INHIBITORS OF PHOSPHOSERINE AND PHOSPHOTHREONINE-PROLINE-SPECIFIC ISOMERASES

Abstract: Peptides and peptide mimetics that inhibit phosphoserine- or phosphothreonine-specific pepidyl prolyl isomerases are described.

Description: INHIBITORS OF PHOSPHOSERINE AND PHOSPHOTHREONINE-PROLINE-SPECIFIC ISOMERASES BACKGROUND Events of the eukaryotic cell cycle are regulated by an evolutionarily conserved set of protein kinases. The cyclin-dependent kinases (Cdks) are important for driving cells through different phases of the cell cycle and their sequential activation and inactivation are tightly regulated. At the G2/M transition, activation of the mitotic Cdk. Cdc2. requires multiple events; these include the synthesis and binding of cyclin B. phosphorylation on Cdc2 at an activating site by Cak. and finally, Cdc25 -dependent dephosphorylation of inactivating sites that have been phosphorylated by Weel and Mytl (P. Nurse. Cell 79:547 ( 1994); R. W. King, P. K. Jackson, M. W. Kirschner. Cell 79:563 (1994); T. R. Coleman, W. G. Dunphy, Curr. Opin. Cell Biol. 6:877 (1994)). How activation of a Cdk elicits the downstream events of cell cycle progression is less well understood. Activation of cyclin B/Cdc2 leads to the phosphorylation of a large number of proteins, mainly on sites containing a Ser/Thr- Pro motif. Protein phosphorylation is believed to alter the functions of proteins to trigger the events of mitosis. In a few cases, mitotic phosphorylation has been shown to regulate mitotic events (R. Heald and F. McKeon. Cell 61 :579 ( 1990); E. Bailly, et al.. Nature 350:715 (1991 ): A. Blangy, et al., Cell 83:1 159 (1995)). However, it is not understood how the rapid changes in mitotic phosphorylation are converted to the sequential events of mitosis. An important experimental tool which has uncovered the general role of phosphorylation in mitotic regulation is the MPM-2 monoclonal antibody (F. M. Davis, et al., Proc. Natl. Acad. Sci. USA 80:2926 (1983)). iVΪPM-2 recognizes a Phospho.Ser/Thr-Pro epitope on approximately 50 proteins which are localized to various mitotic structures (J. M. Westendorf, P. N. Rao, L. Gerace, Proc. Natl. Acad. Sci. USA 91 :714-8 (1994)). Several important mitotic regulators are recognized by this antibody, including Cdc25, Weel , topoisomerase Ila, Cdc27, Map 4, INCENP and NIMA (Stukenberg, P.T.. K.D. Lustig, T.J. McGarry, R.W. King, J. Kuang and M.W. Kirschner, Curr Biol 7:338-348 (1997)). Currently six kinases have been shown to phosphorylate proteins in vitro to produce the MPM-2 epitope: Cdc2, Polo-like kinase (Plkl), NIMA, MAP kinase, a MAP kinase (MEK), and an unidentified activity ME-H (Kuang, J. and C.L. Ashorn., J Cell Biol 123:859-868 (1993); Taagepera et al, Moi Biol Cell 5:1243- 1251 (1994); Kumagai, A. and W.G. Dunphy, Science 273:1377-1380 (1996); Renzi, L., M.S. Gersch, M.S. Campbell, L. Wu, S.A. Osmani and G.J. Gorbsky, J. Cell Sci 110:2013-2025 (1997)). However, these kinases also phosphorylate substrates that do not generate the MPM-2 epitope especially in cell cycle stages other than mitosis. This suggests that there are additional features that are required for the recognition by MPM-2. Determination of the optimal MPM-2 binding sequence have confirmed the importance of amino acid residues flanking the Phospho Ser/Thr-Pro motif for the MPM-2 recognition (Westendorf, J.M., P.N. Rao and L. Gerace. Proc Natl Acad Sci USA 91:714-718 (1994)). Westendorf. et al, 1994). SUMMARY OF THE INVENTION The present invention is based on the discovery that an essential mitotic peptidyl prolyl isomerase specifically recognizes phosphorylated serine/threonine- proline bonds present in mitotic phosphoproteins. As a result of this discovery, a novel class of moleculular compounds are available with activity to act as inhibitors of phosphoserine/phosphothreonine-proline specific peptidyl prolyl isomerases. in particular the peptidyl prolyl isomerase, Pinl, and other Pin 1 -like isomerases. Accordingly, these molecular inhibitors are useful to treat disorders of cell proliferation such as hyperplastic or neoplastic disorders, wherein treatment of the disorder with an inhibitor of the present invention results in the arrest of mitosis and apoptosis (cell death) of the target cells. The inhibitor compounds of the present invention include any molecule that binds into the active site of the phosphoserine- or phosphothreonine-proline specific peptidyl prolyl isomerase and, upon binding to the isomerase, inhibits the isomerase activity. Encompassed by the present invention are inhibitor compounds that mimic the structure and conformation of the substrate moiety when bound to the catalytic site (also referred to herein as the active site) of the isomerase. Molecular inhibitors of the the present invention will typically have an inhibition constant (K,) in the nanomolar to micromolar range. Specifically encompassed herein are organic molecules that mimic the structure and conformation of pSer/pThr and bind to the isomerase of interest, thereby inhibiting its activity. The inhibitor compounds of the present invention inculde inhibitors that comprise a core region that mimics the pSer/pThr-Pro peptide moiety of the phosphoserine- or phosphothreonine-proline peptidyl prolyl isomerase substrate. Encompassed by the present invention are inhibitors that comprise the pSer/pThr mimic moiety with the mimic moiety being flanked on one side by hydrophobic groups and on the other side by hydrophobic or positively charged groups, wherein the groups would contact the active site of the isomerase of interest. The inhibitor compounds of the present invention include compounds that contain a core sequence comprising xSer/xThrY wherein "x" is a negatively charged tetra-or pentavalent moiety and "Y" is a Pro (proline) or a Pro analog. More specifically, the inhibitors of the present invention include compounds that inhibit a phosphoserine- or phosphothreonine-proline specific peptidyl-prolyl isomerase comprising a protein, polypeptide. peptide and/or a peptide mimetic wherein said protein, polypeptide. peptide or peptide mimetic comprises pSer/pThr. Specifically encompassed are inhibitors that have the core sequence of XXXpSer-pProXXX. wherein X is any L-amino acid or D-amino acid. Candidate molecules of the present invention are evaluated for inhibitory activity in competitive inhibition assays. For example, the assay mixture would include the candidate molecule to be tested for inhibiting activity, the isomerase of interest and the intended substrate of the isomerase of interest. This admixture is maintained for a time sufficient and under conditions sufficient for the isomerase of interest to bind and catalyze the isomerization of its intended substrate. The catalytic activity of the isomerase of interest in the presence of the candidate inhibitor is then compared with the activity of the isomerase in the absence of the candidate inhibitor. If the activity of the isomerase in the presence of the inhibitor is less than the activity of the isomerase in the absence of the inhibitor, the candidate inhibitor is suitable for use as an inhibitor of the isomerase of interest. Encompassed by the present invention are inhibitors of interphase-specific pSer/pThr-Pro specific peptidyl prolyl isomerases. Specifically encompassed by the present invention are inhibitors of the essential mitotic peptidyl prolyl isomerase, Pinl, and other PIN 1 -like isomerases. Also encompassed by the present invention are methods of inhibiting mitotic peptidyl-prolyl isomerases comprising administering an effective amount of an inhibitor as described herein For example, a composition compnsing an effective amount of the inhibitor and a pharmaceutically acceptable carrier can be administered to an individual in need thereof Specifically encompassed are methods of inhibiting unwanted cell growth resulting fiom a hyperplastic or neoplastic disoider Also encompassed by the present invention are methods of inhibiting cell growth in target cells, comprising contacting the cells with an mhibitoi as described herein The present invention also relates to libraries of peptides that comprises a mixture of substantially equimolar amounts of peptides comprising the sequence NH,-MAXXXpSXXXAKK. wherein for each peptide X is anv ammo acid The present invention also relates to methods of identifying a phosphorseπne-or phosphothreonine-specific peptidyl prolyl isomeiase mhibitoi comprising the steps of a) providing a hbrarv of compounds that comprises a mixture of substantially equimolar amounts of peptides compiismg the sequence X,X,X J pS-PX X,X ( - wherein for each peptide X is anv amino acid. b) contacting the library of a) with the peptidyl prolyl isomerase of interest undei binding conditions foi time sufficient for the isomerase to bind to the peptides c) determining the ammo acid sequences of peptides bound to the isomerase of interest d) synthesizing the peptides of c), and e) assaying peptides of d) for ci /ti am isomerization by the peptidyl prolyl isomerase of interest to determine which peptides undergo isomeiization by the isomerase of interest, thus identifying peptides that bind to the isomerase and are suitable for use as inhibitors of the isomerase of interest The present invention further relates to methods of identifying a phosphorsenne oi phosphothreonine-specific peptidyl prolyl isomerase inhibitor comprising the steps of a) providing the peptidyl prolyl isomerase of interest, b) mixing the isomerase of interest with I) a candidate molecule and n) the substrate of the isomerase of interest to form an admixture of the isomerase of interest, candidate molecule and substrate; c) maintaining the admixture of b) under conditions sufficient for the isomerase of interest to catalyze the cis/trans isomerization of the substrate; and d) determining the K, of the candidate molecule, wherein a K, of 10 micromolar or less is indicative of an inhibitor of the peptidyl prolyl isomerase of interest. BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts a model for the Pinl -dependent regulation of mitosis- specific phosphoproteins that are phosphorylated by Cdc2 and other mitotic kinases (M kinase). Figure 2 is a graphic representation of the results of an experiment showing that Pinl inhibits mitotic division in Xenopus embryos. Figures 3A-C are graphic representations showing that Pinl, but not the mutant, directly inhibits the ability of Cdc25 to activate cyclin B/Cdc2. DETAILED DESCRIPTION OF THE INVENTION The present invention is related to the discovery that an essential mitotic pepidyl-prolyl isomerase (PPIase) recognizes phosphorylated serine/threonine (pSer/pThr) bonds present in mitotic phosphoproteins. Pinl is an essential peptidyl- prolyl cis-trans isomerase (PPIase). It is distinct from two other well-characterized PPIase families: the cyclophilins and the FK-506-binding proteins (FKBPs), which are targets for the immunosuppressive drugs cyclosporin A and FK506, respectively (reviewed in Schreiber, S.L.. Science 251 :283-287 (1991)). PPIases are ubiquitous enzymes that catalyze rotation about the peptide bond preceding a Pro residue, and may accelerate the folding and trafficking of some proteins (reviewed in Schmid, F.X., Curr. Biol. 5:993-994 (1995)). Interestingly, inhibition of PPIase activity is not required for the immunosuppressive property of cyclosporin A and FK506. Furthermore, neither the cyclophilins nor the FKBPs are essential for normal cell growth. Thus, evidence for the biological importance of PPIase enzymatic activity has been limited. In contrast, Pinl contains a PPIase domain that is essential for cell cycle progression and its subcellular localization is tightly regulated at the G2/M transition (Lu, K.P., S.D. Hanes and T. Hunter, Nature 380:544-547 (1996)). Pinl is localized in a defined nuclear substructure in interphase, but is concentrated to the condensed chromatin, with some staining in other structures, during mitosis. Furthermore, depletion of Pinl protein in HeLa cells or Pinl/Esslp in yeast results in mitotic arrest, whereas overexpression of Pinl induces a G2 arrest. These results suggest that Pinl is an essential mitotic regulator that both negatively regulates entry into mitosis and is required for progression through mitosis. As described herein, Pinl -binding proteins have been identified in human cells and Xenopus extracts. Pinl has been identified in all eukaryotic organisms where examined, including plants, yeast. Aspergillus, and mammals (sequences deposited in GenBank). Results indicate that although Pinl levels are constant throughout the cell cycle, the interaction of Pinl and its targets is cell cycle-regulated and depends upon mitotic phosphorylation of target proteins. Pinl directly interacts with a large subset of mitosis-specific phosphoproteins. which includes Cdc25, Weel, Mytl, Plkl. Cdc27 and E-MAPl 15 as well as some others recently identified by a screen for mitotic phosphoproteins (Stukenberg, P.T., K.D. Lustig, T.J. McGarry, R.W. King. J. Kuang and M.W. Kirschne, Curr Biol 7:338-348 (1997)). Many of these Pinl -interacting proteins are also recognized by the MPM-2 antibody. In functional assays, microinjection of Pinl inhibits mitotic division in Xenopus embryos and entry into mitosis in Xenopus extracts, as is the case in HeLa and yeast cells. Furthermore, Pinl binds the mitotically phosphorylated form of Cdc25 in vitro and in vivo, and it binds Cdc25 on the important phosphorylation sites and inhibits its activity. This characterization of the Pinl-Cdc25 interaction can at least partially explain the ability of Pinl to inhibit the G2/M transition. All these activities of Pinl are dependent upon the ability of Pinl to mitotic phosphoproteins since the activities are disrupted by point mutations which inhibit the ability of Pinl to recognize this unique class of phosphoproteins. Also as described herein. Pinl is a sequence-specific and phosphorylation- dependent PPIase that can specifically recognize the phosphorylated Ser/Thr-Pro bonds present in mitotic phosphoproteins. These results suggest that Pinl acts as a general modulator of mitotic phosphoprotein activity, presumably by catalyzing phosphorylation-dependent Pro isomerization. The crystal structure of human Pinl complexed with an Ala-Pro dipeptide suggests that the isomerization mechanism of Pinl includes general acid-base and covalent catalysis during peptide bond isomerization (Ranganathan et al., Cell 89:875-886 (1997)). More interestingly, Pinl displays a unique substrate specificity. It prefers an acidic residue N-terminal to the isomerized Pro bond due to interaction of the acidic side chain with a basic cluster in Pinl. This basic cluster consists of the highly conserved residues Lys63, Arg68, and Arg69 at the entrance to the active site. In the crystal structure, this conserved triad sequestered a sulfate ion in close proximity to the β methyl group of the Ala residue in the bound Ala-Pro dipeptide. One candidate for this negatively charged residue is Phospho-Ser/Thr. To investigate how Pinl interacts with essential mitotic proteins, a glutathione S-transferase (GST) Pinl fusion protein was used to screen oriented degenerate peptide libraries. The oriented peptide library approach (Z. Songyang et al. Cell 72:767 (1993) was used to screen for optimal peptides. All amino acids except Cys were incorporated at equimolar amounts in each degenerate position, yielding a total theoretical degeneracy for both libraries of 19 6 = 4.7x10 7 distinct peptide sequences. Pinl -GST beads and MPM2 antibody bound to protein-G beads were incubated with the peptide library mixtures and washed extensively. Bound peptides were eluted with 30% acetic acid and sequenced. The crystal structure of Pinl containing an Ala-Pro dipeptide substrate revealed a sulfate ion located 5 A from the C β carbon of Ala (A), suggesting that phosphorylated Ser (pSer) might be preferred at this site (R. Ranganathan. K. P. Lu, T. Hunter. J. P. Noel. Cell 89:875 (1997)). Next a ?S-containing degenerate peptide library of general sequence NH 2 - MAXXX SXXXAKK, where X includes every amino acid except Cys, was prepared. GST-Pinl protein preferentially bound a subset of peptides with Pro (P) immediately COOH-terminal to pSer. To investigate whether peptides containing S-P were preferred substrates for the isomerase activity of Pinl, oligopeptide substrates were synthesized and assayed for cis/trans isomerization by Pinl and by members of the cyclophilin (Cyplδ) and FKBP (FHBP12) families of PPIases. The chromogenic oligopeptides were synthesized (A. Bernhardt, M. Drewello, & M. Schutkowski, Int. J. Peptide Protein Res. 50: 143 (1997) and confirmed by NMR. Standard peptides were purchased from Bachem. PPIase activity was assayed and the bimolecular rate constants k ca /K m were calculated according to the equation k caI /K m = (k obs - k u )/[PPIase], where k u is the first-order rate constant for spontaneous cis/trans isomerization and k obs is the pseudo-first-order rate constant for cis/trans isomerization in the presence of PPIase, as described in G. Fischer, H. Bang, C. Mech, Biomed Biochim Ada 43.1101 (1984) and in J. L. Kofron et al. Biochemistry 30:6127 (1991). Affinity of Pinl for peptides was measured as described in Schutkowski. M . Wδllner, S.. & Fischer, G. Biochemistry 34.13016, (1995)). Neither Cypl δ nor FKBP12 effectively catalyzed isomerization of peptides with pS/pT-P moieties (Table 1) In contrast, either Y-P or/? Y-P bonds were good substrates for both enzymes Thus phosphorylation on S/T-P. but not Y-P, renders the prolyl-peptidyl bond resistant to the catalytic action of conventional PPIases, and suggests the need for a different enzyme to catalyze this reaction. Table Interaction between Pinl and Selected Mitotic Phosphoproteins Phosphoproteins Interphase Mitotic Cdc25 + +-H- Plkl 1 Plxl ++ Weel ++ Mos ++ Cdc27 < +-H- NIMA +τ+ Sox3 H h Xbr-lb + J ~r MP75 (E-MAP-1 15) +++ MP1 10(Cdc5) +-r+ MP68 +++ MP30 ++ MP105 + MP48 Cyclin B* The binding between Pin I and all selected mitotic phosphoproteins was assayed by incubating synthesized proteins with interphase and mitotic Xenopus extiacts, followed by piecipitation with GST- Pinl beads. The Pinl interactions with those proteins indicated with * were also confirmed by GST-Pinl pull-down assay from endogenous interphase and mitotic HeLa cell extracts +, a week but above background interaction, ++, readily detectable interaction, +++, strong interaction In contrast to cyclophilins and FKBPs, Pinl isomerase activity was highly specific for peptides with pS/pT-P bonds (Table 2). Pinl displayed little isomerase activity for substrates containing S/T-P bonds. However, phosphorylation of these peptides on S or T residues increased the k cat /K m values up to 300-fold. Table 2. Sequence-specific and phosphorylation-dependent PPIase activity of Pinl Substrate PPIase Activity kcat/KM (mM-'s "1 ) AAPL-pNA 55 AAAPR-pNA 121 AAPM-pNA 134 ADPY-pNA 220 AEPF-pNA 3410 AYPY-pNA 5 A^YPY-pNA ASPY-pNA 269 A^SPY-pNA 3370 ATPY-pNA 635 A/?TPY-pNA 2480 AAEPF-pNA 220 AASPF-pNA 9 AapSPF-pNA 3760 AATPF-pNA 4 Aa/?TPF-pNA 1370 AASPR-pNA 7 AA SPR-pNA 9300 WypSPRT-pNA 14100 AapTPR-pNA 3700 WFYSPR-pNA 170 WFY/?SPR-pNA 20160 Assays were done as described in Table 1 except that trypsin was used instead of chymotrypsin as an isomer-specific protease when peptides with Pro-Arg-pNA were used as substrates. Pinl had low isomerization activity for peptides containing an Ala-Pro peptide bond, whereas incorporation of Glu (E) or Asp (D) immediately preceding P to mimic the S, increased isomerization activity. Peptides containing a Y oxpY preceding Pro were poor substrates for Pinl. This substrate specificity distinguishes Pinl from the conventional PPIases in the cyclophilin and FKBP families. To further define the sequence specificity of Pinl, a degenerate peptide library containing a fixed pS-P sequence flanked by 3 degenerate positions on each side was used. Pinl selected Arg or aromatic residues at the -1 and +1 positions of fhe/?S-P motif (Table 3). Aromatic amino acids were also selected at the -3, Phe/Ile at the -2, and Leu/Ile at the +2 position of j cS-P. On this basis, several additional peptides were synthesized as Pinl substrates, as described above. Peptides with Arg introduced at the P+l position proved better substrates with specificity constants increased up to 1300 fold compared to their non-phosphorylated counterparts (Table 2). Placing aromatic residues NH 2 -terminal to the/?S-P position made these peptides even better substrates (Table 2). The best substrate identified thus far (Trp-Phe-Tyr- /?Ser-Pro-Arg-pNA) is the optimal sequence selected from the peptide library (Table 2 and 3). The apparent Km of Pinl towards this peptide was 10 μM. GST-Pin l and MPM-2 were incubated with the pS-P oriented degenerate peptide library NH ,- MAXXXpSXXXAKK, where X contains every am o acid except Cys After an extensive wash, peptides bound with GST-Pin l were eluted and sequenced Ammo acids with a significant selection at each degenerate position are shown As described herein, Pml binds a large subset of mitotic phosphoproteins also recognized by the monoclonal antibody MPM-2. Therefore, the sequence specificity for MPM-2 recognition was evaluated When immobilized MPM-2 antibody was probed with a peptide library containing only a pS as the orienting residue, there was a strong selection for peptides with P at the pS+l position. Using the S-P degenerate peptide library, MPM-2 strongly selected peptides with aiomatic and aliphatic ammo acids at the -3, -1 and +1 positions relative to /?S-P (Table 3) This MPM-2 binding motif is similar to the sequence motif selected by Pml (Table 3) and explains the observation that Pinl specifically interacts with MPM-2 antigens. To determine the structural basis for the Pml substrate specificity, molecular model-building was performed and tested by site -directed mutagenesis. A peptide (Trp-Phe-Tyr- Ser-Pro-Arg) was modeled into the Pml structure (R. Ranganathan, K. P. Lu, T. Hunter, J. P. Noel. Cell 89:875 (1997) assuming that the phosphate group ofpS occupies the position of of sulfate in the structure. The phosphate ofpS in the modeled peptide was superimposed on the co-crystallizing S0 4 ion in the original Pinl structure, and P residue displacements minimized with respect to the Ala-Pro ligand in the original Pinl structure using molecular modeling programs GRASP (A. Nicholls, K. Sharp, B. Honig. Proteins 11 :281 (1991). Molscript and Raster3d. In this model, R68 and R69 of Pinl coordinate the_pS phosphate, a hydrophobic groove accepts the preceding aromatic tripeptide. and the side chain of C l 13 and H59 coordinate the isomerizing/?S-P peptide bond with ω=90°, stabilizing the transition state between cis and trans configuration. To test these predictions from the model, site-directed Pinl mutants were generated and their PPIase activity assayed as described herein and in K. P. Lu, S. D. Hanes, T. Hunter, Nature 380:544 (1996); K. P. Lu and T. Hunter. Cell 81 :413 (1995). Substitution of both R68 and R69 by Ala reduced the k. at /K m to 1/ 500 that of wild type Pinl for the phosphorylated substrate. The catalytic activity of Pinl R6 - 69A was the same as wild-type Pinl for the unphosphorylated peptide substrate. Thus, this cluster of basic residues appears to participate in coordinating the phosphate of pS/pT. Parvulin (J. U. Rahfeld. et al.. FEBS Lett. 352: 180 (\ 99 ):idid 343 :65 (1994); K. E. Rudd. et al., TIBS 20: 12 ( 1995). the prototype of the Pinl family of PPIases has R68 and R69 replaced by Glu and failed to catalyze the isomerization of j oS-P peptidyl bonds, though it was very effective in catalyzing the P isomerization of the unphosphorylated peptide. Replacement of the catalytic H59 residue of Pinl with Ala dramatically decreased the PPIase activity for both phosphorylated and unphosphorylated peptides: however, the specificity for phosphorylated over unphosphorylated substrates was unchanged. For Pinl the K,. a /K m for the phosphorylated versus unphosphorylated substrate is 19,400/7, which is approximately equal to the similar ratio of 1120/<1 for Pinl H59A . Thus H59 appears to play an important role in catalyzing P isomerization and/or binding the substrate P residue. On the basis of amino acid preferences deduced in the 6 positions surrounding fhe/?S-P motif for optimal Pinl binding, a weighted screening of the SWISS-PROT sequence database. Protein sequence database screening was performed with the program INDOVINATOR, using an entropy-based weighing technique to score for relative information content at each amino acid position - 1 J- flanking the pS/pT-P motif with the quantitative peptide library results, which are shown qualitatively in Table 3. This scan revealed within the top 5% of highest scores several potentially important mitotic phosphoprotein targets for Pinl. Many of these proteins are involved in regulation of the cell cycle, cytoskeletal/spindle structure, DNA replication, transcription or RNA processing (Table 4). TABLE 4 Piedicted and/or Confirmed Pin l Substrates Based on the ammo acid preference values in each of the 6 positions surrounding the pS/pT-P motif for optimal Pinl binding (Table 3). a weighted screening of the SWISS-PROT sequence database was undertaken. This is a partial list of selected proteins with the top scores, human sequences aie used whenever possible Interactions between P l and some of the identified proteins have been confirmed in vitro Pinl not only binds these two proteins, but also suppresses their functions the interaction between Pinl and SC35 is inferred from their co localization Several of these predicted proteins, such as Rab4, Cdc25 and NIMA. undergo mitosis-specific phosphorylation (R. Heald and F. McKeon, Cell 61 :579 (1990); E. Bailly, et al., Nature 350:715 (1991); A. Blangy, et al., Cell 83:1159 (1995): J. Kuang, et al., Moi. Biol Cell 5:135 (1994); X. S. Ye, et al, EMBO J 14:986 (1995)). Cdc25 and NIMA also binds to Pinl in a phosphorylation-dependent manner, as described herein. Other proteins identified in this search, however, had not been previously suspected of interacting with Pinl ; therefore, a few were further investigated as example cases. Rab4 and ribosomal S6 kinases were found to interact with Pinl specifically in mitotic, but not in interphase extracts. Thus, Pinl binds a wide functional range of mitotic phosphoproteins. Differences in isomerase activity of Pinl and other PPIases result from different organization of the X-P binding pocket. In all PPIases, a hydrophobic pocket sequesters the aliphatic P side chain (S. L. Schreiber, Science 251 :2881 (1991); G. Fischer. Angew Chem. Intl. Ed. Engl. 33: 1415 (1994): F. X. Schmid. Curr. Biol 5:993 (1995)), hence the residues responsible for determining substrate preference must reside at the entrance to the P-binding pocket. In Pinl and its homologues (K. P. Lu. S. D. Hanes. T. Hunter. Nature 380:544 (1996); K. P. Lu and T. Hunter, Cell 81 :413 (1995). S. D. Hanes, et al., Yeast 5:55 (1989); R. Maleszka, et al., Proc. Natl. Acad. Sci. USA 93 :447 (1996)). a cluster of basic residues coordinate the pS phosphate, and determine the specificity of this isomerase. Absence of a basic pocket in the cyclophilins, FKBPs, and other members of the parvulin families of PPIases may explain their failure to isomerize the pS/pT-P bonds. The specificity of Pinl rationalizes Pinl -binding proteins and also predicts a number of novel potential Pinl substrates, some of which have been confirmed as in vitro Pinl targets. Furthermore. Pinl and MPM-2 bind similar sequences and proteins, and have similar phenotypes, indicating that the wide conservation of MPM-2 epitopes across various species (J. Kuang, et al., Moi Biol. Cell 5: 135 (1994); X. S. Ye, et al.. EMBO J. 14:986 (1995). F. M. Davis, et al.. Proc. Natl. Acad. Sci. USA 80:2926 (1983); J. M. Westendorf. P. N. Rao, L. Gerace, idid 91 :714-8 (1994); S. Taagepera, et al, Moi. Biol. Cell 5: 1243 (1994); A. Kumagai, W. G. Dunphy, Science 273: 1377-80 (1996) can be explained by recognition of this epitope by a highly conserved mitotic regulator, Pinl . Based on the determination of specific substrates for Pinl, as described herein, inhibitors of Pinl, Pinl-like isomerases and other phospho-Ser/Thr-specific PPIases can be produced. Thus, the present invention provides compounds that inhibit phosphoserine- and phosphothreonine-specific peptidyl-prolyl isomerases. Specifically encompassed by the present invention are peptidyl prolyl isomerases that recognize phosphorylated serine/threonine-proline bonds present in mitotic phosphoproteins. The inhibitor compounds of the present invention include any molecule that binds into the active site of the phosphoserine- or phosphothreonine-proline specific peptidyl prolyl isomerase and, upon binding to the isomerase, inhibits the isomerase activity. Encompassed by the present invention are inhibitor compounds that mimic the structure and conformation of the substrate moiety when bound to the catalytic site (also referred to herein as the active site) of the isomerase. Molecular inhibitors of the the present invention will typically have an inhibition constant (K,) of ten micromolar. or less. Specifically encompassed are organic molecules that mimic the structure and conformation of pSer/pThr and bind to the isomerase of interest, thereby inhibiting its activity. The inhibitor compounds of the present invention inculde inhibitors that comprise a core region (or moiety) that mimics the pSer/pThr moiety of the phosphoserine- or phosphothreonine-proline peptidyl prolyl isomerase substrate. Encompassed by the present invention are inhibitors that comprise the pSer/pThr mimic moiety with the mimic moiety being flanked on one side by hydrophobic groups and the other side of the mimic moiety being flanked by hydrophobic or positively charged groups, wherein the groups would contact the active site of the isomerase of interest. Specifically encompassed by the present invention are inhibitor compounds comprising proteins, polypeptides and peptides. The proteins, polypeptides and peptides of the present invention comprise naturally-occurring amino acids (e.g., L- amino acids), non-naturally amino acids (e.g., D-amino acids), and small molecules that biologically and biochemically mimic the inhibitor peptides. referred to herein as peptide analogs, derivatives or mimetics. (Saragovi, H.U.. et al . Bio/Technology, 10:773-778 (1992)). The protein, polypeptide or peptide inhibitors of the present invention can be in linear or cvclic conformation. Compounds that have PPIase inhibiting activity can be identified using oriented degenerate peptide libraries as described herein. For example, a library of xSer/Thr- X-containing peptides of a defined length can be screened for specific binding to the PPIase of interest. Peptides that specifically bind to the PPIase of interest can be further evaluated for PPIase inhibiting activity as described herein. The phosphoserine and phosphothreonine-specific peptidyl-prolyl isomerase inhibitors, or PPIase inhibitors, of the present invention can comprise a core sequence of xSer/Thr-Y wherein x can be any negatively charged tetra- or penta- valent moiety and Y can be Pro or any Pro analog. Preferred moieties for x can be phosphate, sulfonate. boronate, phosphonate or a sulfonly amide. The Pro analog can be any nitrogen-containing ring structure, including imidazole, pyrole. tropolone, benzene, camphor, and hetrerocyclic aromatic and non-aromatic ring structures. Typically, the xSer/Thr-Y core sequence is flanked by hydrophobic residues or Arg. where the hydrophobic residues (e.g., Phe. Tyr, Trp and He) typically precede the xSer/Thr residue and Arg follows the Y residue. Specifically encompassed by the present invention are inhibitors comprising the core sequence phosphoserine/phosphothreonine-proline. The inhibitors of the present invention can be anywhere from 2 to 200 amino acid residues in length. The inhibitors are typically 2-20 residues in length, and more typically 2-10 residues in length. Most typically the PPIase inhibitors are about eight residues in length, as represented by the consensus sequence, XXXpSer/pThrXXX, wherein X can be any amino acid residue. Encompassed by the present invention are compounds that are at least eight amino acid residues in length and comprise the core sequence X ] X 2 X 3 pS-PX 4 X 5 X 6 wherein each residue can be independently selected as follows X, is W, Y or F; X 2 is F or I; X 3 is Y, R, F or W; X 4 is R, F, Y or W; X 5 is L or I and Xe is any amino acid. Specifically encompassed by the present invention is the inhibitor of a phosphoserine- or phosphothreonine-proline-specific peptide prolyl isomerase comprising Trp-Phen-Tyr-pSer-Pro- Arg . The inhibitors of the present invention can be synthesized using standard laboratory methods that are well-known to those of skill in the art, including standard solid phase techniques. Inhibitors comprising naturally occurring amino acids can also be produced by recombinant DNA techniques known to those of skill, and subsequently phosphorylated. The inhibitors of the present invention can comprise either the 20 naturally occurring amino acids or other synthetic amino acids. Synthetic amino acids encompassed by the present invention include, for example, naphthylalanine, L- hydroxypropylglycine, L-3,4-dihydroxyphenylalanyl, α-amino acids such as L-α- hydroxylysyl and D-α-methylalanyl, L-α-methyl-alanyl, β amino-acids such as β- analine, and isoquinolyl. D-amino acids and other non-naturally occurring synthetic amino acids can also be incorporated into the inhibitors of the present invention. Such other non-naturally occurring synthetic amino acids include those where the naturally occurring side chains of the 20 genetically encoded amino acids (or any L or D amino acid) are replaced with other side chains, for instance with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclic. In particular, proline analogs in which the ring size of the proline residue is changed from 5 members to 4,6, or 7 members can be employed. As used herein, "lower alkyl" refers to straight and branched chain alkyl groups having from 1 to 6 carbon atoms, such as methyl, ethyl propyl, butyl and so on. "Lower alkoxy" encompasses straight and branched chain alkoxy groups having from 1 to 6 carbon atoms, such as methoxy, ethoxy and so on. Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups typically contain one or more nitrogen, oxygen, and/or sulphur heteroatoms, e.g., furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl (e.g. 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tl iomorpholinyl (e.g. thiomo holino), and triazolyl. The heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl. (U.S. Patent No. 5,654, 276 and U.S. Patent No. 5,643,873). Biologically active derivatives or analogs of the above-described inhibitors, referred to herein as peptide mimetics, can be designed and produced by techniques known to those of skill in the art. (U.S. Patent Nos. 4,612,132; 5,643.873 and 5,654.276) These mimetics are based on a specific peptide PPIase inhibitor sequence and maintain the relative positions in space of the corresponding peptide inhibitor. These peptide mimetics possess biologically activity (i.e., PPIase inhibiting activity) similar to the biological activity of the corresponding peptide compound, but possess a "biological advantage" over the corresponding peptide inhibitor with respect to one, or more, of the following properties: solubility, stability, and susceptibility to hydrolysis and proteolysis. Methods for preparing peptide mimetics include modifying the N-terminal amino group, the C-terminal carboxyl group, and/or changing one or more of the amino linkages in the peptide to a non-amino linkage. Two or more such modifications can be coupled in one peptide mimetic inliibitor. The following are examples of modifications of peptides to produce peptide mimetics as described in U.S Patent Nos: 5,643,873 and 5,654,276; the same techniques are used to prepare mimetics of the PPIase inhibitors described herein. MODIFICATION OF THE N-AMINO TERMINUS After solid phase synthesis of the peptide inhibitor, the blocking group on the N- terminus amino group can be selectively removed so as to provide for a peptide sequence blocked at all positions other than the N-terminal amino group and attached to a solid resin through the C-terminus. One can then modify the amino terminus of the peptides of the invention to produce peptide mimetics of the invention. Amino terminus modifications include alkylating, acetylating, adding a carbobenzoyl group, forming a succinimide group, etc. Specifically, the N-tenτiinal amino group can then be reacted as follows: (1 ) to form an amide group of the formula RC(0)NH- where R is as defined above by reaction with an acid halide (e.g.. RC(O)Cl) or acid anhydride. Typically, the reaction can be conducted by contacting about equimolar or excess amounts (e.g.. about 5 equivalents) of an acid halide to the peptide in an inert diluent (e.g., dichloromethane) preferably containing an excess (e.g., about 10 equivalents) of a tertiary amine. such as diisopropylethylamine. to a scavenge the acid generated during reaction. Reaction conditions are otherwise conventional (e.g.. room temperature for 30 minutes). Alkylation of the terminal amino to provide for a lower alkyl N-substitution followed by reaction with an acid halide as described above will provide for N-alkylamide group of the formula RC(0)NR-: (b) to form a succinimide group by reaction with succinic anhydride. As before, an approximately equimolar amount or an excess of succinic anhydride (e.g.. about 5 equivalents) can be employed and the amino group is converted to the succinimide by methods well known in the art including the use of an excess (e.g., ten equivalents) of a tertiary amine such as diisopropylethylamine in a suitable inert solvent (e.g., dichloromethane). See, for example, Wollenberg, et al., U.S. Pat. No. 4,612.132 which is incorporated herein by reference in its entirety. It is understood that the succinic group can be substituted with, for example, C : -C 6 alkyl or -SR substituents which are prepared in a conventional manner to provide for substituted succinimide at the N-terminus of the peptide. Such alkyl substituents are prepared by reaction of a lower olefin (C > -C 6 ) with maleic anhydride in the manner described by Wollenberg, et al.. supra, and -SR substituents are prepared by reaction of RSH with maleic anhydride where R is as defined above; (c) to form a benzyloxycarbonyl-NH-or a substituted benzyloxycarbonyl-NH-group by reaction with approximately an equivalent amount or an excess of CBZ-C1 (i.e., benzyloxycarbonyl chloride) or a substituted CBZ-C1 in a suitable inert diluent (e.g., dichloromethane) preferably containing a tertiary amine to scavenge the acid generated during the reaction; (d) to form a sulfonamide group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R-S(0) 2 C1 in a suitable inert diluent (dichloromethane) to convert the terminal amine into a sulfonamide where R is as defined above. Preferably, the inert diluent contains excess tertiary amine (e.g., ten equivalents) such as diisopropylethylamine. to scavenge the acid generated during reaction. Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes); (e) to form a carbamate group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R-0C(0)C1 or R-OC(0)OC 6 H 4 -p-NO, in a suitable inert diluent (e.g.. dichloromethane) to convert the terminal amine into a carbamate where R is as defined above. Preferably, the inert diluent contains an excess (e.g.. about 10 equivalents) of a tertiary amine. such as diisopropylethylamine. to scavenge any acid generated during reaction. Reaction conditions are otherwise conventional (e.g.. room temperature for 30 minutes); and (f) to form a urea group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R-N=C=0 in a suitable inert diluent (e.g., dichloromethane) to convert the terminal amine into a urea (i.e., RNHC(O)NH-) group where R is as defined above. Preferably, the inert diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine. Reaction conditions are otherwise conventional (e.g., room temperature for about 30 minutes). MODIFICATION OF THE C-TERMINUS In preparing peptide mimetics wherein the C-terminal carboxyl group is replaced by an ester (i.e., -C(0)OR where R is as defined above), the resins used to prepare the peptide acids are employed, and the side chain protected peptide is cleaved with base and the appropriate alcohol, e.g., methanol. Side chain protecting groups are then removed in the usual fashion by treatment with hydrogen fluoride to obtain the desired ester. In preparing peptide mimetics wherein the C-terminal carboxyl group is replaced by the amide -C(0)NR 3 R\ a benzhydrylamine resin is used as the solid support for peptide synthesis. Upon completion of the synthesis, hydrogen fluoride treatment to release the peptide from the support results directly in the free peptide amide (i.e.. the C-terminus is -C(0)NH 2 ). Alternatively, use of the chloromethylated resin during peptide synthesis coupled with reaction with ammonia to cleave the side chain protected peptide from the support yields the free peptide amide and reaction with an alkylamine or a dialkylamine yields a side chain protected alkylamide or dialkylamide (i.e.. the C-terminus is -C(0)NRR' where R and R 1 are as defined above). Side chain protection is then removed in the usual fashion by treatment with hydrogen fluoride to give the free amides, alkylamides. or dialkylamides. Alternatively, the C-terminal carboxyl group or a C-terminal ester can be induced to cyclize by internal displacement of the -OH or the ester (-OR) of the carboxyl group or ester respectively with the N-terminal amino group to form a cyclic peptide. For example, after synthesis and cleavage to give the peptide acid, the free acid is converted to an activated ester by an appropriate carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride (CH 2 C1 2 ), dimethyl formamide (DMF) mixtures. The cyclic peptide is then formed by internal displacement of the activated ester with the N- terminal amine. Internal cyclization as opposed to polymerization can be enhanced by use of very dilute solutions. Such methods are well known in the art. MODIFICATION TO INCORPORATE A NON-PEPTIDYL LINKAGE Peptide mimetics wherein one or more of the peptidyl linkages [-C(0)NH-] have been replaced by such linkages as a -CH,-carbamate linkage, a phosphonate linkage, a -CH 2 -sulfonamιde linkage, a urea linkage, a secondary amine (-C 2 NH-) linkage, and an alkylated peptidyl linkage [-C(0)NR 6 - where R 6 is lower alkyl] are prepared during conventional peptide synthesis by merely substituting a suitably protected amino acid analogue foi the amino acid reagent at the appropriate point during synthesis Suitable reagents include, for example, ammo acid analogs wherein the carboxyl group of the amino acid has been replaced with a moiety suitable foi forming one of the above linkages For example if one desires to replace a -C(0)NR- linkage in the peptide with a -CH,- carbamate linkage (-CH,OC(0)NR-), then the carboxyl (- COOH) gioup of a suitably protected amino acid is first reduced to the -CH 2 OH group which then converted by conventional methods to a -OC(0)Cl functionality oi a para-mtrocarbonate -0C(0)0-C 6 H 4 -p-N0, functionality Reaction of either of such functional groups with the fiee amine or an alkylated amine on the N-tei minus of the partially fabricated peptide found on the solid support leads to the foimation of a -CH 2 OC(0)NR-lmkage For a more detailed descπption of the foimation of such -CH,-carbamate linkages Similaily, replacement of an amino linkage in the peptide with a phosphonate linkage can be achieved using techniques known to those of skill in the art Replacement of an amino linkage in the peptide with a -CH-,-sulfonamιde linkage can be achieved by reducing the carboxyl (-COOH) gioup of a suitably protected amino acid to the -CH 2 OH group and the hydroxyl group is then converted to a suitable leaving group such as a tosyl group by conventional methods Reaction of the tosylated derivative with, for example, thioacetic acid followed by hydrolysis and oxidative chloπnation will provide for the -CH 2 -S(0) 2 C1 functional group which replaces the carboxyl group of the otherwise suitably protected ammo acid Use of this suitably protected ammo acid analogue in peptide synthesis provides foi mclusion of an -CH 2 S(0) 2 NR- linkage which replaces the amino linkage in the peptide thereby piovidmg a peptide mimetic Replacement of an ammo linkage in the peptide with a uiea linkage can be achieved using techniques known to those of skill in the art Secondary amme linkages wherein a -CH > NH- linkage replaces the amino linkage in the peptide can be prepared by employing, foi example, a suitably protected dipeptide analogue wherein the carbonyl bond of the amino linkage has been reduced to a CH 2 group by conventional methods For example, in the case of diglycme, reduction of the amide to the amme will yield after deportection I LNCH H,NHCH,COOH which is then used in N-protected foim in the next coupling reaction The preparation of such analogues by reduction of the carbonvl group of the ammo linkage in the dipeptide is well known in the art The suitably piotected ammo acid analog is employed in the conventional peptide synthesis in the same manner as would the corresponding amino acid For example, typically about 3 equivalents of the protected amino acid analogue are employed in this reaction An inert organic diluent such as methylene chloride or DMF is employed and, when an acid is generated as a leaction by-product the leaction solvent will typically contain an excess amount of a tertian, amme to scavenge the acid generated during the reaction One particulaily preferred tertiary amine is disopiopylethylamme which is typically employed in about 10 fold excess The reaction lesults in incorporation into the peptide mimetic of an ammo acid analogue having a non-peptidyl linkage Such substitution can be repeated as desired such that from zero to all of the ammo bonds in the peptide have been replaced by non-amino bonds The inhibitors of the present invention can also be cyclic piotem, peptides and cyclic peptide mimetics Such cyclic inhibitors can be produced using known laboratory techniques, e g , as described in U S S N 08/864,392, filed on May 28, 1997 and U S Patent No 5,654,276 Inhibitors of the present invention are evaluated for biological activity as described herein For example, the candidate compounds can be screened m an assay that determines the displacement of a labeled high affinity molecule (e.g., a competitive inhibition asay) in an assay utilizing immobilized molecules on a grid, as well as screening libraries of candidate molecules. These techniques are known to those of skill in the art. As defined herein, biological activity of the PPIase inhibitors include specific binding to the PPIase of interest (e.g., specific binding to Pinl) and/or specific inhibition of the peptidyl prolyl isomerase activity as measured as described in Schutkowski, M. et al. Biochemistry, 34:13016 (1995). Specific binding to the PPIase of interest can be determined as described herein. Further evaluation of candidate inhibitors (e.g., inliibitors that specifically bind to the PPIase of interest, for inhibiting activity can be determined by competitive inhibition assay. Alternatively, candidate moleucules of the present invention can be directly evaluated for their inhibitory activity withour prior determination of their specific binding to the isomerase of interest. Inhibitor compounds of the present invention typically have a K, in the nanomolar or micromolar range. Methods to determine K, are known to those of skill in the art. The inliibitors of the present invention can be used in vitro to study cell cycle regulation and mitotic events. For example, the inliibitors of the present invention can be used to evaluate mitotic events in mammalian cells by inhibiting a specific isomerase and evaluating the effects on the cell cycle. The inhibitors of the present invention can also be used to interfere with eucaryotic cell growth. The inliibitors can be used to inhibit cell growth, and to kill targeted cells. For example, the inhibitors of the present invention can be used to treat fungal and yeast, including Aspergillus, and parasitic infections (e.g., malaria) in mammals. As defined herein, mammals include domesticated animals and humans. Specifically, the inliibitors of the present invention can be used to treat hyperplastic and neoplastic disorders in mammals, including humans. For example, Pinl is an important molecule in controlling the sequential events of mitosis (Figure 1). Entry and exit from mitosis are accompanied by abrupt changes in kinase activities, which lead to changes in the phosphorylation state of numerous proteins that trigger specific events in mitosis. Pinl binding and consequent inhibition of target protein activity may provide a means for temporally synchronizing and/or amplifying the activity of mitotic proteins. Inhibition of Pinl induces mitotic arrest and apoptosis. Thus, the Pinl mediated mechanism of regulating mitotic events is a therapeutic target for cancer. Neoplastic and hyperplastic disorders include all forms of malignancies, psoriasis, retinosis, athrosclerosis resulting from plaque formation, leukemias and benign tumor growth. For example, such disorders include lymphomas, papilomas, pulmonary fibrosis, rheumatoid arthritis and multiple sclerosis. The inhibitors of the present invention can be formulated into compositions with an effective amount of the inhibitor as the active ingredient. Such compositions can also comprise a pharmaceutically acceptable carrier, and are referred to herein as pharmaceutical compositions. The inhibitor compositions of the present invention can be administered intraveneously, parenterally, orally, by inhalation or by suppositoiy. The inhibitor composition may be administered in a single dose or in more than one dose over a period of time to achieve a level of inhibitor which is sufficient to confer the desired effect. Suitable pharmaceutical carriers include, but are not limited to water, salt solutions, alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid esters, hydroxymethylcellulose, poly vinyl pyrolidone, etc. The pharmaceutical preparations can be sterilized and desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active agents, e.g., enzyme inhibitors, to reduce metabolic degradation. For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories. Ampoules are convenient unit dosages. It will be appreciated that the actual effective amounts of the inhibitor in a specific case will vary according to the specific compound being utilized, the particular composition formulated, the mode of administration and the age, weight and condition of the patient, for example. As used herein, an effective amount of inhibitor is an amount of inhibitor which is capable of inhibiting the isomerase activity of the isomerase of interest, thereby inhibiting target cell growth and resulting in target cell death. Dosages for a particular patient can be determined by one of ordinary skill in the art using conventional considerations, (e.g. by means of an appropriate, conventional pharmacological protocol). The present invention is illustrated by the following examples, which are not intended to be limited in any way. Example 1 : Expression, Purification and Kinetic Analysis of Recombinant Pinl Proteins Pinl was expressed and purified by Ni 2+ -NTA agarose column as an N- terminally His 6 -tagged fusion protein, followed by removing the tag using tlirombin. as described in Lu et al.. 1996; and Ranganathan et al., 1997). To generate an N- terminally GST-Pinl fusion protein, Pinl cDNA was subcloned into a pGEX vector and the resulting fusion protein was expressed and purified by glutathione agarose column, as described in Lu et al.. 1993; Lu, et al., 1996. GST-Pinl was stored in the agarose bead at 4 °C for 2 weeks or eluted from the beads and concentrated to 20 mg/ml with a Centricon-10 (Amicon), followed by storing at -80 °C. Both preparations were stored in a buffer containing 20 mM HEPES, pH 7.5, 50 mM NaCl and 1 mM DTT, as described in Ranganathan et al., 1997. All proteins were quantified by the method of Bradford (Biorad) using BSA as a standard. Site-directed mutations of Pinl were introduced using PCR-based techniques and verified by DNA sequencing. The corresponding mutant proteins were expressed and purified using the same procedures as those described for wild-type Pinl . PPIase activity was measured, as described previously (Lu et al., 1996), with the exception that the absorbance of 7-nitroaniline (at 395 nM) was followed every second for 2-10 min. and data were analyzed offline using a kinetic computer program written by G. Tucker-Kellogg in the C. Walsh lab at Harvard Medical School. Example 2: Analysis of Pinl and Its Binding Proteins During Cell Cycle HeLa cells were arrested at the Gl/S boundary using double thymidine and aphidicolin block, and released to enter the cell cycle, as described in Heintz, N., H.L. Sive and R.G. Roeder, Moi Cell Biol 3:539-550 (1983) and Lu, K.P. and T. Hunter, Cell 81 :413-424 (1995)). To accumulate cells at mitosis, nocodazole (50 ng/ml) was added to cells at 8 h after the release for the specified period of time. To obtain a large quantity of interphase and mitotic cells, HeLa cells were incubated with double thymidine and aphidicolin or nocodazole for 16 h, which resulted in over 90% of cells being arrested at the Gl/S boundary or mitosis, respectively. Cells were harvested and a aliquot of cells was subjected to flow cytometry analysis, as described in Lu and Hunter, 1995). The remaining cells were lysed in RIPA buffer (10 mM sodium phosphate pH 7.4. 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM sodium orthovanadate, 10 μg/ml aprotinin, 50 μg/ml pheiiylmethylsulfonyl fluoride and 1 mM DTT) and same amount of total proteins were subjected to immunoblotting analysis using various antibodies or Farwestem analysis using GST- Pinl as a probe. For Farwestem analysis, after blocking with 5% BSA, membranes were incubated with 2 μg/ml GST-Pinl in TBST for 2 hr, followed by incubation with anti-GST monoclonal antibodies (UBI) and the ECL detection procedures. Example 3: Microinjection of Xenopus Embryos Unfertilized eggs were incubated with sperm, dejellied, and 4 μM of the indicated protein (about 10 fold above the estimated endogenous levels) was injected in one cell of two cell stage embryos (30 embryos each protein). The injected embryos were allowed to develop at 18°C to stage 8 and pictures were taken of typical embryos. The titration of Pinl and the mutants was essentially as described above except that the indicated protein was injected into one cell of 4 cell stage (18 embryos each Pinl concentration) to the indicated final concentration and allowed to develop for 3h. The cell cycle blocks by GST-Pinl were not homogeneous as cells that were injected with greater concentrations of GST-Pinl were cleaved fewer times indicating a tighter cell cycle block. To be consistent, cell cycle blocked embryo's were scored as those that contained at least one cell on the injected side that was greater than 5 times larger than uninjected cells (Figure 2). Example 4: Preparation of Xenopus CSF Extracts Xenopus CSF extracts were prepared from unfertilized eggs, as previously described (Murray, 1991) and used immediately. To examine the effect of Pinl on mitotic entry, a fresh CSF extract containing demembranated sperm (150/μl) and rhodamine tubulin (2 μg/ml) was activated by addition of 0.4 mM calcium chloride for 15 min, before the indicated concentrations of various Pinl proteins were added and mitotic entry was followed for 2 h by nuclear morphology, nuclear envelope breakdown, spindle formation and Cdc2 activity, as described previously (Murray. A.W., Methods Cell Biol 36:581-605 (1991)). The cell cycle state of nuclei within the extracts were over 90% synchronous and typical nuclei were photographed. Example 5 : Synthesis of Mitotic Phosphoproteins The mitotic phosphoproteins were translated in vitro using the TNT coupled transcription translation kit (Promega) in a total volume of 10 μl in the presence of 8 μCi [ 35 S]methionine (1000 Ci/mmol) for 2 h at 30 °C. They were then incubated in Xenopus interphase and mitotic extracts as described (Stukenberg et al., 1997). These incubated clones were precipitated by Pinl beads as described below. The Xenopus Mos and Weel clones were a kind gift of M. Murakami, G, F. Woude and J. Cooper; the Xenopus Cdc25 clone was a generous gift of W. Dunphy, T3 and T3S2 Cdc25 mutants were kindly provided by J. Mailer (Izumi and Mailer, 1993). Example 6: Production of Pinl and Cdc25 Antibodies. Since antibodies that we previously raised against C-terminal peptide of Pinl (Lu, et al., 1996) did not have a high sensitivity for detecting Pinl, especially for Xenopus Pinl, rabbits were immunized with His-Pinl as an antigen. After 2 months, antisera specifically recognize a single 18 kDa Pinl protein in human cells and Xenopus extracts. To raise antibodies against Xenopus Cdc25, recombinant GST-Cdc25 (the clone was a kind gift of A. Nebrada and T. Hunt) was affinity purified as described by the manufacture (Pharmacia). The protein was further purified by SDS-PAGE and a gel slice containing Cdc25 was used to immunize rabbits. Example 7: GST Pull-down, Imunoprecipitation and Immunoblotting Analysis To detect Pinl -binding proteins, either HeLa cells were lysed in ox Xenopus extracts were diluted in a buffer (buffer A) containing 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 100 mM NaF, 1 mM sodium orthovanadate, 10% glycerol, 1% Triton XI 00, 10 μg/ml aprotinin, 50 μg/ml phenylmethylsulfonyl fluoride and 1 mM DTT. The ly sates were preclarified with boiled S. aureus bacteria (CalBiochem) and then incubated with 10 μl of agarose beads containing various GST-Pinl proteins or control GST for 2 h at 4°C. The precipitated proteins were washed 5 times in the same buffer and subjected to immunoblotting analysis. Immunoprecipitation and immunoblotting analysis using MPM-2 antibody (Davis et al. 1983), which was kindly provided by J. Kuang, Pinl antibodies (Lu et al., 1996, kindly provided by M. White or newly generated), anti-phospho.Tyr antibody (UBI), anti-Cdc25C (Ogg, et al., 1994) (from H. Piwnica- Worms and Santa Cruz Biotechnology), anti-Cdc27. anti-Plkl (Zymed), anti-Cdc2 (Solomon, M.J., M. Glotzer, T.H. Lee, M. Philippe and M.W. Kirschner. Cell 63:1013-1024 (1991), anti-human Mytl, anti-human cyclin Bl and anti-Xenopus cyclin B were performed, as described previously in Lu and Hunter, 1995; Lu et al., 1996). Example 8: Coimmunoprecipitation of Pinl and Cdc25 To detect Pinl and Cdc25 interaction during the Xenopus cell cycle, about 500 eggs were fertilized in a minimal volume of MMR (100 m NaCl, 2 mM KCl, 1 mM MgC12, 2m CaC12, 0.1 mM EDTA, 5 mM HEPES, pH 7.8), diluted in 0.1X MMR for 10 minutes, dejellied as described in Murray, (1991) and incubated in CSF-XB (100 mM KCl, 0.1 mM CaC12, 2 mM MgC12, 10 mM K-HEPES, pH 7.7, 50 mM Sucrose 5 mM EGTA, pH 7.7). At the indicated time after fertilization 15 eggs were crushed into 150 μl of ice cold CSF-XB with 1 μM okadeic Acid, microcenfuged for 20 seconds, the layer between the yolk and the pellet was removed to a fresh chilled tube. This solution was mixed well and 5 μl was frozen in liquid nitrogen for future HI kinase assays, and 30 μl was diluted in 10 μl of either α-cdc25 or control rabbit sera beads in 100 μl of buffer A (containing 5 mM EDTA and 1 μM microcystein but not vanadate). The immunoprecipitation reactions were rotated for approximately 40 minutes at 4°C, washed 4 times in and subjected to immunoblotting with anti-Pinl antibodies. The associated Pinl was quantified as described (Stukenberg et al., 1997). Example 9: Cdc2 and Cdc25 Assays Cdc2 was assayed using histone HI as a substrate, as previously described in Murray, 1991; Lu and Hunter, (1995). Cdc25 activity was assayed by using the activation of its endogenous substrate, Cdc2/cyclin B complex phosphorylated on Thrlβl, Tyrl5, Tlιrl4 as an indicator using a variation of an established protocol (Kumagai, A. and W.G. Dunphy. 1996. Science 273:1377-1380 (1996). When cyclin B is added to a Xenopus interphase extract at levels insufficient to activate mitosis (referred to as a "subthreshold cyclin concentration"), the added cyclin B binds Cdc2 and the Cdc2 in the complex is phosphorylated by CAK, Weel and Myt 1 to accumulate in an inactive form (Solomon et al. 1991). A subthreshold concentration of GST cyclin B (10 μg) was added to 1 ml of Xenopus interphase extract for thirty min at room temperature (Solomon, M.J., M. Glotzer, T.H. Lee, M. Philippe and M.W. Kirschner, Cell 63:1013-1024 (1990). This was diluted 8 fold in XB+ 3 mM DTT, rotated for 1 h with 3 ml of GST agarose beads, washed 3 times in XBIP (XB+ 500 mM NaCl and 1% NP40 + 2 mM DTT), washed 2 times (once overnight) in EB (80 mM β glycerol phosphate, 15 mM EGTA, 15 mM MgC12)+ 2 mM DTT, 500 mM NaCl and 1% NP40, and finally twice with EB +10 mM DTT. These Cdc25 assay beads were stored at 4°C for up to 1 month. Mitotic GST-Cdc25 was purified by incubating 22 μg of GST-Cdc25, in a Xenopus mitotic extract for 30 min at 23 °C, this was diluted 8 fold in XB and rotated with 50 μl of glutathione-Agarose beads (Sigma) for 1 hr at 4°C. The beads were washed 5 times in XB-IP, twice in XB+2 mM DTT and eluted in 25 μl XB + 2 mM reduced GSH. The final concentration of Mitotic GST-Cdc25 was 0.36 mg/ml. A 27 fold dilution of this mitotic GST-Cdc25 could fully activate Cdc2 in the assay below, while GST-cdc25 isolated from Interphase extracts in parallel lost activity after a 3 fold dilution. Thus the mitotic extract stimulated the Cdc25 at least 9 times over interphase extracts. To assay Cdc25 activity 1 μM mitotic GST-cdc25, and the indicated concentration of either Pinl , Pinl R68 - 69A 0 r BSA were incubated in a 20 μl reaction in XB + 1 mM ATP for 10 minutes at room temperature. These reactions were sequentially diluted (1/1 , 1/3, 1/9, 1/27) into XB + lmM ATP and 10 μl of each was mixed with 10 μl of cdc25 assay beads for 10 minutes at room temperature with constant shaking. The Cdc25 assay beads were washed 3 times in XB-IP, 2 times in EB+lmM DTT and assayed for HI kinase activity. Phosphoimager analysis of the HI kinase assays were quantified by the Molecular Dynamics ImageQuant 3.3 software. As described herein an assay with 1 μM mitotic GST-cdc25, 0.67 μM of either Pinl, pi n l R68 - 69A or 16 μM BSA then diluted 27 fold before being mixed with the Cdc25 assay beads and the amount of HI kinase activity is relative to the amount of activity of the beads without cdc25 being zero and the BSA reaction being 100%. The most reproducible way to quantify the Cdc25 activity in this assay was by determining the endpoint dilution of Cdc25 which could activate Cdc2. Therefore the Cdc25 activity is quantified by the endpoint dilution of the mitotic GST-Cdc25 at which Cdc2 on the beads could still be significantly activated. Example 10: Pinl Levels are Constant through the Cell Cycle Whereas overexpression of Pinl results in G2 arrest, depletion of Pinl induces mitotic arrest without affecting DNA synthesis. To determine the basis for this cell cycle-specificity, it was determined whether Pinl protein level fluctuated during the cell cycle. To address this question. HeLa cells were synchronized at the Gl/S boundary. At different times following the release from the block, cells were harvested and analyzed by flow cytometry or lysed and analyzed for protein expression by immunoblotting. Analysis of DNA content and cyclin B l levels indicated that the HeLa cells synchronously progressed through different phases of the cell cycle. However, total Pinl levels did not change significantly during the cell cycle. Example 12: Pinl Directly Binds a Subset of Conserved Mitotic Phosphoproteins Since the levels of Pinl do not fluctuate during the cell cycle, its mitosis-specific function is likely conferred by some other mechanisms. There are many such possibilities. Pinl could be subjected to post-translational modifications, such as phosphorylation. or allosteric interactions with a transiently appearing subunit, like a cyclin which regulates its activity. Alternatively, the interaction of Pinl and its targets may be cell cycle-regulated. Initial experiments suggested no evidence for Pinl phosphorylation or for interaction of Pinl with a regulatory subunit. A cell cycle-dependent interaction of Pinl with its binding proteins was tested for. A glutathione-S-transferase (GST) fusion protein containing full length Pinl was bacterially expressed, purified, and then used to probe for interacting proteins in S-phase, mitosis or Gl -phase by Farwestem analysis. The ability of Pinl to interact with cellular proteins remained relatively low during S, increased when cells progressed though G2/M ( 10 h point), and was almost completely lost when cells moved to the next Gl (14 h point). However, if cells were not allowed to progress into the next cell cycle, but rather were blocked at mitosis by adding nocodazole (14+Noc), Pinl -binding activity increased even further. Since the binding activity was detected using denatured proteins, the protein-protein interaction between Pinl and these proteins must be direct. To examine whether this Pinl interaction with its target proteins occur under nondenaturing conditions and to estimate the number of Pinl -interacting proteins, glutathione beads containing GST and GST-Pinl were incubated with interphase and mitotic extracts, and beads were extensively washed and proteins bound to beads were separated on SDS-containing gels and stained with Coomassie blue. Whereas GST did not precipitated any detectable proteins from either interphase or mitotic extracts, GST-Pinl specifically precipitated about 30 clearly Coomassie-stainable bands from mitotic extracts, but only 4-7 minor bands from interphase extracts. Together, these two results indicate that Pinl mainly interacts with a subset of proteins in a mitosis-specific manner. The crystal structure of Pinl suggests that Pinl could strongly interact with a Phospho.Ser/Thr-Pro motif (Ranganathan. et al, Cell 89:875-886 (1997)). A large number of proteins have been shown to be phosphorylated at such a motif specifically during mitosis and many of these phosphoproteins are recognized by the MPM-2 antibody. Therefore, interactions between Pinl and MPM-2 antigens were examined. After incubation with soluble proteins prepared from interphase and mitotic HeLa cells. GST-Pinl and control GST glutathione beads were washed extensively and the interacting proteins are detected by immunoblotting with the MPM-2 antibody. Many of the GST-Pinl -binding proteins reacted with MPM-2 only in the mitotic extracts, including a strong band of 55 kDa (p55). p55 has been previously shown to be the most prominent MPM-2 antigen in HeLa cells (Zhao et al., FEBSLett 249:389-395 (1989), although its identity remains to be determined. In contrast, control GST glutathione beads precipitated just two proteins (p58/60) from either lysate. In addition, when MPM-2 immunoprecipitates were subjected to Farwestem analysis using GST-Pinl as a probe, Pinl directly bound MPM-2 antigens on membranes. To determine whether GST-Pinl can deplete MPM-2 antigens and to estimate what concentrations of Pinl are required to completely deplete MPM-2 antigens, mitotic extracts were incubated with different amounts of GST-Pinl, followed by analyzing MPM-2 antigens remaining in the depleted supernatants. The total cellular Pinl concentration in HeLa cells was estimated to be about 0.5 μM, based on immunoblotting analysis using anti-Pinl antibodies with recombinant Pinl protein as a standard. At a concentration (8 μM) that was about 15 fold higher than the endogenous level, Pinl depleted the majority of MPM-2 antigens, indicating that Pinl strongly interacts with most MPM-2 antigens. The above results demonstrate that Pinl interacts with MPM-2 antigens in vitro. To determine if endogenous Pinl interacts with MPM-2 antigens in the cell, Pinl was immuno precipitated from either interphase or mitotic HeLa extracts using anti-Pinl antibodies in the presence of various phosphatase inhibitors. The resulting Pinl immunoprecipitates were probed with MPM-2. As described above, several MPM-2 antigens were co-immunoprecipitated with anti-Pinl antibodies. These results indicate that stable complexes between Pinl and MPM-2 antigens exist in the cell and that Pinl does not form complexes with all Pinl -binding proteins at the same time in vivo. Since Pinl and MPM-2 antigens are highly conserved, it is possible that Pinl- binding proteins are also conserved. To examine this possibility, the interaction between human Pinl and mitotic phosphoproteins in Xenopus extracts was observed. When GST-Pinl was incubated with interphase or mitotic egg extracts, Pinl specifically precipitated a subset of MPM-2 antigens from mitotic extracts, with molecular weights similar, although not identical, to those present in human cells. Again, this interaction between Pinl and Xenopus MPM-2 antigens was specific as it was not detected if the precipitation was performed with control GST glutathione beads. These results demonstrate that Pinl also interacts with a subset of conserved mitosis-specific phosphoproteins in Xenopus. Example 13 : Mutations in the Binding Pocket Abolish the Ability of Pin 1 to Interact with Most Mitotic Phosphoproteins The above results demonstrate that Pinl directly binds numerous conserved mitotic phosphoproteins in a mitosis-dependent manner. To insure that this interaction is highly specific for Pinl, site-specific mutations were introduced into Pin 1. A high resolution X-ray structural and preliminary functional analysis of Pinl suggest that a basic cluster consisting of Lys63, Arg68, and Arg69 is likely to be coordinate the putative phosphate group in the substrate. Ala substitutions at these residues (Pinl R68 69A ) should cause a reduction in the ability to bind phosphorylated residues N-terminal to the target Pro residue in the substrate. In addition, His59 has been shown to have an intimate contact with the cyclic side chain of the catalyzing Pro residue. An Ala substitution at His-59 of Pinl (Pinl H:>9A ) should therefore disrupt the interaction between Pinl and the substrate Pro residue. The mutant proteins were expressed and purified as GST fusion proteins, and both their PPIase activity and their ability to bind mitotic phosphoproteins were determined. PPIase activity was assayed with two peptide substrates: AEPF. which has an acidic residue at the position N-terminal to the catalytic Pro residue, and AAPF, which does not. Pinl had a strong preference for the AEPF substrate. The PPIase activity of Pinl R68 ά9A was reduced more than 90% against AEPF, whereas the reduction was very small against AAPF. Moreover, pi n l R68 ' 69A had little preference for either substrate. These results confirm that residues Arg68 and Arg69 are critical for promoting strong selection for a negatively charged residue at the position N- terminal to the substrate Pro residue. The PPIase activity of Pinl H " 9A was barely detectable against either peptide substrate, confirming the importance of His59 in Pinl substrate binding and/or catalysis. To determine if the Pinl mutants interact with mitotic phosphoproteins, GST- Pinl , -Pinl R68 ' 69A and -Pin 1 H:,9A fusion proteins were incubated with interphase or mitotic HeLa cell extracts and associated proteins subjected to MPM-2 immunoblotting analysis. Pinl specifically interacted with MPM-2 antigens in two independently prepared mitotic extracts, but the binding activity of both pi n ι R6869A and Pinl H59A was significantly reduced compared to the wild-type protein. A few proteins including the most strongly reacting p55 band could still be recognized. The two Pinl mutants also failed to bind most mitotic phosphoproteins from Xenopus extracts. Thus, mutating the residues that are implicated in binding either the substrate's putative phosphate group or the substrate's Pro residue abolish the ability of Pinl to bind MPM-2 antigens. This suggests that Pinl must recognize both the Phospho Ser/Thr and the Pro residues to bind MPM-2 antigens. These results also demonstrate that mitotic phosphoproteins specifically interact with active site residues of Pinl Example 14: Identification of Several Mitotic Regulators as Pinl Targets Several known mitotic regulators such as cyclin B, Cdc25, Mytl , Plkl and Cdc27 are phosphorylated at mitosis. To identify at least a few of the many Pinl binding proteins. Pinl -binding proteins were precipitated from HeLa cells, or Xenopus extracts and probed with antibodies specific for known mitotic phosphoproteins. As shown previously, levels of Plkl and cyclin B l increased at mitosis, whereas similar amounts of Cdc25C were present in interphase and mitotic HeLa cells. Moreover, a significant fraction of Cdc25C. Plkl. Mytl, Cdc27 and PTP- 1 B became hyperphosphorylated during mitosis and exhibited a shift in electrophoretic mobility by SDS-PAGE. Although cyclin B l and PTP-1B were not precipitated by Pinl in either inteφhase or mitotic extracts, Pinl bound selectively only to the mitotically hypeφhosphorylated form of Cdc25C, Plkl, Mytl and Cdc27. Furthermore, neither mutant Piιτl Rfc8 - 69A n0 r Pinl l b9A interacted with Cdc25 or Cdc27, indicating that the residues that are implicated in binding either the substrate's putative phosphate group or the substrate's Pro residue are necessary for Pinl to bind Cdc25 and Cdc27. Similarly, only the mitotic, but not the inteφhase form of Xenopus Cdc27 was precipitated by Pinl. Moreover, pretreatment of the mitotic extract with calf intestine phosphatase (CIP) completely dephosphorylated Cdc27 and abolished the interaction between Pinl and Cdc27, demonstrating a phosphoryla ion-dependent interaction. These results indicate that the interaction between Pinl and Cdc25 or Cdc27 is likely to be mediated by a Phospho. Ser/Thr- Pro motif. To gain a sense of the generality of the interaction between Pinl and mitotic phosphoproteins and to confirm the Pinl interaction with target proteins is indeed mediated by phosphorylation, the ability of Pinl to bind other known mitotic 58- phosphoproteins and a set of mitotic phosphoproteins identified by a systematic phosphoprotein screen (Stukenberg et al., Curr Biol 7:338-348 (1997). Proteins synthesized in vitro were phosphorylated in a cell cycle specific manner by incubating them in either Xenopus inteφhace or mitotic extracts. These labeled protein were subsequently incubated with GST-Pinl beads that were extensively washed and the bound proteins analyzed by SDS-PAGE. To validate this method, Cdc25 was first tested. Again, the mitotically phosphorylated form of in vitro translated Cdc25 could be precipitated by GST-Pinl beads. However, Cdc25 was not recognized by Pinl if it was incubated in inteφhase extracts. Moreover. Pinl did not interact with Cdc25 if the mitotically phosphorylated Cdc25 was treated with phosphatase prior to the GST-Pinl incubation. These results demonstrate that this method can be used to detect mitosis-specific and phosphorylation-dependent interactions between Pinl and phosphoproteins. Out of the 13 mitotic phosphoproteins examined, Pinl bound 10 in a mitosis and phosphorylation- dependent manner (summarized in Table 1), including Weel, MP75 and MPl 10, MP75 and MPl 10 are Xenopus proteins related to microtubule-associated protein E- MAP-115 and the fission yeast G2 transcription factor Cdc5, respectively. These results indicate that Pinl may target many mitosis-specific phosphoproteins. Example 15: Pinl Blocks Cell Cycle Progression inXenopus Embryos and Entry into Mitosis in Xenopus Extracts Since Pinl is conserved from yeast to humans, it is likely that Pinl exists in Xenopus. To confirm this, Xenopus egg extracts were immunoblotted with two separate anti-human Pinl antisera. Both antibodies, but not their respective preimmune sera, specifically recognized a band which comigrated with human Pinl at 18 kDa, indicating that Pinl is present in Xenopus. Overexpression of Pinl has been shown to inhibit cell cycle progression in both yeast and HeLa cells. To examine whether increasing the concentration of Pinl has similar biological effects in Xenopus, Pinl or Pinl mutants were injected into one cell of 2 cell stage embryos and allowed the embryos to develop for 3 h (about 5 divisions). Wild-type Pinl injected cells failed to cleave or cleaved slowly when compared to the cells in the uninjected side. A similar concentration (4 μM final) of either Pinl mutant had little effect on the cell cycle. In a separate experiment Pinl blocked cleavage of the injected cells in a concentration-dependent manner, and at a concentration approximately 20 fold above the estimated endogenous levels (10 μM), completely inhibited the cell cycle (Figure 1). In contrast, higher concentrations of the mutant proteins were needed to block the cell cycle (Figure 1). Injection of control BSA had no obvious effect on cell cycle progression. These results suggest that Pinl must bind mitotic phosphoproteins in order to block cell cycle progression. To determine the nature of the cleavage block in Xenopus. GST- Pinl was added to Xenopus egg extracts that had been arrested in second meiotic metaphase due to the activity of cytostatic factor. These extracts are arrested in mitosis (meiosis II) and reenter the cell cycle in response to the addition of Ca 2+ . Extracts containing demembranated sperm to monitor nuclear morphology and rhodamine-tubulin to monitor microtubule spindle assembly, were activated with Ca 2 \ Pinl was added after the extracts had entered inteφhase (15 min after the addition of Ca 2+ ). and the subsequent entry of the extracts into mitosis was followed by nuclear morphology and Cdc2 kinase activity using histone HI as a substrate. Addition of either 10 or 40 μM Pinl. approximately 20 or 80 fold higher than endogenous levels, completely blocked entry into mitosis as detected by the persistence of inteφhase nuclei and low Cdc2 kinase activity. In contrast, the same extracts containing 40 μM of either BSA or the mutant Pinl proteins entered mitosis by 70 to 80 min as detected by nuclear envelope breakdown, spindle formation and high histone HI kinase activity. Thus, as was shown previously in HeLa cells, increasing the Pinl concentration causes a cell cycle block in G2. More importantly, Pinl must bind mitotic phosphoproteins to elicit this phenotype. Example 16: Pinl Binds and Inhibits Mitotically Phosphorylated Cdc25 The above results indicate that overexpression of Pinl inhibits mitotic entry in Xenopus, as is the case in HeLa cells and yeast. Entry into mitosis is regulated by dephosphorylation of Cdc2 by the phosphatase Cdc25, and Cdc25 is activated by mitosis-specific phosphorylation at the MPM-2 epitope at the G2/M transition. Earlier results indicated that it is the mitotically phosphorylated form of Cdc25 that interacts with Pinl in vitro. Therefore, it is conceivable that the inhibitory effects of Pinl on entry into mitosis could at least partially explained through inhibition of Cdc25 activity. To test this possibility, it was determined whether Pinl interacts with Cdc25 in vivo and if so. whether this interaction is cell cycle regulated. Xenopus eggs were collected at various times after fertilization and subjected to immunoprecipitation using anti-Xenopus Cdc25 antibodies as well as histone HI kinase assay to monitor cell cycle progression. When the resulting Cdc25 immunoprecipitates were immunoblotted with anti-Pinl antibodies, we found that endogenous Pinl was precipitated by anti-Cdc25 antibodies. Furthermore, this interaction between Pinl and Cdc25 was cell cycle-regulated, significantly increased just prior to mitosis. Similar results were also obtained using synchronized HeLa cells using anti-human Cdc25C. Unfortunately, we were not able to detect Cdc25 in anti-Pinl immunoprecipitates. probably because the amount of Cdc25 precipitated is below the detection of the Cdc25 antibodies. It is worth of pointing out that the percentage of coimmunoprecipitatable Pinl and phosphorylated Cdc25 is not high. This might be expected because the complex might not be stable to the stringent immunoprecipitation conditions, the amount of Cdc25 phosphorylated on Pinl- binding sites might be low at this point, and/or the complex might have a high off rate, since the phosphorylated Cdc25 is a substrate of Pinl. Nevertheless, these results suggest that Pinl is associated with Cdc25 at a time when Cdc25 is partially phosphorylated and yet its activity is low. Since the interaction between Pinl and Cdc25 is mediated by phosphorylation of Cdc25, it was determined whether Pinl interacts with Cdc25 on important phosphorylation sites. At entry into mitosis, Cdc25 is phosphorylated at multiple Thr/Ser-Pro (Peng, C. Y. Graves, P. R., Thoma, R. S. Wu, Z. Shaw, A. S. and Piwnica-Worms, H. Science, 277:1501-1505 (1997)). Izumi and Mailer (Izumi, T. and J.L. Mailer, Moi Biol Cell 4:1337-1350 (1993)) have shown that the triple mutation of conserved Thr48, Thr67 and Thrl38 (T3 Cdc25), and the quintuple mutation of the three Thr residues plus Ser205 and Ser285 (T3S2 Cdc25) prevent most of the shift in electrophoretic mobility of Cdc25 after incubation with mitotic extracts. When they measured the ability of the Cdc25 mutants to activate Cdc2 in the Cdc25-depleted oocyte extracts and to initiate mitotic entry in oocyte extracts, the activities of T3 and T3S2 mutants were reduced about 70% and 90%. respectively (Izumi and Mailer, 1993). These results indicate that these Thr/Ser residues are essential for the Cdc25 function. We examined the ability of Pinl to bind the T3 and T3S2 Cdc25 mutants. As shown previously (Izumi and Mailer, 1993), the T3 and T3S2 Cdc25 mutants failed to undergo the mobility shift after incubation with mitotic extracts. Although Pinl strongly bound mitotically phosphorylated form of Cdc25, Pinl almost (T3) or completely (T3S2) failed to bind the Cdc25 mutants which were incubated with either inteφhase or mitotic extracts. Although further experiments are required to pinpoint which phosphorylation site(s) play(s) the major role in mediating the Pinl and Cdc25 interaction, these results show that Pinl interacts with the phosphorylation sites on Cdc25 that are essential for its mitotic activation. The above results indicate that Pinl interacts with Cdc25 both in vitro and in vivo. Therefore, we tested whether Pinl could affect the physiological activity of Cdc25, which is to dephosphorylate and activate the cyclin B/Cdc2 complex. To generate the mitotically phosphorylated form of Cdc25, GST-Cdc25 was incubated in Xenopus mitotic extracts, affinity purified on glutathione agarose beads and eluted. This mitotic Cdc25 was at least 9 fold more active than GST-Cdc25 purified in parallel from inteφhase extracts (data not shown). This mitotic GST-Cdc25 activated cyclin B/Cdc2 complex that was kept inactive due to inhibitory phosphorylations on Tyrl5 and Thrl4. If Pinl (0.67 μM) was included in the assay at amounts approximately stoichiometric to mitotic Cdc25 (1 μM), mitotic Cdc25 failed to activate the Cdc2 complex. In contrast, neither the mutant Pinl R6869 at the same concentration (0.67 μM), or BSA at a 25 fold higher concentration (15 μM) had a significant inhibitory effect on Cdc25 activity (Figure 3 A). Five fold higher concentrations of Pinl R6869A could partially inhibit mitotic Cdc25 activity (Figure 3B), a result which is consistent with the requirement for higher concentrations of this mutant protein to arrest the Xenopus cell cycle. To rule out the possibility that Pinl could directly inhibit the cyclin B/Cdc2 complex itself, we examined the effect of Pinl and its mutants on the activity of dephosphorylated-active cyclin B/Cdc2 under same conditions. Neither Pinl nor the Pinl mutant had any effect on Cdc2 activity (Figure 3C). Taken together, these results indicate that Pinl could inhibit premature mitotic activation of Cdc25 by interacting with the phosphorylation sites on Cdc25 that are essential for its activation. This offers one explanation for the ability of Pinl to inhibit mitotic entry. Example 17: Screening of Peptide Libraries The oriented peptide library approach (Z. Songyang et al. Cell 72:767 (1993) was used to screen for optimal peptides. All amino acids except C were incoφorated at equimolar amounts in each degenerate position, yielding a total theoretical degeneracy for both libraries of 19 6 = 4.7x10 7 distinct peptide sequences. Pinl -GST beads and MPM2 antibody bound to protein-G beads were incubated with the peptide library mixtures and washed extensively. Bound peptides were eluted with 30% acetic acid and sequenced. The chromogenic oligopeptides were synthesized (A. Bernhardt, M. Drewello, & M. Schutkowski, Int. J. Peptide Protein Res. 50:143 (1997)) and confirmed by NMR. Standard peptides were purchased from Bachem. PPIase activity were assayed and the bimolecular rate constants k cat /K m were calculated according to the equation k cat /K m = (k obs -k u )/[PPIase], where k u is the first-order rate constant for spontaneous cis/trans isomerization and k obs is the pseudo-first-order rate constant for cis/trans isomerization in the presence of PPIase, as described in G. Fischer, H. Bang, C. Mech, Biomed. Biochim. Ada 43:1101 (1984); J. L. Kofron et al. Biochemistry 30:6127 (1991). Affinity of Pinl for peptides was measured as described in Schutkowski, M., Wδllner, S., & Fischer, G. Biochemistry 34:13016, (1995). EQUIVALENTS While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the claims.

Claims: CLAIMS What is claimed is: 1. An inhibitor of a phosphoserine- or phosphothreonine-proline specific peptidyl- prolyl isomerase comprising a molecule that mimics the structure and conformation of the pSer/pThr-Pro peptide moiety of the isomerase substrate when the substrate is bound into the active site of the isomerase. 2. The inhibitor of Claim 1 wherein the structure surrounding the mimic moiety is flanked on one side by hydrophobic residues and on the other side by hydrophobic or positively charged groups wherein the groups contact the active site of the isomerase. J . An inliibitor of a phosphoserine- or phosphothreonine-proline specific peptidyl- prolyl isomerase comprising a protein, peptide or peptide mimetic comprising xSer/ThrY wherein x is a negatively charged tetra or pentavalent moiety and Y is a Pro or a Pro analog. 4. The inhibitor of Claim 3 wherein x is selected from the group consisting of phosphate, sulfonate. boronate, phosphonate and sulfonyl amide. 5. The inhibitor of Claim 3 wherein the proline analog is a nitrogen-containing ring structure selected from the group consisting of imidazole, pyrole, tropolone, benzene, camphor and heterocyclic aromatic and non-aromatic ring structures. 6. Tl e inhibitor of Claim 3 wherein the K, of the inhibitor is ten micromolar or less. 7. The inliibitor of Claim 3 wherein xSer/Thr-Y is flanked on one side by hydrophobic residues and on the other side by hydrophobic residues or positively charged residues. 8. The compound of Claim 3 wherein the protein, peptide or peptide mimetic is linear or cyclic. 9. A method of inhibiting cell growth comprising inhibiting a mitotic peptidyl- prolyl isomerase in the cell comprising contacting the cell with an effective amount of the inhibitor of Claim 3. 10. The method of Claim 9 wherein the mitotic peptidyl-prolyl isomerase is Pinl. 11. The method of Claim 9 wherein the cell is in an individual. 12. The method of Claim 9 wherein the cell growth results from a hypeφlastic or neoplastic disorder. 13. The method of Claim 9 wherein the cells are eukaryotic cells. 14. The method of Claim 9 wherein the cells are selected from the group consisting of: mammalian cells, yeast cells and fungal cells. 15. A composition comprising an inhibitor of Claim 3 and a pharmaceutically - acceptable carrier. 16. A compound that inhibits a phosphoserine- or phospho threonine-proline specific peptidyl-prolyl isomerase comprising a protein, peptide and or a peptide mimetic wherein said protein, peptide or peptide mimetic has a core sequence of XXXpSer-ProXXX, wherein X is any L-amino acid, or D-amino acid. 17. The compound of Claim 16 wherein the ptrotein, peptide or peptide mimetic is at least eight amino acid residues in length and comprises the sequence X,X 2 X 3 pS-PX 4 X 5 X 6 wherein each residue can be independently selected as follows X, is W, Y or F; X 2 is F or I; X 3 is Y, R, F or W; X 4 is R, F, Y or W; X 5 is L or I and Xg is any amino acid. 18. The compound of Claim 16 wherein the inhibitor has a K, often micromolar or less. 19. A peptide inhibitor of a phosphoserine- or phospho threonine-proline-specific peptide prolyl isomerase comprising Tφ-Phen-Tyr-pSer-Pro-Arg. 20. A library of peptides that comprises a mixture of substantially equimolar amounts of peptides comprising the sequence NH 2 -MAXXXpSXXXAKK. wherein for each peptide X is any amino acid. 21. A library of compounds that comprises a mixture of substantially equimolar amounts of peptides comprising the sequence X,X 2 X 3 pS-PX 4 X 5 X 6 , wherein for each peptide X is any amino acid. 22. A method of identifying a phosphorserine- or phosphothreonine-specific peptidyl prolyl isomerase inhibitor comprising the steps of: a) providing a library of compounds that comprises a mixture of substantially equimolar amounts of peptides comprising the sequence X,X 2 X 3 pS-PX 4 X 5 X 6 , wherein for each peptide X is any amino acid; b) contacting the library of a) with the peptidyl prolyl isomerase of interest under binding conditions for time sufficient for the isomerase to bind to the peptides; c) determining the amino acid sequences of peptides bound to the isomerase of interest; d) synthesizing the peptides of c); and e) determining the I , of the peptide wherein a peptide with a K; often micromolar or less indicates that the peptide is suitable for use as an inhibitor of the isomerase of interest. 23. A method of identifying a phosphorserine- or phosphothreonine-specific peptidyl prolyl isomerase inhibitor comprising the steps of: a) providing the peptidyl prolyl isomerase of interest; b) mixing the isomerase of interest with: i) a candidate inhibitor molecule and ii) the substrate of the isomerase of interest to form an admixture of the isomerase of interest, candidate molecule and substrate; c) maintaining the admixture of b) under conditions sufficient for the isomerase of interest to catalyze the cis/trans isomerazation of the substrate; and d) determining the K ; of the candidate molecule, wherein a K ; of 10 micromolar or less is indicative of an inhibitor of the peptidyl prolyl isomerase of interest.

Inventors: CANTLEY LEWIS C, FISCHER GUNTER, LU KUN PING, YAFFEE MICHAEL, CANTLEY, LEWIS, C., FISCHER, GUNTER, LU, KUN, PING, YAFFEE, MICHAEL
Applicants: BETH ISRAEL HOSPITAL, MAX PLANCK GESELLSCHAFT, BETH ISRAEL DEACONESS MEDICAL CENTER, MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN E.V.
Application year: 1998
Main classification: C07K14
IPC codes: A61K38, A61P35, C07K1, C07K7, C07K14, C12N9