non-ATP Competitive Inhibitor Research Articles

Computational Insights for the Discovery of Non-ATP Competitive Inhibitors of MAP Kinases

Due to their role in cellular signaling mitogen activated protein (MAP) kinases represent targets of pharmaceutical interest. However, the majority of known MAP kinase inhibitors compete with cellular ATP and target an ATP binding pocket that is highly conserved in the 500 plus representatives of the human protein kinase family. Here we review progress toward the development of non-ATP competitive MAP kinase inhibitors for the extracellular signal regulated kinases (ERK1/2), the c-jun N-terminal kinases (JNK1/2/3) and the p38 MAPKs (α, β, γ, and δ). Special emphasis is placed on the role of computational methods in the drug discovery process for MAP kinases. Topics include recent advances in X-ray crystallography theory that improve the MAP kinase structures essential to structurebased drug discovery, the use of molecular dynamics to understand the conformational heterogeneity of the activation loop and inhibitors discovered by virtual screening. The impact of an advanced polarizable force field such as AMOEBA used in conjunction with sophisticated kinetic and thermodynamic simulation methods is also discussed.

Small Molecule Kinase Inhibitors as Anti-Cancer Therapeutics

Protein kinases have emerged as the most important class of targets in oncology drug discovery because of their major roles in regulating cellular growth and survival. At least, 11 kinase inhibitors have received FDA approval to be used as cancer treatments, and there are continuous efforts to bring more candidates from laboratory benches to the clinic. Although many protein kinase inhibitors directly interact with the ATP binding site, other can alter the kinase conformation to prevent productive ATP binding. Herein we discuss the different mechanisms of action of kinase inhibitors and provide classification of the inhibitors according to their binding sites. Some of these are allosteric inhibitors, ATP competitive inhibitors, protein substrate competitive inhibitors, and covalent bond forming inhibitors. This review provides a broad overview of the relation between mechanism of action and the issues of target selectivity and resistance. Special attention was given to the kinase inhibitors currently in clinical trials.

Computational Insights for the Discovery of Non-ATP Competitive Inhibitors of MAP Kinases

Computational Insights for the Discovery of Non-ATP Competitive Inhibitors of MAP Kinases

Pyrrolo[3,2-d]pyrimidine Derivatives as Type II Kinase Insert Domain Receptor (KDR) Inhibitors: CoMFA and CoMSIA Studies

Kinase insert domain receptor (KDR) inhibitors have been proved to be very effective anticancer agents. Molecular docking, 3D-QSAR methods, CoMFA and CoMSIA were performed on pyrrolo[3,2-d]pyrimidine derivatives as non-ATP competitive KDR inhibitors (type II). The bioactive conformation was explored by docking one potent compound 20 into the active site of KDR in its DFG-out inactive conformation. The constructed CoMFA and CoMSIA models produced statistically significant results with the cross-validated correlation coefficients q2 of 0.542 and 0.552, non-cross-validated correlation coefficients r2 of 0.912 and 0.955, and predicted correction coefficients r2pred of 0.913 and 0.897, respectively. These results ensure the CoMFA and CoMSIA models as a tool to guide the design of a series of new potent KDR inhibitors.

Evidence for Irreversible Inhibition of Glycogen Synthase Kinase-3β by Tideglusib

Tideglusib is a GSK-3 inhibitor currently in phase II clinical trials for the treatment of Alzheimer disease and progressive supranuclear palsy. Sustained oral administration of the compound to a variety of animal models decreases Tau hyperphosphorylation, lowers brain amyloid plaque load, improves learning and memory, and prevents neuronal loss. We report here that tideglusib inhibits GSK-3β irreversibly, as demonstrated by the lack of recovery in enzyme function after the unbound drug has been removed from the reaction medium and the fact that its dissociation rate constant is non-significantly different from zero. Such irreversibility may explain the non-competitive inhibition pattern with respect to ATP shown by tideglusib and perhaps other structurally related compounds. The replacement of Cys-199 by an Ala residue in the enzyme seems to increase the dissociation rate, although the drug retains its inhibitory activity with decreased potency and long residence time. In addition, tideglusib failed to inhibit a series of kinases that contain a Cys homologous to Cys-199 in their active site, suggesting that its inhibition of GSK-3β obeys to a specific mechanism and is not a consequence of nonspecific reactivity. Results obtained with [(35)S]tideglusib do not support unequivocally the existence of a covalent bond between the drug and GSK-3β. The irreversibility of the inhibition and the very low protein turnover rate observed for the enzyme are particularly relevant from a pharmacological perspective and could have significant implications on its therapeutic potential.

Activity of Allosteric, Switch-Pocket, ABL/FLT3 Kinase Inhibitor DCC2036 Against Cultured and Primary AML Progenitors with FLT-ITD or FLT3 Kinase Domain Mutations

Abstract 2611Approximately 30% of acute myeloid leukemia (AML) patients have activating mutations in FLT3, commonly internal tandem duplication (ITD) mutations, which are associated with poor survival. Although FLT3 tyrosine kinase inhibitors (TKI) such as AC220 can induce remissions, resistance-causing mutations in FLT3-ITD are known to impair the in vitro activity of first and second generation FLT3 TKIs. DCC2036 is a unique switch-pocket, non-ATP competitive (allosteric) inhibitor with low nanomolar inhibitory concentration 50 (IC50) activity against a number of tyrosine kinases including FLT3 (1.7 nM), TRKA (7.0 nM), TIE-2 (2.7 nM) and BCR-ABL (2.0 nM). DCC2036 has shown promising activity in a phase I/II clinical trial in chronic myeloid leukemia (CML), where plasma concentrations of 350 nM of DCC2036 have been safely achieved. DCC-2036 has induced clinical and molecular remissions in patients with TKI-resistant CML expressing the ‘gate-keeper' T315I BCR-ABL mutation, as well as demonstrated activity against mutations that cause BCR-ABL conformational escape resistant (Cancer Cell. 2011;19:556). Here, we evaluated the in vitro activity of DCC2036 against FLT3-ITD in cell line model systems. In the FLT3-ITD expressing human leukemia MV4-11 and MOLM-13 cells, treatment with DCC2036 (20 to 500 nM for 24 hours) dose-dependently induced cell cycle G1-phase accumulation with decline in the S and G2/M phases. Exposure to 50 to 500 nM DCC2036 for 48 to 72 hours also dose-dependently induced apoptosis of 30 to 80 % of MV4-11 and MOLM-13, as well as induced 30 to 50% apoptosis of patient-derived primary AML cells with FLT3-ITD (n =4). This was associated with dose-dependent decline in the levels of p-FLT3, p-STAT5, p-AKT, p-ERK1/2 and Bcl-xL levels but increase in the levels of BIM and p27. In contrast, treatment with DCC2036 induced significantly lower level of apoptosis (<15%) in either normal CD34+ progenitor cells or AML cells without FLT3-ITD. We next evaluated the in vitro activity of DCC2036 against AC220-resistant mutations in FLT3-ITD (F691L, D835V/Y and Y842C/H) that we identified in a pre-clinical screen and in patients with loss of response to AC220 (Smith et al, ASH 2011, submitted), as well as against FLT3-ITD/F691I, analogous to the BCR-ABL/T315I mutation. DCC2036 potently inhibited proliferation of FLT3-ITD transduced Ba/F3 cells with an IC50 of 14.5 nM (IC50 of parental Ba/F3 cells in the presence of IL-3 >1000nM). DCC2036 retained some activity against the clinically relevant FLT3-ITD gatekeeper mutation F691L and F691I (IC50 49 nM and 34 nM), and was similarly active against the activation loop mutations Y842C and Y842H (IC50 26–28 nM). The activation loop mutations D835V and D835Y, which are commonly detected in patients with loss of response to AC220 and are hypothesized to destabilize the kinase inactive “DFG-out” conformation, were substantially less sensitive to DCC2036 (IC50 233 nM and 196 nM, respectively). Based on our previous findings (Blood. 2005;105:1768) that FLT3-ITD is a heat shock protein (hsp) 90 client oncoprotein, we also determined the effect of co-treatment with the non-geldanamycin hsp90 inhibitor AUY922 (5 to 10 nM) (Novartis Pharmaceuticals) against the cultured and primary FLT3-ITD expressing AML cells. Co-treatment with AUY922 significantly improved the activity of DCC2036 against primary AML cells (p < .05). These findings demonstrate that DCC2036 exhibits potent activity against cultured and primary AML cells with FLT-3-ITD, as well as against cellular models of FLT3-ITD with AC220-resistant gatekeeper and select activation loop mutations. The molecular basis of resistance to DCC2036 conferred by activation loop mutations at D835 is under investigation. Co-treatment with DCC-2036 and the hsp90 inhibitor AUY922 exerted higher lethal activity against cultured and primary AML cells with FLT3-ITD. Disclosures:Wise:Deciphera Pharmaceuticals LLC: Employment. Reyes:Millennium, Sanofi Aventis: Consultancy. Berger:Deciphera Pharmaceuticals: Employment. Rutkoski:Deciphera Pharmaceuticals: Employment.

A Phase II Study of the MEK 1/2 Inhibitor AZD6244 (Selumetinib, ARRY-142866) in Relapsed or Refractory Multiple Myeloma

Abstract 2931AZD6244 is a potent, selective, oral, non-ATP competitive small molecule inhibitor of the mitogen-activated protein kinase, MEK 1/2 that has shown significant pre-clinical activity in multiple myeloma (MM) cells, both in vitro and in vivo, as well as a favorable clinical profile. The present phase II study was designed to determine the response rate for AZD6244 in patients with relapsed or refractory MM.The study utilized a two-stage Simon design to allow for early termination if there was strong evidence of regimen inactivity. Eligible patients were restricted to those with MM who have had at least 2 prior regimens. AZD6244 capsules (75 mg) were administered orally twice daily continuously for 28 day cycles. Response was evaluated after 3 cycles.To date, 37 patients have been enrolled (13 in the 1st stage and 24 in the 2nd stage). One subject enrolled in the 1st stage was not treated. Gender enrollment was balanced (male/female 18/19). The median age of treated patients was 65 years [range 43–81]. ECOG performance scores ranged from 0–2. The median number of prior therapies was 5 [range 2–11].The most common treatment-related adverse events (occurring in 10–50% of patients) were leukopenia, acneiform rash and other skin/subcutaneous tissue manifestations, fatigue, limb edema, increased aspartate aminotransferase (AST), neutropenia, nausea, facial edema, vomiting, thrombocytopenia, increased creatine phosphokinase, and diarrhea. The most common grade 3 and 4 toxicities (CTCAE v4) included fatigue, peripheral sensory neuropathy, increased AST, neutropenia, nausea, hypotension, thrombocytopenia, increased alanine aminotransferase, and diarrhea. Five deaths have occurred: 2 associated with sepsis, 1 associated with acute kidney injury, all deemed possibly related to AZD6244; and 2 due to disease progression after discontinuation of study treatment.Two objective partial responses have been reported, the first of which justified expansion of the study to the 2nd stage. Twelve patients have had a best response of stable disease, 11 patients have had progressive disease, 1 patient withdrew after cycle 1 (unrelated to toxicity) and did not have response assessed, 3 patients died before response was assessed, and 7 patients are too early to evaluate. Accrual is ongoing to determine if the response threshold in the 2nd stage can be met.Correlative studies are ongoing and are designed to identify potential mechanisms of response/resistance to AZD6244, and to determine the effect of AZD6244 on the bone marrow microenvironment. These include, among others, assessment of pre- and post-treatment expression of phospho-MEK 1/2 and -ERK 1/2, and total levels of Bim. Fifteen patients consented to correlative sampling of bone marrow, blood and/or urine. Results and sample analysis are pending.It is concluded that AZD6244 has modest activity as a single agent in relapsed or refractory MM. This trial also provides a foundation for successor studies employing the MEK 1/2 inhibitor AZD6244 in combination with other agents in patients with MM. Disclosures:Voorhees:Pfizer: Research Funding; Centocor Ortho Biotech: Research Funding; Celgene: Research Funding; MedImmune: Consultancy; Merck: Research Funding.

Exploring the Binding Sites of Glycogen Synthase Kinase 3. Identification and Characterization of Allosteric Modulation Cavities

Glycogen synthase kinase 3 (GSK-3) is an important drug target for human severe unmet diseases. Discovery and/or design of allosteric kinase modulators are gaining importance in this field not only for the increased selectivity of this kind of compounds but also for the subtle modulation of the target. This last point is of utmost importance for the GSK-3 inhibition as a therapeutic approach. GSK-3 activity is completely necessary for life, and only the aberrant overactivity found in the pathologies should be inhibited with its inhibitors treatment. We performed here a search for the druggable sites on the enzyme using the fpocket algorithm with the aim to provide allosteric potential binding sites on it and new clues for further drug discoveries. Moreover, our results allowed us to determine the binding sites of different GSK-3 ATP noncompetitive inhibitors, such as manzamine A and the new small molecule VP 0.7, providing evidence for potential allosteric inhibition of GSK-3.

Examining Docking Interactions on ERK2 with Modular Peptide Substrates

ERK2 primarily recognizes substrates through two recruitment sites, which lie outside the active site cleft of the kinase. These recruitment sites bind modular-docking sequences called docking sites and are potentially attractive sites for the development of non-ATP competitive inhibitors. The D-recruitment site (DRS) and the F-recruitment site (FRS) bind D-sites and F-sites, respectively. For example, peptides that target the FRS have been proposed to inhibit all ERK2 activity (Galanis, A., Yang, S. H., and Sharrocks, A. D. (2001) J. Biol. Chem. 276, 965-973); however, it has not been established whether this inhibition is steric or allosteric in origin. To facilitate inhibitor design and to examine potential coupling of recruitment sites to other ligand recognition sites within ERK2, energetic coupling within ERK2 was investigated using two new modular peptide substrates for ERK2. Modeling shows that one peptide (Sub-D) recognizes the DRS, while the other peptide (Sub-F) binds the FRS. A steady-state kinetic analysis reveals little evidence of thermodynamic linkage between the peptide substrate and ATP. Both peptides are phosphorylated through a random-order sequential mechanism with a k(cat)/K(m) comparable to Ets-1, a bona fide ERK2 substrate. Occupancy of the FRS with a peptide containing a modular docking sequence has no effect on the intrinsic ability of ERK2 to phosphorylate Sub-D. Occupancy of the DRS with a peptide containing a modular docking sequence has a slight effect (1.3 ± 0.1-fold increase in k(cat)) on the intrinsic ability of ERK2 to phosphorylate Sub-F. These data suggest that while docking interactions at the DRS and the FRS are energetically uncoupled, the DRS can exhibit weak communication to the active site. In addition, they suggest that peptides bound to the FRS inhibit the phosphorylation of protein substrates through a steric mechanism. The modeling and kinetic data suggest that the recruitment of ERK2 to cellular locations via its DRS may facilitate the formation of F-site selective ERK2 signaling complexes, while recruitment via the FRS will likely inhibit ERK2 through a steric mechanism of inhibition. Such recruitment may serve as an additional level of ERK2 regulation.

Development of a time-resolved fluorescence resonance energy transfer assay for cyclin-dependent kinase 4 and identification of its ATP-noncompetitive inhibitors

Protein kinases are recognized as important drug targets due to the pivotal roles they play in human disease. Many kinase inhibitors are ATP competitive, leading to potential problems with poor selectivity and significant loss of potency in vivo due to cellular ATP concentrations being much higher than Km. Consequently, there has been growing interest in the development of ATP-noncompetitive inhibitors to overcome these problems. There are challenges to identifying ATP-noncompetitive inhibitors from compound library screens because ATP-noncompetitive inhibitors are often weaker and commonly excluded by potency-based hit selection criteria in favor of abundant and highly potent ATP-competitive inhibitors in screening libraries. Here we report the development of a time-resolved fluorescence resonance energy transfer (TR–FRET) assay for protein kinase cyclin-dependent kinase 4 (CDK4) and the identification of ATP-noncompetitive inhibitors by high-throughput screening after employing a strategy to favor this type of inhibitors. We also present kinetic characterization that is consistent with the proposed mode of inhibition.

Phase Ib results of c-MET inhibitor ARQ 197 in combination with gemcitabine in a cohort of patients (pts) with advanced breast, ovarian, and uterine tumors.

3077 Background: ARQ 197 is a non-ATP competitive selective inhibitor of c-MET. In vitro data suggest its synergy with gemcitabine. This study evaluates safety, pharmacokinetics (PK), biomarkers, and preliminary efficacy of the combination treatment in pts with advanced solid tumors. Here we present results of the cohort of pts with metastatic breast, ovarian, and uterine carcinoma. Methods: Multicenter, phase Ib 3+3 dose escalation trial. Oral ARQ 197 administered at doses of 120-360 mg bid across different schedules (continuous vs. continuous with 1 week [wk] break every 2 or 3 wks) in combination with a fixed gemcitabine at 1,000 mg/m2/weekly 3 every 4 wks. Results: As of January 4, 2011, a total of 32 pts with metastatic breast (12), ovarian (14), and uterine (6) carcinoma were enrolled in the study (ECOG 0:1 = 21:11; mean age 61, range 35-77 yrs; with median number of prior systemic therapies = 4 [1–9]). Seven pts had progressed to prior gemcitabine. Adverse events (AEs) considered at least possibly treatment-related were reported in 29 (91%) of these pts. Most commonly observed AEs (>10%) included thrombocytopenia (21; 66%, 4 G 3-4), anemia (21; 66%, 6 G 3-4), neutropenia (20; 63%, 7 G 3-4), fatigue (11; 34%, 3 G 3-4), nausea (9; 31%, 2 G 3-4), vomiting (6; 19%, 1 G 3-4), and leucopenia (4; 13%, 2 G 3-4). Three pts experienced treatment-related serious AEs (pancytopenia, thrombocytopenia, hypotension and interstitial lung disease, grade 3). Post-baseline tumor assessment was done in 27 evaluable pts; of them, 12 remained on study for >10 wks (mean 22; range 10-36), 5 (2 breast, 1 uterine, 2 ovarian) attained partial response (response rate 19%), and 15 demonstrated decline (4-87%) in tumor markers (CA15.3, CA125, CEA). In this study, 2 pts (ovarian and breast) with PR and 2 with SD (breast) had failed prior gemcitabine. Details on efficacy and scheduling will be presented. Conclusions: Orally administered ARQ 197 at the MTD of 360 mg bid in combination with gemcitabine is generally well tolerated and shows encouraging signs of antitumor activity in the presented tumor types.

Efficacy in selected tumor types in a phase I study of the c-MET inhibitor ARQ 197 in combination with sorafenib.

3034 Background: ARQ 197 (A) is a selective, oral, non-ATP competitive inhibitor of c-MET, a RTK implicated in tumor cell proliferation, invasion and metastasis, currently in phase II/III clinical ...

The effect of CYP2C19 polymorphism on the tolerability of ARQ 197: Results from phase I trial in Japanese patients with metastatic solid tumors.

2516 Background: ARQ 197 is a selective, non-ATP competitive inhibitor of c-MET, a receptor tyrosine kinase implicated in tumor cell migration, invasion and proliferation. ARQ 197 is mainly metabolized by CYP2C19. The ratio of the poor metabolizer (PM) of CYP2C19 in Asians is reported to be around 20 % while it is very low in Caucasians. Recommended phase II dose (RP2D) of ARQ 197 in western countries was determined to be 360 mg bid.In this monotherapy phase I study in Japanese patients (pts) with metastatic solid tumors, we examined the drug's safety profile, pharmacokinetics and preliminary antitumor activity and determined RP2D in PM pts as well as extensive metabolizer (EM) pts. Methods: ARQ 197 was administered orally at 8 dose levels from 70 to 360 mg bid in a standard 3+3 dose escalation design. We enrolled PM and EM pts separately from 120 mg bid. Results: Forty-seven pts consisted of 33 EM pts (median age: 61.0 yrs; ECOG PS 0/1: 15/18; median prior therapies: 4) and 14 PM pts (median age: 59.5 yrs; ECOG PS 0/1: 6/8; median prior therapies: 4) were treated. The most common drug-related adverse events were fatigue, leukopenia, anemia, neutropenia and anorexia. Gr.4 neutropenia occurred in 1 of 6 EM pts at 360 mg bid and 3 of 7 PM pts at 240 mg bid, which identified each dosage as RP2D for EM and PM respectively. Cmax and AUC0-12 increased in a dose-dependent manner up to 300 mg bid in the EM cohorts and up to 240 mg bid in the PM. At 240 mg bid, the ARQ 197 Cmax and AUC0-12 of PM pts were 1.5-fold and 1.9-fold higher than those of EM pts respectively. Pts who had a relatively high exposure of ARQ 197 such as Cmax > 4,000 ng/mL or AUC0-12 > 40,000 ng.h/mL on day 1 tended to experience Gr.4 neutropenia regardless of CYP2C19 genotype. Two NSCLC pts, both of whom were PM at 240 mg bid, achieved partial response and 16 pts (34.0%) had stable disease (SD) for >= 16 weeks, especially 6 of 25 pts (24.0 %) in NSCLC had SD for >= 28 weeks. Conclusions: ARQ 197 was well tolerated in PM pts as well as EM pts. CYP2C19 genotype clearly affected the exposure to ARQ 197 which led different RP2Ds, 360 mg bid for EM pts and 240 mg bid for PM pts. ARQ 197 monotherapy showed a clear sign of activity. Further study is warranted.

Abstract LB-198: Design and synthesis of non-ATP competitive inhibitors of CDK4/Cyclin D using REPLACE

Abstract CDK4/Cyclin D is an established anticancer drug target closely associated with breast and non-small cell lung cancers. Conventionally, the ATP binding site of CDKs has been the focus of inhibitor design. Here we present an alternative strategy to target CDK4 which allows us to design non-ATP competitive inhibitors by exploring the cyclin binding groove (CBG) present in Cyclin D, its positive regulatory subunit. The CBG of Cyclin D recognizes a cyclin binding motif (CBM) possessed by both natural inhibitors and substrates of CDK4/Cyclin D and is composed of primary and secondary hydrophobic pockets separated by a polar subsite; In addition, we recently described a hydrophobic extension to the major pocket and which is unique to Cyclin D1. The REPLACE method has been previously applied, through which determinants in known potent peptide inhibitors (binding in an extended conformation) are replaced with non-peptidic surrogates. In this study, a new series of non-peptidic surrogates to replace the original C-terminal residues (C-capping groups) that are key to binding to the primary hydrophobic pocket have been ligated to truncated peptides. Also using this approach, additional series of N-capping groups, (surrogates for N-terminal residues) which are important for the binding to the secondary hydrophobic pocket have been ligated to peptides. Both N- and C-capping groups have been selected to obtain more potent binding to their specific subsite. In addition, through guided docking of a small set of compounds into the hydrophobic extension unique to the cyclin D1 CBG, we have identified compounds with high predicted affinity to cyclin D, suggesting that appropriate modification of an inhibitor could lead to an increase in potency and selectivity. In order to do this, a virtual library composed of low molecular weight fragments has been modeled. Fragments were selected based on several criteria: 1) Log P ≤ 3; 2) Rotational bonds ≤ 3; 3) MW between 170 and 280. After docking, formation and energy minimization of the protein-fragment peptide complex enabled the prediction of capping groups with the highest potential for binding to the Cyclin D pocket. In order to evaluate these compounds in a facile and straightforward manner and to eliminate ATP competitive inhibitors, a competitive binding assay based on fluorescence polarization, has been developed for the first time for cyclin D1. The FP assay for Cyclin D provided insights into the binding of peptidic inhibitors to cyclin D1, as well as SAR for both N- and C-capping groups. By virtue of the establishment of computational, synthetic and in vitro testing work, the prediction of more potent capping groups will be facilitated. These experiments will serve as a useful guide for future direction of drug development for the design of inhibitors with selectivity towards Cyclin D and with potential as anti-tumor therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-198. doi:10.1158/1538-7445.AM2011-LB-198

Abstract 1351: Computational design and synthesis of non-peptidic inhibitors targeting the cyclin binding groove of CDK2/Cyclin A

Abstract Inhibition of CDK2/Cyclin A (CDK2/CA) at G1/S phase of cell cycle controls cell proliferation and induces selective apoptosis of cancer cells. Neutralization of E2F-1by CDK2/CA phosphorylation is required prior to S-phase exit or apoptosis is triggered. E2F-1 is deregulated in many types of cancer and inhibition of CDK2/CA leads to selective cell death in these tumors. In our present study we target the substrate recruitment site knows as cyclin binding groove (CBG) for inhibition of CDK2/CA. The CBG recognizes a consensus sequence present on cyclin interacting proteins and the endogenous tumor suppressors knows cyclin binding motif (CBM) which has been minimized to the pentapeptide, RRLIF. It has been shown that the drug likeliness of the peptide can be improved by ligation of small molecules to truncated sequences. This was achieved using a unique strategy for designing inhibitors of protein-protein interactions known as REPLACE, in which small molecule fragments were identified by in silico methods. These were then ligated onto a truncated peptide sequence in order to replace critical residues, including the N-terminal Arginine moiety important for cyclin binding. Small molecule fragments for in silico docking were selected based on molecule weight, drug like features and possession of a carboxylic acid group for the formation of the peptide link. Docking was carried out using LigandFit and the crystal structure of 1-phenyl-1H-[1,2,4]triazole N-terminally capped peptides (Ncaps) in the CBG provided insight into molecular design. The 3,5 dichloro1-phenyl-1H-[1,2,4]triazole derivative linked to Arg-Leu-Asn-pF-Phe was previously shown to be a potent inhibitor with micromolar activity. In our present study we have replaced the N-terminal Arginine using different heterocyclic isosteres capable of interactions similar to critical amino acids of the parent peptide and the triazole. In order to probe the structure-activity relationship, we synthesized pyrazole, furan, pyrrole and thiazole systems and explored various substitutions of the phenyl ring. A number of Ncaps were then ligated to the tetra peptide, RLIF using solid phase synthesis, purified by reverse phase HPLC and characterized by MS. In vitro binding and functional assays were performed in order to study the inhibitory effect of compounds on CDK2/Cyclin A prior to further evaluation in cell viability assays to determine anti-tumor effects. On the basis of the results, further high throughput docking of potential heterocyclic fragments was carried out to identify N-capping groups of varying chemical diversity for synthesis and in vitro testing. The results presented demonstrate the utility of the REPLACE method in the development of protein-protein interaction inhibitors and non-ATP competitive CDK2 inhibitors as anti-tumor therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1351. doi:10.1158/1538-7445.AM2011-1351

Abstract 3240: Development of non-ATP competitive, PLK1 selective inhibitors

Abstract Through a better understanding of the molecular mechanisms that control normal and cancer cell proliferation, new therapeutic targets that regulate the cell cycle have been identified. One of these, Polo like kinase 1 (PLK1) is a member of a conserved family of serine/threonine kinases. Its activity and cellular levels play an important role in mitotic entry, DNA-damage checkpoint activation, and centrosome duplication and maturation. Because other PLK family members such as PLK3 act as putative tumor suppressors, it is imperative that a PLK1 inhibitor be exclusive for PLK1, a potential draw back for ATP competitive inhibitors. The polo box domain (PBD) of PLK1 plays an important role in sub-cellular localization and mitotic functions of PLK1. Importantly, it has been shown that inhibiting the PBD site leads to selective PLK1 inhibition and can lead to mitotic arrest and inhibit proliferation of tumor cells. The structure-guided discovery strategy called REPLACE can be used to target protein-protein interactions otherwise considered undruggable. REPLACE uses structure activity relationships of peptide inhibitors to generate a pharmaceutically acceptable lead molecule by replacing individual amino acid residues with non-peptide fragments. REPLACE has been used to identify fragment mimetics for determinants from the endogenous phosphopeptide sequence, LLCSpTPNGL (CDC25c), known to specifically bind to the PBD of PLK1. Using this approach, FLIPs (Fragment Ligated Inhibitory Peptides) were generated and tested in a fluorescence polarization (FP) assay to determine binding potential for the PBD. Small molecule fragment alternatives for the N-terminal tripeptide (LLC) were identified through this approach and shown to possess increased binding relative to the truncated peptide. Modeling and amino acid replacement was also used to elucidate essential binding sites and specificity determinants needed by the endogenous peptide to maintain affinity and selectivity for the PBD. This was confirmed through comparisons of binding for peptide analogs in the FP assay relative to that of the endogenous peptide. As part of the development process, peptides have been examined for anti-proliferative activity in cancer cells and to determine if a PLK1 inhibitory phenotype is observed. Mitotic events were evaluated by flow cytometry and by direct visualization, utilizing cells expressing GFP-labeled histone H2B. Conditions to transduce peptides have been established and these will be used to characterize FLIPs with sufficient affinity for the PBD. By targeting the PBD of PLK1 using REPLACE, we believe that we can develop a non-ATP competitive inhibitor that is not only PLK1 specific but is less toxic to normal cells and safer than current drugs on the market. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3240. doi:10.1158/1538-7445.AM2011-3240

Discovery of the first non-ATP competitive IGF-1R kinase inhibitors: Advantages in comparison with competitive inhibitors

A new series of IGF-1R inhibitors related to hydantoins were identified from a lead originating from HTS. Their noncompetitive property as well as their slow binding characteristics provided a series of compounds with unique selectivity and excellent cellular activities.

An application of two MIFs-based tools (Volsurf+ and Pentacle) to binary QSAR: The case of a palinurin-related data set of non-ATP competitive Glycogen Synthase Kinase 3β (GSK-3β) inhibitors

VolSurf+ and GRIND descriptors extract the information present in MIFs calculated by GRID: the first are simpler to interpret and generally applied to ADME-Tox topics, whereas the latter are more sophisticated and thus more suited for pharmacodynamics events. Here we present a study which compares binary QSAR models obtained with VolSurf+ descriptors and GRIND for a data set of non-ATP competitive GSK-3β inhibitors chemically related to palinurin for which the biological activity is expressed in binary format. Results suggest not only that the simpler Volsurf+ descriptors are good enough to predict and chemically interpret the investigated phenomenon but also a bioactive conformation of palinurin which may guide future design of ATP non-competitive GSK-3 inhibitors.

Synergistic Effects of a Novel Water-Soluble Small Molecule, ON 013105, and Rituximab on Mantle Cell Lymphoma In Vitro and In Vivo

AbstractAbstract 771Mantle cell lymphoma (MCL) is a well-defined subtype of B-cell non-Hodgkin's lymphoma characterized by a t(11;14)(q13;q32) chromosomal translocation, and associated with constitutive over-expression of cyclin D1. MCL generally has poor clinical outcome marked by relapse. There is considerable need for novel and more effective agents against MCL. ON 013105 belongs to the styryl benzylsulfones, a novel family of non-ATP competitive kinase inhibitors with potent antitumor activity. Here, we report that ON 013105 induced cell death in a dose-dependent manner in two well-characterized MCL cell lines, Granta 519 and Z138C. In vitro cell death was preceded by the activation of caspases 3 and 9 and cleavage of PARP, indicating induction of apoptosis. In addition, ON 013105-treated cells exhibited reduced expression of cyclin D1 and c-myc. These effects on expression and apoptosis were not evident in cells treated with ON 013101, an inactive (non-cytotoxic) isomer of ON 013105. Since it is common clinical practice to combine Rituximab (RTX) with chemotherapy regimens in treating CD20+ B cell-lymphoma, we studied ON 013105 combined with rituximab, and found ON 013105-induced apoptosis more efficiently than when employed as a single agent. The combination effect on cell death was synergistic in nature. To further study this activity, we focused on Mcl-1, a member of the anti-apoptotic Bcl-2 family known to inhibit apoptosis induced by cytotoxic stimuli through antagonizing pro-apoptotic Bcl-2 family members. We observed a dramatic decrease in Mcl-1 expression in cells treated with ON 013105 (but not with ON 013101) in combination with RTX, compared to ON 013105 alone. We also evaluated the effects of ON 013105 in combination with Doxorubicin or Vincristine and found that both these compounds also significantly enhanced the cytotoxic effects of ON 013105. In vivo pharmacokinetics studies in a mouse model system revealed that plasma concentrations up to 50 μM could be safely achieved by administering ON 013105 at 100 mg/kg via i.v or i.p routes. Significant levels of ON 013100 (30-40% of the peak levels of ON 013105), an active metabolite, were also detected in the circulation, presumably due to the in vivo dephosphorylation of ON 013105 by phosphatase action. ON 013105 was well tolerated in mice, both as a single agent and when used in combination with rituximab, and there were no systemic toxic effects to the host and no loss in body weight. In vivo efficacy studies in mouse xenograft models employing transplanted MCL cells demonstrated that ON 013105 effectively inhibited tumor growth in a dose-dependent manner. ON 013015 at 25 mg/kg (Q2D) and 75mg/kg (Q7D) induced 46% and 80 % reduction of tumor volume, respectively, compared to controls, over 4 weeks of treatment. Moreover, ON 013105 at 25 mg/kg (Q2D) in a combination regimen with RTX (2.5 mg/kg, Q3D) induced over 85% reduction of tumor volume. Though in vivo efficacy studies of ON013015 (25 mg/kg, Q2D) in combination with Doxorubicin (3.5mg/kg, Q7D) or Vincristine (0.3mg/kg, Q2D) showed drastic decrease in tumor growth in mouse models, this effect was accompanied by severe side effects to the host, including mortality. In sum, ON 013105, alone and in combination with RTX may be a potent therapeutic agent against MCL. A Phase I dose escalation trial of ON 013105 as a single agent is underway in patients with relapsed/refractory lymphoma including MCL. Disclosures:No relevant conflicts of interest to declare.

Inhibition of glioblastoma growth by the thiadiazolidinone compound TDZD-8.

BackgroundThiadiazolidinones (TDZD) are small heterocyclic compounds first described as non-ATP competitive inhibitors of glycogen synthase kinase 3β (GSK-3β). In this study, we analyzed the effects of 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), on murine GL261 cells growth in vitro and on the growth of established intracerebral murine gliomas in vivo.Methodology/Principal FindingsOur data show that TDZD-8 decreased proliferation and induced apoptosis of GL261 glioblastoma cells in vitro, delayed tumor growth in vivo, and augmented animal survival. These effects were associated with an early activation of extracellular signal-regulated kinase (ERK) pathway and increased expression of EGR-1 and p21 genes. Also, we observed a sustained activation of the ERK pathway, a concomitant phosphorylation and activation of ribosomal S6 kinase (p90RSK) and an inactivation of GSK-3β by phosphorylation at Ser 9. Finally, treatment of glioblastoma stem cells with TDZD-8 resulted in an inhibition of proliferation and self-renewal of these cells.Conclusions/SignificanceOur results suggest that TDZD-8 uses a novel mechanism to target glioblastoma cells, and that malignant progenitor population could be a target of this compound.