 5 , 004 , 758 . these compounds are water soluble camptothecin analogs useful for inhibiting growth of animal tumor cells . synthesis of cytotoxic indenoisoquinoline topoisomerase i poisons . are described in , strumberg , d ., et al ., j med chem , 11 , pp . 446 - 457 ( 1999 ), and the synthesis of new indeno [ 1 , 2 - c ] isoquinolines : cytotoxic non - camptothecin topoisomerase i inhibitors are described in , cushman , m ., et al ., j med chem , 5 , pp . 3688 - 3698 ( 2000 ). [ heading - 0066 ] d . combinatorial crystallization screening to identify ternary topo i - dna - inhibitor crystallization conditions . in order to identify crystallization conditions that generate crystals comprised of topo70 in covalent complex with dna and bound to anti - cancer compounds such as topotecan , numerous crystallization conditions that had salt concentrations less than 400 mm and buffered phs between 4 and 9 were screened . the crystallant buffer ; salt ( cbs ) cross optimization strategy is shown in disclosed in u . s . pat . no . 6 , 039 , 804 and is incorporated herein by reference . the screening system utilized a combinatorial approach involving the set up of parallel crystallization conditions asdescribed in u . s . pat . no . 6 , 039 , 804 . issued mar . 21 , 2000 . the screened mixtures contained topo i ( topo70 or topo58 / 6 . 3 ), suicide substrate 5 ′- bridging oligonucleotide duplex , and various inhibitors . in order to identify crystallization conditions that depended on the presence of topotecan or other compounds , a novel approach to crystallization screening wherein was developed . a large number of novel crystallization conditions using all combinations of crystallants , buffers , and salts from all known crystallization conditions for topo70 and topo58 / 6 . 3 . these recombinant crystallization conditions were screened with enzyme ( topo70 or topo58 / 6 . 3 ), topotecan , and suicide substrate that contained a 5 - bidging phosphorothiolate at the site of topo i breakage wherein the base immediately downstream of the break site on the cleaved strand was a guanine ( g ) which was base paired to its complementary cytosine ( c ) on the complementary strand . this approach proved to be successful in producing novel crystal forms of human topo70 , wherein the crystal growth absolutely depended on the presence of the topotecan . tris base ( sigma cat . # t1503 , cas # 77 - 86 - 1 ) stock solutions were made ph 7 . 0 or 8 . 0 with concentrated hcl ( sigma cat . # h7020 , cas # 7647 - 01 - 0 ), and the volumes adjusted to 1 m final concentration of tris base . 0 . 5 m na 2 hpo 4 ( sigma cat . # s7907 , cas # 7558 - 79 - 4 ) and 0 . 5 m kh 2 po 4 ( sigma cat . # po662 , cas # 7778 - 77 - 0 ) solutions were mixed together to make a ph 6 . 2 na / k phosphate stock solution . a mes ( sigma cat . # m8250 , cas # 4432 - 31 - 9 ) stock solution was made ph 6 . 4 with 50 % naoh ( sigrna cat . # s0899 , cas # 1310 - 73 - 2 ), and the volume adjusted to 1 m mes . a ada ( sigma cat . # a9883 , cas # 26239 - 55 - 4 ) stock solution was made ph 6 . 5 with 50 % naoh ( sigma cat . # s0899 , cas # 1310 - 73 - 2 ), and the volume adjusted to 1 m ada . table i table i lists the crystal form space group parameters for crystals made in accordance with the present invention . crystal form space group parameters alternative space ######## unit cell parameters ########## crystal form protein oligo oligo drug group a b c alpha beta gamma crystal form 7 topo70 cl22 - st : cp22 - a cl22 - sa : cp22 - t none p32 72 . 0 72 . 8 185 . 5 90 . 0 90 . 0 120 . 0 crystal form 8 topo70 cl22 - sg : cp22 - c yet to be attempted topotecan p1 76 . 2 76 . 2 103 . 8 107 . 8 96 . 1 113 . 0 crystal form 9 ttc topo70 cl22 - sg : cp22 - c cl22 - sc : cp22 - g topotecan p21 57 . 7 115 . 9 75 . 4 90 . 0 97 . 3 90 . 0 crystal form 9 ag260 topo70 cl22 - sg : cp22 - c yet to be attempted ag260 p21 57 . 7 115 . 9 75 . 4 90 . 0 97 . 3 90 . 0 crystal form 10 topo70 cl22 - sg : cp22 - c yet to be attempted mj - 11 - 38 c2 260 . 9 74 . 6 57 . 5 90 . 0 96 . 9 113 . 0 crystal form 11 topo70 cl22 - sa : cp22t cl22sc : cp22g hoechst - p21212 270 . 9 71 . 1 57 . 6 90 . 0 90 . 0 90 . 0 33342 detailed coordinate for various crystal forms are set - out in fig1 - 5 the x - ray diffraction data collected on the various crystal forms of human topoisomerase i have been obtained at the x25 beamline of the national synchrotron light source ( nsls ) at brookhaven national laboratory ( bnl ) in upton , n . y . ; or at the com - cat beam line of the advanced photon source ( aps ) of the argonne national laboratory ( anl ) in argonne , ill . all x - ray diffraction experiments were performed with crystals held in a gaseous nitrogen cryo stream at 100 degrees kelvin as described in , rodgers , d . w ., structure , 2 , pp . 1135 - 1140 ( 1994 ). x - ray diffraction data was processed using the software package hkl - 2000 . this software has been reported in the following reference , otwinoski , z . and minor , w ., meths . enzymol ., 276 , pp . 307 - 326 ( 1997 ) structure determinations have been performed using molecular replacement , navaza , j ., acta . crystallogr ., a50 , pp . 157 - 163 ( 1994 ), in conjunction with cnx , brlnger , a . t ., et al ., acta . crystallogr ., d54 , pp . 905 - 921 ( 1998 )), and xtalview crystallographic computing packages under license to emerald biostructures , inc . mcree , d . e ., j . struct . biol ., 125 , pp . 156 - 165 ( 1999 ). oligonucleotide duplexes ( 22 - mer suicide substrates ) at 0 . 05 mm were mixed with crystallization solution ( referred to as “ crystallant ”) in the drop chambers of patented clover plates described in u . s . pat . no . 6 , 029 , 804 , followed by the addition of drug compound , and then protein solution at 2 - 5 mg / ml ( as determined by a bradford assay , relative to a bovine serum albumin standard ). the reservoir chambers of the clover plates contained 0 . 4 to 1 . 0 ml of crystallant . after set up of the crystallization drops at room temperature , the clover chambers were sealed with crystal clear tape and incubated at 15 - 16 degrees c . crystals appeared within 2 - 5 days but sometimes crystallization required incubation of up to 7 months . on certain occasions , the tape from one quarter of a combinatorial crystallization clover was removed , thereby exposing the crystallization drops to the outside air environment causing evaporation crystallization drops and promotion of crystal growth . crystallizations are preferably set up and conducted in accordance with the methods and apparatus described in u . s . pat . no . 6 , 039 , 804 . however , crystallizations could also be performed in other crystallization apparatuses that accommodate vapor diffusion techniques . in order that the invention described herein may be more fully understood , the following examples are set forth . it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner . [ heading - 0090 ] this crystal structure contains the first example of a fully active human topoisomerase i ( topo70 ) in covalent complex with the duplex 5 ′- bridging phosphorthiolate dna . the crystal form 7 crystallant was composed of 10 % ( w / v ) peg - 8000 ( sigma , cat .# p4463 , cas # 25322 - 68 - 3 ) 100 mm tris - hcl ph 8 . 0 , 100 mm na / k phosphate ph 6 . 2 , 100 mm kcl ( sigma , cat . # p9333 , cas # 7447 - 40 - 7 ) 10 mm dithiothreitol ( sigma cat . # d5545 , cas 27565 - 41 - 9 ). the internal reference code for this crystallant is “ vii - 6 - 1 # 4 )” the crystallization set up that produces crystal form 7 was prepared at 25 degrees c . ( room temperature ) in drop chambers of combinatorial clover plates as follows . a milliliter ( 1 ml ) of crystal form 7 crystallant was placed into the reservoir chamber of a combinatorial clover . three microliters ( 3 ul ) of crystal form 7 crystallant was removed from the reservoir chamber and placed into one of the four surrounding drop chambers . one and a half microliter ( 1 . 5 ul ) of 22 - mer cl22 - st : cp22 - a suicide substrate oligonucleotide duplex at 0 . 05 mm in 3 mm nacl ( sigma cat . # s7653 , cas 7647 - 14 - 5 ) was then added to the 3 ul drop of crystal form 7 crystallant in the drop chamber . after allowing the suicide substrate to mix with the crystallant for approximately 1 minute , 2 . 5 microliter ( 2 . 5 ul ) of topo70 wild type ( tyr723 ) at 2 . 5 milligrams per milliliter ( 2 . 5 mg / ml ) was added to the drop . after allowing the mixture of crystallant , suicide substrate , and topo70 wild type to sit for approximately 1 minute . the combinatorial clover reservoir was sealed with crystal clear tape ( manco ), and the crystallization sample was maintained at 16 degrees c . for approximately two to four weeks . crystals typically grew between the first and third weeks after set up . the form 7 crystal growth specifications and the following cryopreservation specifications are based on emerald &# 39 ; s internal reference code of “ bnl - 63 ” cryopreservation of form 7 crystals was achieved by transferring individual form 7 crystals ( at room temperature , using glass capillary pipettes or by looping the crystal out of its liquid crystallization drop ) into a cryoprotectant solution comprised of 20 microliters of ( 20 ul ) of form 7 cryoprotectant solution [ 30 % ( v / v ) peg - 400 ( sigma cat . # p3265 , cas # 25322 - 68 - 3 ), 100 mm tris - hcl ph 8 . 0 , 100 mm na / k phosphate ph 6 . 2 , 100 mm kcl ( sigma , cat . # p9333 , cas # 7447 - 40 - 7 )]. the transferred crystal was incubated in the cryoprotectant solution for approximately one minute , looped up in a nylon loop of approximately 700 micrometers in diameter , and plunged into liquid nitrogen for cryopreservation . [ heading - 0097 ] crystal structure of fully active human topoisomerase i ( topo70 ) in ternary complex with 22 mer phosphorthiolate duplex dna and the anti - cancer compound topotecan . the crystal form 8 crystallant was composed of 15 % ( w / v ) peg - 3000 ( fluka , cat .# 81227 , cas # 25322 - 68 - 3 ) 100 mm tris - hcl ph 7 . 0 , 100 mm na / k phosphate ph 6 . 2 , 10 mm beta - mercaptoethanol ( sigma cat . # m6250 , cas 60 - 24 - 2 ). the internal reference code for this crystallant is “ vii - 10 # 23 ” the crystallization set up that produces crystal form 8 was prepared at 25 degrees c . ( room temperature ) in drop chambers of emerald &# 39 ; s combinatorial clover plates as follows . a milliliter ( 1 ml ) of crystal form 8 crystallant was placed into the reservoir chamber of a combinatorial clover . two microliters ( 2 ul ) of crystal form 8 crystallant was removed from the reservoir chamber and placed into one of the four surrounding drop chambers . one microliter ( 1 ul ) of 22 - mer cl22 - sg : cp22 — c suicide substrate oligonucleotide duplex at 0 . 05 mm in 3 mm nacl ( sigma cat . # s7653 , cas 7647 - 14 - 5 ) was then added to the 2 ul drop of crystal form 8 crystallant in the drop chamber . after allowing the suicide substrate to mix with the crystallant for approximately 1 minute , 0 . 3 microleter ( 0 . 3 ul ) of 5 mm topotecan ( obtained from the drug synthesis branch of the national cancer institute , nsc609699 ) was added to the drop . after allowing the topotecan to mix with the crystallant and suicide substrate for approximately 1 minute , i microleter ( 1 ul ) of topo70 wild type ( tyr723 ) at 3 milligrams per milliliter ( 3 mg / ml ) was added to the drop . after allowing the mixture of crystallant , suicide substrate , topotecan and topo70 wild type to sit for approximately 1 minute . the combinatorial clover reservoir was sealed with crystal clear tape ( manco ), and the crystallization sample was maintained at 15 degrees c . for approximately 7 months . crystals grew sometime between the first and seventh month of incubation . the form 8 crystal growth specifications and the following cryopreservation specifications are based on emerald &# 39 ; s internal reference code of “ bnl - 91 ” cryopreservation of form 8 crystals was achieved by transferring individual form 8 crystals ( at room temperature , using glass capillary pipettes or by looping the crystal out of its liquid crystallization drop ) into a cryoprotectant solution comprised of 20 microliters of ( 20 ul ) of form 8 cryoprotectant solution [ 30 % ( v / v ) peg - 400 ( sigma cat . # p3265 , cas # 25322 - 68 - 3 ) 100 mm tris - hcl ph 7 . 0 , 100 mm na / k phosphate ph 6 . 2 ] plus 1 . 5 microliter ( 1 . 5 ul ) of 1 mm topotecan . the transferred crystal was incubated in the cryoprotectant solution for approximately one minute , during which time , the crystal was observed to crack and therefore a small chunk of the crystal that displayed no visible cracking was looped up in a nylon loop of approximately 300 micrometers in diameter and plunged into liquid nitrogen for cryopreservation . [ heading - 0103 ] crystal structure of fully active human topoisomerase i ( topo70 ) in ternary complex with 22 mer phosphorthiolate duplex dna and the anti - cancer compound topotecan . this example demonstrates the utility of using the said invention to crystallize one compound in multiple crystal forms ( see example 2 above ). the crystal form 9 crystallant was composed of 10 % ( w / v ) peg - 8000 ( fluka , cat .# 81268 , cas # 25322 - 68 - 3 ) 100 mm mes - naoh ph 6 . 4 ( or alternatively ada - naoh ph 6 . 5 ), 200 mm lithuim sulfate ( sigma cat . # l8158 , cas # 10102 - 25 - 7 ). the internal reference code for this crystallant is “ t80p # 9 or # 10 )” the crystallization set up that produces crystal form 9 was prepared at 25 degrees c . ( room temperature ) in drop chambers of emerald &# 39 ; s combinatorial clover plates as follows . a milliliter ( 1 ml ) of crystal form 9 crystallant was placed into the reservoir chamber of a combinatorial clover . two microliters ( 2 ul ) of crystal form 9 crystallant was removed from the reservoir chamber and placed into one of the four surrounding drop chambers . one and a half microliter ( 1 . 5 ul ) of 22 - mer cl22 - sg : cp22 — c suicide substrate oligonucleotide duplex at 0 . 05 mm in 3 mm nacl ( sigma cat . # s7653 , cas 7647 - 14 - 5 ) was then added to the 2 ul drop of crystal form 9 crystallant in the drop chamber . after allowing the suicide substrate to mix with the crystallant for approximately 1 minute , 0 . 3 microleter ( 0 . 3 ul ) of 1 mm topotecan ( obtained from the drug synthesis branch of the national cancer institute , nsc609699 ) was added to the drop . after allowing the topotecan to mix with the crystallant and suicide substrate for approximately 1 minute , i microleter ( 1 . 5 ul ) of topo70 wild type ( tyr723 ) at 4 milligrams per milliliter ( 4 mg / ml ) was added to the drop . after allowing the mixture of crystallant , suicide substrate , topotecan and topo70 wild type to sit for approximately 1 minute , the combinatorial clover reservoir was sealed with crystal clear tape ( manco ), and the crystallization sample was maintained at 16 degrees c . for approximately two to four weeks . crystals typically grew between the first and third weeks after set up . the form 9 crystal growth specifications and the following cryopreservation specifications are based on emerald &# 39 ; s internal reference code of “ topo - 104 ” cryopreservation of form 9 crystals was achieved by transferring individual form 9 crystals ( at room temperature , using glass capillary pipettes or by looping the crystal out of its liquid crystallization drop ) into a cryoprotectant solution comprised of 10 microliters of ( 10 ul ) of form 9 cryoprotectant solution [ 30 % ( v / v ) peg - 400 ( sigma cat . # p3265 , cas # 25322 - 68 - 3 ), 100 mm mes - naoh ph 6 . 4 ( or alternatively ada - naoh ph 6 . 5 ), 200 mm lithuim sulfate ( sigma cat . # l8158 , cas # 10102 - 25 - 7 )], plus i microliter ( 1 ul ) of 1 mm topotecan . the transferred crystal was incubated in the cryoprotectant solution for approximately one minute , during which time , the crystal was looped up in a nylon loop of approximately 300 micrometers in diameter and plunged into liquid nitrogen for cryopreservation . crystal structure of fully active human topoisomerase i ( topo70 ) in ternary complex with 22 mer phosphorthiolate duplex dna and the anti - cancer compound ag260 . this example demonstrates the utility of using said invention to crystallize and solve the three - dimensional structure of different compounds with the same crystal form . this example also demonstrates the utility of using said invention to determine the three dimensional structure of camptothecin derivative compounds such the silatecan , ag - 260 . crystals of ag260 were grown and the structure was solved exactly as detailed in example 3 above . crystal unit cell parameters were determined to be similar to the form - 9 topotecan crystal . see table 1 . crystal structure of fully active human topoisomerase i ( topo70 ) in ternary complex with 22 mer phosphorthiolate duplex dna and the anti - cancer compound mj - ii - 38 . this example demonstrates the utility of using said invention to determine the three dimensional structure of non - camptothecin derivatives such the indenoisoquinoline compound mj - ii - 38 . the crystal form 10 crystallant was composed of 10 % ( w / v ) peg - 8000 ( fluka , cat .# 81268 , cas # 25322 - 68 - 3 ) 100 mm mes - naoh ph 6 . 4 ( or alternatively ada - naoh ph 6 . 5 ), 200 mm lithuim sulfate ( sigma cat . # l8158 , cas # 10102 - 25 - 7 ). the internal reference code for this crystallant is “ t80p # 9 or # 10 the crystallization set up that produces crystal form 10 was prepared at 25 degrees c . ( room temperature ) in drop chambers of emerald &# 39 ; s combinatorial clover plates as follows . a milliliter ( 1 ml ) of crystal form 9 crystallant was placed into the reservoir chamber of a combinatorial clover . two microliters ( 2 ul ) of crystal form 9 crystallant was removed from the reservoir chamber and placed into one of the four surrounding drop chambers . one and a half microliter ( 1 . 5 ul ) of 22 - mer cl22 - sg : cp22 — c suicide substrate oligonucleotide duplex at 0 . 05 mm in 3 mm nacl ( sigma cat . # s7653 , cas 7647 - 14 - 5 ) was then added to the 2 ul drop of crystal form 7 crystallant in the drop chamber . after allowing the suicide substrate to mix with the crystallant for approximately 1 minute , 0 . 3 microleter ( 0 . 3 ul ) of 1 mm mj - ii - 38 ( see fig1 for the structure of mj - ii - 38 ) in 90 % ( v / v ) dmso ( sigma cat . # d5879 , cas 67 - 68 - 5 ) was added to the drop . after allowing the mj - 1 ′- 38 to mix with the crystallant and suicide substrate for approximately 1 minute , i microleter ( 1 . 5 ul ) of topo70 wild type ( tyr723 ) at 4 milligrams per milliliter ( 4 mg / ml ) was added to the drop . after allowing the mixture of crystallant , suicide substrate , mj - ii - 38 , and topo70 wild type to sit for approximately 1 minute , the combinatorial clover reservoir was sealed with crystal clear tape ( manco ), and the crystallization sample was maintained at 16 degrees c . for approximately two to four weeks . crystals typically grew between the first and third weeks after set up . note : the form 10 crystallization condition first produces large transamerica building shaped crystals . however , these crystals are found not to diffract x - rays to beyond 8 angstrom resolution . however , crystals with form 9 morphology will grow out of the conditions if one of the four drop chambers of the combinatorial clover is unsealed ( by removal of the tape above the drop ) and evaporation is allowed to occur at 16 degrees c . over a period of two weeks . the resulting crystals that have form 9 morphology are the form 10 crystals . the form 10 crystal growth specifications and the following cryopreservation specifications are based on internal reference code of “ bart - com - cat - from 10 ” cryopreservation of form 10 crystals was achieved by transferring individual form 10 crystals ( at room temperature , using glass capillary pipettes or by looping the crystal out of its liquid crystallization drop ) into a cryoprotectant solution comprised of 10 microliters of ( 10 ul ) of form 10 cryoprotectant solution [ 30 % ( v / v ) peg - 400 ( sigma cat . # p3265 , cas # 25322 - 68 - 3 ), 100 mm mes - naoh ph 6 . 4 ( or alternatively ada - naoh ph 6 . 5 ), 200 mm lithuim sulfate ( sigma cat . # l8158 , cas # 10102 - 25 - 7 )], plus i microliter ( 1 ul ) of 1 mm mj - ii - 38 in 90 % ( v / v ) dmso ( sigma cat . # d5879 , cas 67 - 68 - 5 ). the transferred crystal was incubated in the cryoprotectant solution for approximately one minute , during which time , the crystal was looped up in a nylon loop of approximately 300 micrometers in diameter and plunged into liquid nitrogen for cryopreservation . crystal structure of fully active human topoisomerase i ( topo70 ) in ternary complex with 22 mer phosphorthiolate duplex dna and the dna minor - groove binding compound hoecsht - 33342 . this example demonstrates the utility of using said invention to crystallize and solve the structure of dna binding compounds which do not bind to the active site of topoisomerase 1 . crystals of form - 11 were grown and the structure was solved similarly as detailed in example 3 above . while we have described a number of the embodiements of this invention , it is apparent that our basic examples may be altered to provide other embodiements which utilize the products and processes of this invention . therefore , it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by specific embodiements which have been represented by way of example . d &# 39 ; arpa , p ., et al ., “ cdna cloning of human dna topoisomerase i : catalytic activity of a 67 . 7 - kda carboxyl - terminal fragment ”, proc natl acad sci usa , 85 , pp . 2543 - 2547 ( 1988 ). burgin jr ., a . b ., huizenga , b . n ., nash , h . a ., “ a novel suicide substrate for dna topoisomerases and site - 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