Active Small-molecule Inhibitor Research Articles

A Small-Molecule Antagonist of HIF2α Is Efficacious in Preclinical Models of Renal Cell Carcinoma

More than 90% of clear cell renal cell carcinomas (ccRCC) exhibit inactivation of the von Hippel-Lindau (pVHL) tumor suppressor, establishing it as the major underlying cause of this malignancy. pVHL inactivation results in stabilization of the hypoxia-inducible transcription factors, HIF1α and HIF2α, leading to expression of a genetic program essential for the initiation and progression of ccRCC. Herein, we describe the potent, selective, and orally active small-molecule inhibitor PT2385 as a specific antagonist of HIF2α that allosterically blocks its dimerization with the HIF1α/2α transcriptional dimerization partner ARNT/HIF1β. PT2385 inhibited the expression of HIF2α-dependent genes, including VEGF-A, PAI-1, and cyclin D1 in ccRCC cell lines and tumor xenografts. Treatment of tumor-bearing mice with PT2385 caused dramatic tumor regressions, validating HIF2α as a pivotal oncogenic driver in ccRCC. Notably, unlike other anticancer agents that inhibit VEGF receptor signaling, PT2385 exhibited no adverse effect on cardiovascular performance. Thus, PT2385 represents a novel class of therapeutics for the treatment of RCC with potent preclincal efficacy as well as improved tolerability relative to current agents that target the VEGF pathway. Cancer Res; 76(18); 5491-500. ©2016 AACR.

'Up with the LRRK': a phosphorylated Rab10 assay for evaluation of LRRK2 activity and inhibitor engagement.

Protein kinases catalyse the addition of phosphate groups to Ser/Thr and Tyr residues in cognate substrates and are mutated or hyperactive in a variety of diseases, making them important targets for rationally designed drugs. A good example is the Parkinson's disease-associated kinase, leucine-rich repeat kinase 2 (LRRK2), which is mutated (and probably hyperactive) in a small, but significant, subset of patients. An exciting new approach for personalised therapy is the development of central nervous system (CNS)-active small-molecule kinase inhibitors, which could be employed to ‘normalise’ LRRK2 signalling in affected cell types. However, the development of such drugs requires validated assays for the analysis of target engagement and the assembly of a set of tools for interrogating LRRK2, and its substrates, both in vitro and in vivo. A new study published in the Biochemical Journal by Ito et al. establishes that a ‘Phos-tag’™-binding assay can be exploited to measure phosphorylation of a recently identified LRRK2 substrate (Ras-related protein in brain 10 (Rab10)), and to compare and contrast relative catalytic output from disease-associated LRRK2 mutants. Powerful in vivo chemical genetic approaches are also disclosed, in which the catalytic activity of LRRK2 is unequivocally linked to the extent of Rab10 phosphorylation and the effects of chemically distinct LRRK2 inhibitors are matched with on-target inhibition mechanisms mediated through LRRK2 and its substrate Rab10. These important findings should simplify the generic analysis of Rab10 phosphorylation in model biological systems and are likely to be applicable to other substrates of LRRK2 (or indeed other kinases) for which phospho-specific antibodies are either absent or unsatisfactory.

Finding a Potential Dipeptidyl Peptidase-4 (DPP-4) Inhibitor for Type-2 Diabetes Treatment Based on Molecular Docking, Pharmacophore Generation, and Molecular Dynamics Simulation.

Dipeptidyl peptidase-4 (DPP-4) is the vital enzyme that is responsible for inactivating intestinal peptides glucagon like peptide-1 (GLP-1) and Gastric inhibitory polypeptide (GIP), which stimulates a decline in blood glucose levels. The aim of this study was to explore the inhibition activity of small molecule inhibitors to DPP-4 following a computational strategy based on docking studies and molecular dynamics simulations. The thorough docking protocol we applied allowed us to derive good correlation parameters between the predicted binding affinities (pKi) of the DPP-4 inhibitors and the experimental activity values (pIC50). Based on molecular docking receptor-ligand interactions, pharmacophore generation was carried out in order to identify the binding modes of structurally diverse compounds in the receptor active site. Consideration of the permanence and flexibility of DPP-4 inhibitor complexes by means of molecular dynamics (MD) simulation specified that the inhibitors maintained the binding mode observed in the docking study. The present study helps generate new information for further structural optimization and can influence the development of new DPP-4 inhibitors discoveries in the treatment of type-2 diabetes.

Design, synthesis and biological evaluation of 3′-benzylated analogs of 3′-epi-neoponkoranol as potent α-glucosidase inhibitors

A group of 3′-epi-neoponkoranol analogs with different hydrophobic substituents attached at 3′-position of side chain moiety were designed and synthesized in order to further improve the inhibitory activities against α-glucosidases. Biological evaluation of these compounds revealed that sulfonium salts attached with ortho-substituted benzyl groups showed best α-glucosidase inhibitory activities. The most potent compound 10i showed greater inhibitory activities than all natural products. Moreover, docking studies on 10i with ntMGAM presented a new binding mode, indicating that amino residue Asp542 should be the key interacting point for strong inhibitory activity of small molecules against α-glucosidase enzymes. The strongest α-glucosidase inhibitory activity of 10i could be rationalized by van der Waals interactions between the 3′-attached substituent and particularly the ortho-substituted trifluoromethyl on benzyl group with the adjacent hydrophobic amino residues. Cytotoxicity evaluation assay demonstrated a high level of safety profile of the synthesized sulfonium salts against normal cell line. The enzyme kinetic studies showed a fully competitive inhibition of these sulfonium salts on each α-glucosidase.

Abstract B48: Identification of potent, cell active MTH1 inhibitors and their use in target validation studies

Abstract MTH1 is a protein that sanitizes oxidized dNTPs in the cell. It preferentially hydrolyzes 8-oxo-dGTP and 2-OH-dATP to their corresponding monophosphates and thereby prevents the incorporation of oxidized nucleotides into DNA or RNA. The functional result is a reduction in down-stream mutations, or DNA damage, thus preventing cell death. Recent publications suggest that MTH1 is a non-essential enzyme in normal cells, but is required for the survival of cancer cells as a consequence of being subjected to high levels of oxidative stress; hence its relevance as an oncology target. Gad et al., 2014 The interesting target rationale combined with a perceived high chemical tractability of the target resulting from availability of x-ray crystallographic information for the protein, led to a decision to undertake, in parallel, target validation, assay development and drug discovery. This effort resulted in the structure-based design of potent, cell active MTH1 small molecule inhibitors. This poster will describe the discovery and optimization of these inhibitors, and their use to evaluate the potential of MTH1 as an oncology target. This work was complemented by parallel genetic studies. Pharmacodynamic evaluation of target engagement using proximal biomarkers will be presented, as will phenotypic responses across a range of cancer cell lines. Citation Format: Elisabetta Leo, Alessia Petrocchi, Jennifer Bardenhagen, Maria Alimova, Xi Shi, Connor Parker, Naphtali Reyna, Matthew Hamilton, Edward Felix, Andrzej Mazan, Christian Dillon, Faika Mseeh, Joseph R. Marszalek, Carlo Toniatti, Giulio Draetta, Phil Jones, Richard T. Lewis. Identification of potent, cell active MTH1 inhibitors and their use in target validation studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B48.

Functional mapping of multiple myeloma kinome with library of small molecule inhibitors

The functional role of individual kinases in multiple myeloma (MM) cells has been primarily interrogated using RNAi approaches, which are mechanistically dissimilar to the pharmacological action of small molecule inhibitors used in the clinic; and in stroma-free cultures, which don’t account for stroma-induced signaling events. To address these dissimilarities, we performed functional mapping of the kinome in 16 MM cell lines, cultured in the presence vs. absence of stromal cells, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure) targeting a total of 43 known primary oncogenic targets. The cell-autonomous activity of the various inhibitors was broadly potent for Aurora, PLK, and mTORC1/2 inhibitors (consistent with high proliferative rates of MM cell lines in vitro); modest to minimal for selective PDK1, PI3K (excluding those that also inhibit mTOR), and Akt inhibitors; virtually none for selective inhibitors of c-met, ALK, EGFR, HER2, c-kit, PDGFR, VEGFR, Flt3, FAK, Syk, Src, and BTK (even in cell lines with detectable transcripts for the respective kinases). We also observed modest activity for FGFR3 inhibitors against FGFR3expressing cell lines; and none for BRAF inhibitors in our BRAF wild-type cell lines (some of which had increased proliferation upon treatment; consistent with known adaptive activation of signaling through C-RAF). Testing 8 different MEK1/2 inhibitors identified lines with low, intermediate vs. high response; the latter being KRAS-mutant and BRAF-wild-type cells with high baseline p-ERK levels (e.g. due to MEK2-activating mutation in one cell line). Importantly, comparison of results obtained in the presence vs. absence of stroma, indicate heterogeneous effect of bone marrow stromal cells on MM cell response to inhibitors across different targets and cell lines, including stroma-induced resistance to the same kinase inhibitor for some cell lines vs. sensitization for others. Our studies demonstrate the feasibility of functional annotations of the kinome in MM using libraries of small-molecule kinase inhibitors in phenotypic assays against panels of cell lines. Our results provide insight into the relative role of different classes of kinases in MM, and how the genetic (e.g. MEK2 or BRAF mutation status) and microenvironmental context can influence the role of these kinases and possible future individualized administration of inhibitors against these kinases in MM. BP-032

Abstract 4126: CSF-1R inhibition blocks rhabdomyoscarcoma metastasis by polarizing macrophage differentiation

Abstract Colony Stimulating Factor 1 (CSF-1) is overexpressed by many tumors and predicts poor clinical outcome. CSF-1 expression results in the recruitment of CSF-1R (Fms)-expressing cells, which have been shown to suppress tumor immunity, thereby promoting cancer progression. PLX3397 is a novel, orally active small molecule inhibitor that selectively targets CSF-1R, KIT, and oncogenic FIT3. CSF-1R and KIT regulate the tumor, tumor microenvironment, and the pre-metastatic niche. However, it is unknown if targeting the myeloid CSF-1R expressing component of the tumor microenvironment regulates metastasis of pediatric tumors, such as rhabdomyosarcoma. Using the M3-9-M murine syngeneic orthotopic rhabdomyosarcoma model, we evaluated the effects of PLX3397 on primary tumor growth and metastatic progression. This highly metastatic rhabdomyosarcoma model has high infiltration of CSF-1R-expressing bone marrow-derived cells, which includes inflammatory monocytes, M2 macrophages, and myeloid-derived suppressor cells. Therefore, we hypothesized PLX3397 would have a direct effect on CSF-1R expressing M2 macrophages and inflammatory monocytes, resulting in decreased metastasis. Using both in vitro and in vivo models, we found that PLX3397 treatment decreased tumor-infiltrating M2 macrophages and skewed the differentiation of inflammatory monocytes toward M1 macrophages and development of immunocompetent mature dendritic cells. This resulted in impairment of spontaneous M3-9-M metastasis to secondary sites. In addition, we have found that the polarization of inflammatory monocytes reversed the T cell suppressive capability of tumor-derived myeloid cells. In conclusion, CSF-1R blockage with PLX3397 inhibited metastatic progression by decreasing the infiltration of M2 macrophages in the primary tumor and pre-metastatic niche. The polarization of inflammatory monocytes to dendritic cells restored the immune recognition and decreased metastasis. This data supports our rationale for testing PLX3397 in patients with high M2 infiltration and suggests value in this agent as an adjuvant setting for metastasis prevention in high-risk patients. Note: This abstract was not presented at the meeting. Citation Format: Justin Evans, Amber Giles, Caitlin Reid, Rosandra Kaplan. CSF-1R inhibition blocks rhabdomyoscarcoma metastasis by polarizing macrophage differentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4126. doi:10.1158/1538-7445.AM2015-4126

Discovery of a 1-isopropyltetrahydroisoquinoline derivative as an orally active N-type calcium channel blocker for neuropathic pain

N-type calcium channel blockade is a promising therapeutic approach for the treatment of neuropathic pain. Starting from lead compound (S)-1, we focused our optimization efforts on potency for N-type calcium channel inhibition and improvement of CYP inhibition profile. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-(1R)-(1-isopropyl-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone oxalate ((R)-5r) was identified as a novel orally active small-molecule N-type calcium channel inhibitor with reduced CYP inhibition liability. Oral administration of (R)-5r improved mechanical allodynia in a spinal nerve ligation model of neuropathic pain in rats with an ED50 value of 2.5mg/kg.

QSAR Study of the Inhibitors of the Acetyl-CoA Carboxylase 1 and 2 using Bayesian Regularized Genetic Neural Networks: A Comparative Study

Linear and non-linear quantitative structure-activity relationship (QSAR) models were presented for modeling and predicting anti-diabetic activities of a set of inhibitors of acetyl-CoA carboxylase 1 and 2 (ACC1 and ACC2). Different algorithms were utilized to choose the best variables among large numbers of descriptors and then these selected descriptors were used for non-linear (artificial neural network) and linear (multiple linear regression) modeling. The variable selection methods were consisted of stepwise-multiple linear regression (stepwise-MLR), successive projections algorithm (SPA), genetic algorithm-multiple linear regression (GA-MLR) and Bayesian regularized genetic neural networks (BRGNNs). The prediction abilities of the models were evaluated by Monte Carlo cross validation (MCCV) in variable selection and modeling steps. The results revealed that the best variables for describing the inhibition mechanism of ACC were among topological charge indices, radial distribution function, geometrical, and autocorrelation descriptors. The statistical parameters of R2 and root mean square error (RMSE) indicated that BRGNNs is superior for modeling the inhibitory activity of ACC modulators over the other methods. The sensitivity analysis together with the frequency of the selected molecular descriptors in this work can establish an understanding to the mechanism of ACC inhibitory activity of small molecules.

Preclinical Evaluation of Orally Bioavailable Small-Molecule Inhibitors of Complement Factor D As a Potential Treatment for Paroxysmal Nocturnal Hemoglobinuria

BACKGROUNDComplement factor D, a serine protease, plays an essential role in the activation of the alternative complement pathway and provides important amplification of the classical and lectin complement pathways. Cleavage of factor B by factor D generates C3 convertase that leads to opsonization of targeted surfaces with complement activation fragments and to the formation of the terminal complement complex (TCC); both events lead to cell lysis. Complement dysregulation underlies multiple hematological disorders including paroxysmal nocturnal hemoglobinuria (PNH), which is characterized by complement-mediated lysis of clonal populations of erythrocytes that lack glycophosphatidylinositol-anchored complement regulators. The current treatment for PNH is intravenous infusion of the anti-C5 monoclonal antibody eculizumab. Although eculizumab lessens intravascular hemolysis, it does not prevent opsonization of erythrocytes and subsequent extravascular hemolysis by immune cells. In contrast, factor D inhibitors are expected to inhibit both terminal complement pathway activation as well as opsonization and should, therefore, be well-positioned to potentially serve this unmet medical need. Herein, we present the preclinical evaluation of our small-molecule inhibitors of factor D including potency, off-target activities, metabolism, and pharmacokinetic properties.METHODS & RESULTS: Initial inhibitors were discovered through laboratory and virtual screening efforts. A high-resolution (1.5 Å) X-ray structure of an early proprietary inhibitor co-crystallized with factor D aided our optimization campaign that culminated in compounds with IC50 values below 100 nM in biochemical protease assays using natural and non-specific substrates. The potent inhibitory effect of the compounds on factor D protease activity translated to inhibition of cell lysis in an alternative pathway hemolytic assay with EC50 values as low as single-digit nM. These small molecules are highly selective for factor D and displayed no significant inhibitory effect on a panel of human serine proteases. In accordance with the high selectivity, the inhibitors showed minimal cellular toxicity, no effect on multiple human receptor-ligand interactions, and no inhibition of hERG potassium channel current. Finally, oral and intravenous administration of selected lead compounds to preclinical animal species showed pharmacokinetic properties that suggest their suitability for oral dosing in humans. CONCLUSIONSWe have discovered highly active small-molecule inhibitors of factor D that demonstrate oral bioavailability and low off-target activities. These key attributes, and the potential to inhibit both intravascular and extravascular hemolysis through inhibition of both terminal complement pathway and opsonization, position these inhibitors as promising development candidates for the oral treatment of PNH. DisclosuresWiles:Achillion Pharmaceuticals: Employment. Podos:Achillion Pharmaceuticals : Employment. Thanassi:Achillion Pharmaceuticals: Employment. Phadke:Achillion Pharmaceuticals: Employment. Gadhachanda:Achillion Pharmaceuticals: Employment. Pais:Achillion Pharmaceuticals: Employment. Hashimoto:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Chen:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Agarwal:Achillion Pharmaceuticals: Employment. Rivera:Achillion Pharmaceuticals: Employment. Elliot:Achillion Pharmaceuticals: Employment. Marlor:Achillion Pharmaceuticals: Employment. Zhang:Achillion Pharmaceuticals: Employment. Deshpande:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment.

Abstract 1322: Novel CDK4/6 inhibitors provide robust antitumor activity in a murine model of luminal breast cancer

Abstract Introduction: Selective CDK4/6 inhibitors have emerged as attractive antineoplastic agents due to the importance of CDK4/6 activity in regulating cell proliferation. We have identified a series of orally available, potent and selective dual CDK4/6 inhibitors (G1T100182, G1T100183, G1T101551). Preclinical characterization of these compounds indicates that they inhibit CDK4 and CDK6 with an IC50 of 0.7-1.0 nM and 5-6 nM, respectively. In Rb competent tumor cells, these compounds potently inhibit Rb phosphorylation resulting in a G1 arrest. Here we assess the in vivo efficacy of these compounds in a genetically engineered mouse model of luminal breast cancer. Methods: MMTV-Neu mice were housed and therapeutic response assessed in the UNC Lineberger Comprehensive Cancer Center's Mouse Phase I Unit (MP1U). Compounds were supplied by G1 Therapeutics, Inc. Tumors were serially assessed weekly using caliper measurements. Therapeutic intervention began once tumors reached 40-64mm3 and continued for 28 consecutive days. Tumor volume was calculated using the formula ((Width2) x Length)/2). All compounds were administered orally via medicated diets. RECIST criteria were used to assess objective response rates. Results: Significant objective responses were noted in mice treated with G1T100182, G1T100183, G1T101551. Treatments produced modest reversible myelosuppression, but where otherwise well tolerated with no other clinical signs of toxicity or deaths associated with toxicity. A linear regression t-test was used to determine statistical significance of tumor volume changes over time. All treatment cohorts demonstrated statistically significant improvement when compared to un-treated mice; G1T100182, p<0.0001; G1T100183, p=0.0001; G1T101551, p<0.0001. Conclusions: In summary, we have identified a series of orally active, potent, and selective small molecule inhibitors of CDK4/6 that may provide therapeutic benefit to cancer patients with tumors that have functional Rb protein. Complete ResponsePartial ResponseStable DiseaseProgressive DiseaseORR % (CR+PR+SD/PD)Mean % Change in Tumor VolNo Treatment00090 (0/9)500G1T1001821510100 (7/0)-70G1T100183141185 (6/1)-67G1T1015511250100 (8/0)-40 Citation Format: Patrick J. Roberts, David B. Darr, John E. Bisi, Jay C. Strum. Novel CDK4/6 inhibitors provide robust antitumor activity in a murine model of luminal breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1322. doi:10.1158/1538-7445.AM2014-1322

Oral epidermal growth factor receptor tyrosine kinase inhibitors for the treatment of non-small cell lung cancer: comparative pharmacokinetics and drug-drug interactions.

The development of orally active small molecule inhibitors of the epidermal growth factor receptor (EGFR) has led to new treatment options for non-small cell lung cancer (NSCLC). Patients with activating mutations of the EGFR gene show sensitivity to, and clinical benefit from, treatment with EGFR tyrosine kinase inhibitors (EGFR-TKls). First generation reversible ATP-competitive EGFR-TKls, gefitinib and erlotinib, are effective as first, second-line or maintenance therapy. Despite initial benefit, most patients develop resistance within a year, 50-60% of cases being related to the appearance of a T790M gatekeeper mutation. Newer, irreversible EGFR-TKls - afatinib and dacomitinib - covalently bind to and inhibit multiple receptors in the ErbB family (EGFR, HER2 and HER4). These agents have been mainly evaluated for first-line treatment but also in the setting of acquired resistance to first-generation EGFR-TKls. Afatinib is the first ErbB family blocker approved for patients with NSCLC with activating EGFR mutations; dacomitinib is in late stage clinical development. Mutant-selective EGFR inhibitors (AZD9291, CO-1686, HM61713) that specifically target the T790M resistance mutation are in early development. The EGFR-TKIs differ in their spectrum of target kinases, reversibility of binding to EGFR receptor, pharmacokinetics and potential for drug-drug interactions, as discussed in this review. For the clinician, these differences are relevant in the setting of polymedicated patients with NSCLC, as well as from the perspective of innovative anticancer drug combination strategies.

Preclinical characterization of the CDK4/6 inhibitor LY2835219: in-vivo cell cycle-dependent/independent anti-tumor activities alone/in combination with gemcitabine.

SummaryThe G1 restriction point is critical for regulating the cell cycle and is controlled by the Rb pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). This pathway is important because of its inactivation in a majority of human tumors. Transition through the restriction point requires phosphorylation of retinoblastoma protein (Rb) by CDK4/6, which are highly validated cancer drug targets. We present the identification and characterization of a potent CDK4/6 inhibitor, LY2835219. LY2835219 inhibits CDK4 and CDK6 with low nanomolar potency, inhibits Rb phosphorylation resulting in a G1 arrest and inhibition of proliferation, and its activity is specific for Rb-proficient cells. In vivo target inhibition studies show LY2835219 is a potent inhibitor of Rb phosphorylation, induces a complete cell cycle arrest and suppresses expression of several Rb-E2F-regulated proteins 24 hours after a single dose. Oral administration of LY2835219 inhibits tumor growth in human tumor xenografts representing different histologies in tumor-bearing mice. LY2835219 is effective and well tolerated when administered up to 56 days in immunodeficient mice without significant loss of body weight or tumor outgrowth. In calu-6 xenografts, LY2835219 in combination with gemcitabine enhanced in vivo antitumor activity without a G1 cell cycle arrest, but was associated with a reduction of ribonucleotide reductase expression. These results suggest LY2835219 may be used alone or in combination with standard-of-care cytotoxic therapy. In summary, we have identified a potent, orally active small-molecule inhibitor of CDK4/6 that is active in xenograft tumors. LY2835219 is currently in clinical development.Electronic supplementary materialThe online version of this article (doi:10.1007/s10637-014-0120-7) contains supplementary material, which is available to authorized users.

Cathepsin S inhibition lowers blood glucose levels in mice.

Cathepsin S (CatS) belongs to a family of proteases that have been implicated in several disease processes. We previously identified CatS as a protein that is markedly overexpressed in adipose tissue of obese individuals and downregulated after weight loss and amelioration of glycaemic status induced by gastric bypass surgery. This prompted us to test whether the protease contributes to the pathogenesis of type 2 diabetes using mouse models with CatS inactivation. CatS knockout mice and wild-type mice treated with orally active small-molecule CatS inhibitors were fed chow or high-fat diets and explored for change in glycaemic status. CatS deletion induced a robust reduction in blood glucose, which was preserved in diet-induced obesity and with ageing and was recapitulated with CatS inhibition in obese mice. In vivo testing of glucose tolerance, insulin sensitivity and glycaemic response to gluconeogenic substrates revealed that CatS suppression reduced hepatic glucose production despite there being no improvement in insulin sensitivity. This phenotype relied on downregulation of gluconeogenic gene expression in liver and a lower rate of hepatocellular respiration. Mechanistically, we found that the protein 'regulated in development and DNA damage response 1' (REDD1), a factor potentially implicated in reduction of respiratory chain activity, was overexpressed in the liver of mice with CatS deficiency. Our results revealed an unexpected metabolic effect of CatS in promoting pro-diabetic alterations in the liver. CatS inhibitors currently proposed for treatment of autoimmune diseases could help to lower hepatic glucose output in obese individuals at risk for type 2 diabetes.

Structure-Based Design, Synthesis and Validation of CD4-Mimetic Small Molecule Inhibitors of HIV-1 Entry: Conversion of a Viral Entry Agonist to an Antagonist

ConspectusThis Account provides an overview of a multidisciplinary consortium focused on structure-based strategies to devise small molecule antagonists of HIV-1 entry into human T-cells, which if successful would hold considerable promise for the development of prophylactic modalities to prevent HIV transmission and thereby alter the course of the AIDS pandemic.Entry of the human immunodeficiency virus (HIV) into target T-cells entails an interaction between CD4 on the host T-cell and gp120, a component of the trimeric envelope glycoprotein spike on the virion surface. The resultant interaction initiates a series of conformational changes within the envelope spike that permits binding to a chemokine receptor, formation of the gp41 fusion complex, and cell entry. A hydrophobic cavity at the CD4–gp120 interface, defined by X-ray crystallography, provided an initial site for small molecule antagonist design. This site however has evolved to facilitate viral entry. As such, the binding of prospective small molecule inhibitors within this gp120 cavity can inadvertently trigger an allosteric entry signal.Structural characterization of the CD4–gp120 interface, which provided the foundation for small molecule structure-based inhibitor design, will be presented first. An integrated approach combining biochemical, virological, structural, computational, and synthetic studies, along with a detailed analysis of ligand binding energetics, revealed that modestly active small molecule inhibitors of HIV entry can also promote viral entry into cells lacking the CD4 receptor protein; these competitive inhibitors were termed small molecule CD4 mimetics. Related congeners were subsequently identified with both improved binding affinity and more potent viral entry inhibition. Further assessment of the affinity-enhanced small molecule CD4 mimetics demonstrated that premature initiation of conformational change within the viral envelope spike, prior to cell encounter, can lead to irreversible deactivation of viral entry machinery. Related congeners, which bind the same gp120 site, possess different propensities to elicit the allosteric response that underlies the undesired enhancement of CD4-independent viral entry.Subsequently, key hotspots in the CD4–gp120 interface were categorized using mutagenesis and isothermal titration calorimetry according to the capacity to increase binding affinity without triggering the allosteric signal. This analysis, combined with cocrystal structures of small molecule viral entry agonists with gp120, led to the development of fully functional antagonists of HIV-1 entry. Additional structure-based design exploiting two hotspots followed by synthesis has now yielded low micromolar inhibitors of viral entry.

Differential response of orthologous l,l-diaminopimelate aminotransferases (DapL) to enzyme inhibitory antibiotic lead compounds

l,l-Diaminopimelate aminotransferase (DapL) is an enzyme required for the biosynthesis of meso-diaminopimelate (m-DAP) and l-lysine (Lys) in some bacteria and photosynthetic organisms. m-DAP and Lys are both involved in the synthesis of peptidoglycan (PG) and protein synthesis. DapL is found in specific eubacterial and archaeal lineages, in particular in several groups of pathogenic bacteria such as Leptospira interrogans (LiDapL), the soil/water bacterium Verrucomicrobium spinosum (VsDapL) and the alga Chlamydomonas reinhardtii (CrDapL). Here we present the first comprehensive inhibition study comparing the kinetic activity of DapL orthologs using previously active small molecule inhibitors formerly identified in a screen with the DapL of Arabidopsis thaliana (AtDapL), a flowering plant. Each inhibitor is derived from one of four classes with different central structural moieties: a hydrazide, a rhodanine, a barbiturate, or a thiobarbituate functionality. The results show that all five compounds tested were effective at inhibiting the DapL orthologs. LiDapL and AtDapL showed similar patterns of inhibition across the inhibitor series, whereas the VsDapL and CrDapL inhibition patterns were different from that of LiDapL and AtDapL. CrDapL was found to be insensitive to the hydrazide (IC50 >200μM). VsDapL was found to be the most sensitive to the barbiturate and thiobarbiturate containing inhibitors (IC50 ∼5μM). Taken together, the data shows that the homologs have differing sensitivities to the inhibitors with IC50 values ranging from 4.7 to 250μM. In an attempt to understand the basis for these differences the four enzymes were modeled based on the known structure of AtDapL. Overall, it was found that the enzyme active sites were conserved, although the second shell of residues close to the active site were not. We conclude from this that the altered binding patterns seen in the inhibition studies may be a consequence of the inhibitors forming additional interactions with residues proximal to the active site, or that the inhibitors may not act by binding to the active site. Compounds that are specific for DapL could be potential biocides (antibiotic, herbicide or algaecide) that are nontoxic to animals since animals do not contain the enzymes necessary for PG or Lys synthesis. This study provides important information to expand our current understanding of the structure/activity relationship of DapL and putative inhibitors that are potentially useful for the design and or discovery of novel biocides.

Discovery of a Novel Orally Active Small-Molecule gp130 Inhibitor for the Treatment of Ovarian Cancer

Interleukin (IL)-6 and Stat3 play key roles in ovarian cancer progression. However, the role of glycoprotein 130 (gp130), the signal transducer of this signaling axis, is not well-established. Currently, there are no small-molecule inhibitors of gp130 under clinical development. In this study, we show that gp130 is an attractive drug target in ovarian cancer due to its role in promoting cancer progression via the activation of its downstream Stat3 signaling. We also present preclinical studies of SC144, the first-in-class orally active small-molecule gp130 inhibitor. SC144 shows greater potency in human ovarian cancer cell lines than in normal epithelial cells. SC144 binds gp130, induces gp130 phosphorylation (S782) and deglycosylation, abrogates Stat3 phosphorylation and nuclear translocation, and further inhibits the expression of downstream target genes. In addition, SC144 shows potent inhibition of gp130 ligand-triggered signaling. Oral administration of SC144 delays tumor growth in a mouse xenograft model of human ovarian cancer without significant toxicity to normal tissues.

Abstract 5513: Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase (PDI) for ovarian cancer treatment.

Abstract Protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) chaperone protein, catalyzes disulfide bond breakage, formation and rearrangement. The effect of PDI inhibition on ovarian cancer progression is not yet clear, and there is a need for potent, selective and safe small-molecule inhibitors of PDI. Here, we report a new class of novel propynoic acid carbamoyl methyl amides (PACMAs) that are active against a panel of human ovarian cancer cell lines. Using fluorescent derivatives, 2D gel electrophoresis, and mass spectrometry, we established that PACMA 31, one of the most active analogues, acts as an irreversible small-molecule inhibitor of PDI, forming a covalent bond with PDI's active site cysteines. We also demonstrated that PDI activity is essential for the survival and proliferation of human ovarian cancer cells. In vivo, PACMA 31 showed tumor targeting ability and significantly suppressed ovarian tumor growth without causing toxicity to normal tissues. These irreversible small-molecule PDI inhibitors represent a new approach for the development of targeted anticancer agents for ovarian cancer therapy, and can also serve as useful probes for investigating the biology of PDI-implicated pathways. Citation Format: Shili Xu, Alexey N. Butkevich, Roppei Yamada, Yu Zhou, Bikash Debnath, Roger Duncan, Ebrahim Zandi, Nicos A. Petasis, Nouri Neamati. Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase (PDI) for ovarian cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5513. doi:10.1158/1538-7445.AM2013-5513

In silico Screening for Identification of Novel Aurora Kinase Inhibitors by Molecular Docking, Dynamics Simulations and Ligand-Based Hypothesis Approaches

Aurora kinase family members are involved in a wide variety of cell cycle events including centrosome separation, cytokinesis, kinetochore formation, spindle assembly, chromosomal segregation and microtubule dynamics. Typically, dysfunction of Aurora proteins is associated with aneuploidy, cell death and mitotic arrest, leading to tumorigenesis. This spurred a vast interest to identify pharmacologically active small-molecule inhibitors of Aurora proteins. In this study, we isolated four novel inhibitors through virtual screening and docking analyses. These hits were subsequently characterized by molecular dynamic simulations to monitor their binding stabilities at ATP binding site. To aid characterizing novel and more potent inhibitors for Aurora kinases, we explored selective features of our ligand dataset by ligand-based pharmacophore modeling approach. The best pharmacophore model was then employed for performing virtual screenings of libraries isolated from Princeton and Uorsy databases. On the basis of common pharmacophore features, Lipinski’s rule of five, absorption, distribution, metabolism and elimination properties, hits were short listed and refined by molecular dockings. Finally, the selected compounds were validated on the basis of binding capabilities, consensus scoring and activity values. We propose that the novel inhibitors described in this study may warrant characterization in designing active lead for clinical studies that may serve as anticancer drugs in future.

Targeting the IGF-IR system in pituitary tumors in vitro: antiproliferative action & pitfalls

IGF-I receptors (IGF-IR) and their aberrant signaling cascade contribute to the pathogenesis of several solid cancers. Pituitary adenomas express IGF-IR and present with overactivation of the IGF-IR pathway. The aim of the study was to identify the potential antiproliferative action of small molecule inhibitors targeting IGF-IR signaling cascade in pituitary adenomas. The IGF-IR tyrosine kinase inhibitor NVP-TAE226, the PI3K/mTOR inhibitor NVP_BEZ235 and the allosteric mTOR inhibitor everolimus were used on acromegalic (n=11) and nonfunctioning pituitary adenomas (NFPA; n=15) in primary cell culture. Changes in cell viability were determined by thymidine incorporation and a nonradioactive colorimetric assay. TAE226at the 100nM concentration suppressed cell viability by more than 20% in 9 out of 15 NFPA and in 10 acromegalic tumors, while at 10nM it was efficacious in 8 NFPA and 4 acromegalic tumors. Inhibiting the PI3K pathway in the same tumors, using BEZ235 suppressed cell viability at 10nM concentration in all NFPA and in two acromegalic tumors. In contrast, mTOR inhibition with everolimus was efficacious in all but one acromegalic cases, but in only 4 out of 15 NFPA. IGF-IR inhibition in acromegalic tumors in vitro was accompanied by increased GH secretion, which was evidenced when GH got normalized to cell viability assay counts. Similar GH increase was observed after mTOR but not in the same extend after PI3K inhibition (P=0.043), indicating a previously unsuspected role for the mTOR pathway on GH synthesis. Altogether, these data indicate a tumor specific dependency to the different IGF-IR signaling branches, with NFPA being sensitive to PI3K inhibition and acromegalic tumors to the mTOR inhibition. Targeting IGF-IR pathways have a potential as antiproliferative agents for the treatment of NFPA. In contrast their potent antiproliferative action in acromegalic tumors is compromised by the concomitant increase in GH secretion.