Discovery of OICR12694: A Novel, Potent, Selective, and Orally Bioavailable BCL6 BTB Inhibitor.

B cell lymphoma 6 (BCL6), a highly regulated transcriptional repressor, is deregulated in several forms of non-Hodgkin lymphoma (NHL), most notably in diffuse large B-cell lymphoma (DLBCL). The activities of BCL6 are dependent on protein-protein interactions with transcriptional co-repressors. To find new therapeutic interventions addressing the needs of patients with DLBCL, we initiated a program to identify BCL6 inhibitors that interfere with co-repressor binding. A virtual screen hit with binding activity in the high micromolar range was optimized by structure-guided methods, resulting in a novel and highly potent inhibitor series. Further optimization resulted in the lead candidate 58 (OICR12694/JNJ-65234637), a BCL6 inhibitor with low nanomolar DLBCL cell growth inhibition and an excellent oral pharmacokinetic profile. Based on its overall favorable preclinical profile, OICR12694 is a highly potent, orally bioavailable candidate for testing BCL6 inhibition in DLBCL and other neoplasms, particularly in combination with other therapies.

Discovery of Potent and Orally Bioavailable Pyridine N-Oxide-Based Factor XIa Inhibitors through Exploiting Nonclassical Interactions.

Activated factor XI (FXIa) inhibitors are promising novel anticoagulants with low bleeding risk compared with current anticoagulants. The discovery of potent FXIa inhibitors with good oral bioavailability has been challenging. Herein, we describe our discovery effort, utilizing nonclassical interactions to improve potency, cellular permeability, and oral bioavailability by enhancing the binding while reducing polar atoms. Beginning with literature-inspired pyridine N-oxide-based FXIa inhibitor 1, the imidazole linker was first replaced with a pyrazole moiety to establish a polar C-H···water hydrogen-bonding interaction. Then, structure-based drug design was employed to modify lead molecule 2d in the P1' and P2' regions, with substituents interacting with key residues through various nonclassical interactions. As a result, a potent FXIa inhibitor 3f (Ki = 0.17 nM) was discovered. This compound demonstrated oral bioavailability in preclinical species (rat 36.4%, dog 80.5%, and monkey 43.0%) and displayed a dose-dependent antithrombotic effect in a rabbit arteriovenous shunt model of thrombosis.

BCL10 Mutations Define Distinct Dependencies Guiding Precision Therapy for DLBCL.

Activated B cell-like diffuse large B-cell lymphomas (ABC-DLBCL) have unfavorable outcomes and chronic activation of CARD11-BCL10-MALT1 (CBM) signal amplification complexes that form due to polymerization of BCL10 subunits, which is affected by recurrent somatic mutations in ABC-DLBCLs. Herein, we show that BCL10 mutants fall into at least two functionally distinct classes: missense mutations of the BCL10 CARD domain and truncation of its C-terminal tail. Truncating mutations abrogated a motif through which MALT1 inhibits BCL10 polymerization, trapping MALT1 in its activated filament-bound state. CARD missense mutations enhanced BCL10 filament formation, forming glutamine network structures that stabilize BCL10 filaments. Mutant forms of BCL10 were less dependent on upstream CARD11 activation and thus manifested resistance to BTK inhibitors, whereas BCL10 truncating but not CARD mutants were hypersensitive to MALT1 inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL, and further precision can be achieved by selecting therapy based on specific biochemical effects of distinct mutation classes. SIGNIFICANCE: ABC-DLBCLs feature frequent mutations of signaling mediators that converge on the CBM complex. We use structure-function approaches to reveal that BCL10 mutations fall into two distinct biochemical classes. Both classes confer resistance to BTK inhibitors, whereas BCL10 truncations confer hyperresponsiveness to MALT1 inhibitors, providing a road map for precision therapies in ABC-DLBCLs. See related commentary by Phelan and Oellerich, p. 1844. This article is highlighted in the In This Issue feature, p. 1825.

Discovery and optimization of a series of small-molecule allosteric inhibitors of MALT1 protease.

We describe a series of potent and highly selective small-molecule MALT1 inhibitors, optimized from a High-Throughput Screening hit. Advanced analogues such as compound 40 show high potency (IC50: 0.01 µM) in a biochemical assay measuring MALT1 enzymatic activity, as well as in cellular assays: Jurkat T cell activation (0.05 µM) and IL6/10 secretion (IC50: 0.10/0.06 µM) in the TMD8 B-cell lymphoma line. Compound 40 also inhibited cleavage of the MALT1 substrate RelB (IC50: 0.10 µM). Mechanistic enzymology results suggest that these compounds bind to the known allosteric site of the protease.

Design, synthesis and structure activity relationships of indazole and indole derivatives as potent glucagon receptor antagonists.

A novel series of indazole/indole derivatives were discovered as glucagon receptor (GCGR) antagonists through scaffold hopping based on two literature leads: MK-0893 and LY-2409021. Further structure-activity relationship (SAR) exploration and optimization led to the discovery of multiple potent GCGR antagonists with excellent pharmacokinetic properties in mice and rats, including low systemic clearance, long elimination half-life, and good oral bioavailability. These potent GCGR antagonists could be used for potential treatment of type II diabetes.

Design and synthesis of a series of bioavailable fatty acid synthase (FASN) KR domain inhibitors for cancer therapy.

We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC50 = 28 nM) that effectively inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement.

Thioredoxin 1 is associated with the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synoviocytes.

We aimed to investigate the possible effects of thioredoxin 1 (Trx1) on the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) and elucidate the possible mechanisms involved. We investigated the distribution and expression of Trx1 in synovial tissues from RA and osteoarthritis (OA) patients by immunohistochemistry and real-time polymerase chain reaction (RT-PCR) analyses. RA-FLSs were isolated and cultured under normoxic (21% oxygen) or hypoxic (3% oxygen) concentrations. Transfection of Trx1-siRNAs and a Trx1 overexpression construct was conducted to manipulate the expression of Trx1. Protein expression was detected by Western blot. Doxorubicin (Adriamycin, ADR) was used to induce apoptosis. LY-294002 was used for the inhibition of PI3K-Akt. Cell proliferation and apoptosis were determined by MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay and flow cytometry, respectively. The mRNA and protein expression of Trx1 in RA tissues was higher than that in OA tissues. The expression levels of Trx1 and cell proliferation in RA-FLSs were increased under hypoxia in comparison to those under normoxia. In hypoxia, downregulation of Trx1 significantly suppressed FLS proliferation, and the expression of PI3Kp85, phospho-Akt, and Bcl-2, while notably increased FLS apoptosis and the expression of active Caspase3 and Bax. In normoxia, Trx1 overexpression promoted the FLS proliferation and the expression of PI3Kp85, phospho-Akt, and Bcl-2, but inhibited FLS apoptosis and the expression of active Caspase3 and Bax in FLSs. Such effects were partially repressed by LY-294002 treatment. Trx1 may play an important role in regulating the proliferation and apoptosis of RA-FLSs by modulating PI3K-Akt activation.

Glucose-6-Phosphate Isomerase (G6PI) Mediates Hypoxia-Induced Angiogenesis in Rheumatoid Arthritis.

The higher level of Glucose-6-phosphate isomerase (G6PI) has been found in both synovial tissue and synovial fluid of rheumatoid arthritis (RA) patients, while the function of G6PI in RA remains unclear. Herein we found the enrichment of G6PI in microvascular endothelial cells of synovial tissue in RA patients, where a 3% O2 hypoxia environment has been identified. In order to determine the correlation between the high G6PI level and the low oxygen concentration in RA, a hypoxia condition (~3% O2) in vitro was applied to mimic the RA environment in vivo. Hypoxia promoted cellular proliferation of rheumatoid arthritis synovial fibroblasts (RASFs), and induced cell migration and angiogenic tube formation of human dermal microvascular endothelial cells (HDMECs), which were accompanied with the increased expression of G6PI and HIF-1α. Through application of G6PI loss-of-function assays, we confirmed the requirement of G6PI expression for those hypoxia-induced phenotype in RA. In addition, we demonstrated for the first time that G6PI plays key roles in regulating VEGF secretion from RASFs to regulate the hypoxia-induced angiogenesis in RA. Taken together, we demonstrated a novel pathway regulating hypoxia-induced angiogenesis in RA mediated by G6PI.

Discovery of novel potent imidazo[1,2-b]pyridazine PDE10a inhibitors.

Design and optimization of a novel series of imidazo[1,2-b]pyridazine PDE10a inhibitors are described. Compound 31 displays excellent pharmacokinetic properties and was also evaluated as an insulin secretagogue in vitro and in vivo.

Functional role of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in NSCLC.

Eukaryotic translation initiation factor 4 gamma 1(EIF4G1) is related to tumorigenesis and tumor progression. However, its role and the underlying mechanisms in the regulation of tumor development in non-small cell lung cancers (NSCLC) remain largely unknown. Here we report that the levels of EIF4G1 expression are much higher in NSCLC cell lines and tumor tissues than those in the normal lung cells and adjacent normal tissues from the same patients. Using shRNA to knock down EIF4G1 expression stably, we found EIF4G1 required for NSCLC cell proliferation, anchorage-independent growth, migration and invasion. Furthermore, silencing of EIF4G1 induces NSCLC cell apoptosis and causes G0/G1 cell cycle arrest. To identify the partner protein network of EIF4G1 in NSCLC cells, we found that Ubiquitin-specific protease 10 (USP10) can directly interacts with EIF4G1, while acting as a negative regulator for EIF4G1-mediated functions. Together, our results indicate that EIF4G1 functions as an oncoprotein during NSCLC development, which may represent a novel and promising therapeutic target in lung cancer.

Glucose-6-phosphate isomerase promotes the proliferation and inhibits the apoptosis in fibroblast-like synoviocytes in rheumatoid arthritis.

INTRODUCTION: Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to investigate the role of glucose 6-phosphate isomerase (GPI) in the proliferation of RA-FLS. METHODS: The distribution of GPI in synovial tissues from RA and osteoarthritis (OA) patients was examined by immunohistochemical analysis. FLS were isolated and cultured, cellular GPI level was detected by real-time polymerase chain reaction (PCR) and Western blot analysis, and secreted GPI was detected by Western blot and enzyme-linked immunosorbent assay (ELISA). Doxorubicin (Adriamycin, ADR) was used to induce apoptosis. Cell proliferation was determined by MTS assay. Flow cytometry was used to detect cell cycle and apoptosis. Secreted pro-inflammatory cytokines were measured by ELISA. RESULTS: GPI was abundant in RA-FLS and was an autocrine factor of FLS. The proliferation of both RA and OA FLS was increased after GPI overexpression, but was decreased after GPI knockdown. Meanwhile, exogenous GPI stimulated, while GPI antibody inhibited, FLS proliferation. GPI positively regulated its receptor glycoprotein 78 and promoted G1/S phase transition via extracellular regulated protein kinases activation and Cyclin D1 upregulation. GPI inhibited ADR-induced apoptosis accompanied by decreased Fas and increased Survivin in RA FLS. Furthermore, GPI increased the secretion of tumor necrosis factor-α and interleukin-1ß by FLS. CONCLUSIONS: GPI plays a pathophysiologic role in RA by stimulating the proliferation, inhibiting the apoptosis, and increasing pro-inflammatory cytokine secretion of FLS.

Discovery and clinical evaluation of 1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-[2,2-difluoro-2-phenylethylamino]pyrazinone (RWJ-671818), a thrombin inhibitor with an oxyguanidine P1 motif.

We have identified RWJ-671818 (8) as a novel, low molecular weight, orally active inhibitor of human alpha-thrombin (K(i) = 1.3 nM) that is potentially useful for the acute and chronic treatment of venous and arterial thrombosis. In a rat deep venous thrombosis model used to assess antithrombotic efficacy, oral administration of 8 at 30 and 50 mg/kg reduced thrombus weight by 87 and 94%, respectively. In an anesthetized rat antithrombotic model, where electrical stimulation of the carotid artery created a thrombus, 8 prolonged occlusion time 2- and 3-fold at 0.1 and 1.0 mg/kg, i.v., respectively, and more than doubled activated clotting time and activated partial thromboplastin time at the higher dose. This compound had excellent oral bioavailability of 100% in dogs with an estimated half-life of approximately 3 h. On the basis of its noteworthy preclinical data, 8 was advanced into human clinical trials and successfully progressed through phase 1 studies.

[Association of the PADI4 gene polymorphism and HLA-DRB1 shared epitope alleles with rheumatoid arthritis].

OBJECTIVE: To investigate the association of single nucleotide polymorphisms (SNPs) of the peptidylarginine deiminase IV (PADI4) and HLA-DRB1 shared epitope (SE) alleles with rheumatoid arthritis(RA) in a Chinese population. METHODS: Four exonic SNPs of the PADI4 gene (PADI 4_89*A/G, PADI 4_90*C/T, PADI 4_92*C/G and PADI 4_104*C/T) were genotyped in 67 unrelated patients with RA and 81 healthy controls, using cDNA sequencing and T vector cloning. HLA-DRB 1*01, *04 and *10 subtypes were determined by polymerase chain reaction with sequence specific primers (PCR-SSP). RESULTS: The distributions of the 4 SNPs were different in the two groups, and increased RA susceptibility was significantly associated with the minor alleles of PADI 4_89*G (P was 0.023), PADI 4_90*T (P was 0.004), PADI 4_104*T (P was 0.003), and the haplotypes carrying the 4 minor alleles (P was 0.008). HLA-DRB1 SE alleles are composed of HLA-DRB 1*0101, *0102, *0401, *0404, *0405, *0408, *0409, *0410 and *1001. Individuals carrying the SE alleles were associated with increased RA susceptibility (P was 0.002). Individuals carrying both the SE alleles and minor alleles of the 4 SNPs were more susceptible to RA than individuals carrying neither the minor SNP alleles nor the SE alleles. CONCLUSION: The PADI4 SNPs and haplotypes are associated with RA susceptibility in Chinese. HLA-DRB1 shared epitope is also an important risky factor for RA. There may exist certain synergistic effect between the PADI4 minor alleles and the HLA-DRB1 shared epitope.

Orally efficacious thrombin inhibitors with cyanofluorophenylacetamide as the P2 motif.

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K(i)=1.2nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.

7-fluoroindazoles as potent and selective inhibitors of factor Xa.

We have developed a novel series of potent and selective factor Xa inhibitors that employ a key 7-fluoroindazolyl moiety. The 7-fluoro group on the indazole scaffold replaces the carbonyl group of an amide that is found in previously reported factor Xa inhibitors. The structure of a factor Xa cocrystal containing 7-fluoroindazole 51a showed the 7-fluoro atom hydrogen-bonding with the N-H of Gly216 (2.9 A) in the peptide backbone. Thus, the 7-fluoroindazolyl moiety not only occupied the same space as the carbonyl group of an amide found in prior factor Xa inhibitors but also maintained a hydrogen bond interaction with the protein's beta-sheet domain. The structure-activity relationship for this series was consistent with this finding, as the factor Xa inhibitory potencies were about 60-fold greater (DeltaDelta G approximately 2.4 kcal/mol) for the 7-fluoroindazoles 25a and 25c versus the corresponding indazoles 25b and 25d. Highly convergent synthesis of these factor Xa inhibitors is also described.

2-(2-Chloro-6-fluorophenyl)acetamides as potent thrombin inhibitors.

2-(2-Chloro-6-fluorophenyl)acetamides having 2,2-difluoro-2-aryl/heteroaryl-ethylamine P3 and oxyguanidine P1 substituents are potent thrombin inhibitors (K(i)=0.9-33.9 nM). 2-(5-Chloro-pyridin-2-yl)-2,2-difluoroethylamine was the best P3 substituent, yielding the most potent inhibitor (K(i)=0.7 nM). Replacing the P3 heteroaryl group with a phenyl ring or replacing the difluoro substitution with dimethyl or cyclopropyl groups in the linker reduced the affinity for thrombin significantly. The aminopyridine P1s also provided an increase in potency.

Exploration of potential prodrugs of RWJ-445167, an oxyguanidine-based dual inhibitor of thrombin and factor Xa.

Compound 2 (RWJ-445167; 3DP-10017), a dual inhibitor of thrombin and factor Xa, was advanced into human clinical studies. However, its oral bioavailability in humans proved to be below acceptable limits. To address this issue, we explored a prodrug approach involving numerous guanidine derivatives. Prodrug candidates of classes A (carbamate derivatives), B (imidate derivatives), and C (alkyl and acyl derivatives), compounds 3-6, were synthesized and evaluated for anticoagulant activity at 2 h after oral administration to rats. In comparison to the parent drug (2), little worthwhile improvement was observed for the prodrug candidates.

Novel 1,4-benzodiazepine-2,5-diones as Hdm2 antagonists with improved cellular activity.

The disruption of the p53-Hdm2 protein-protein interaction induces cell growth arrest and apoptosis. We have identified the 1,4-benzodiazepine-2,5-dione scaffold as a suitable template for inhibiting this interaction by binding to the Hdm2 protein. Several compounds have been made with improved potency, solubility, and cell-based activities.

Substituted 1,4-benzodiazepine-2,5-diones as alpha-helix mimetic antagonists of the HDM2-p53 protein-protein interaction.

Small molecule antagonists of protein-protein interactions represent a particular challenge for pharmaceutical discovery. One approach to finding molecules that can disrupt these interactions is to seek mimics of common protein structure motifs. We present an analysis of how molecules based on the 1,4-benzodiazepine-2,5-dione scaffold serve to mimic the side-chains presented by the hydrophobic face of two turns of an alpha-helix derived from the tumor suppressor protein p53, and thus antagonize the HDM2-p53 protein-protein binding interaction.

Enantiomerically pure 1,4-benzodiazepine-2,5-diones as Hdm2 antagonists.

The 1,4-benzodiazepine-2,5-dione is a suitable template to disrupt the interaction between p53 and Hdm2. The development of an enantioselective synthesis disclosed the stereochemistry of the active enantiomer. An in vitro p53 peptide displacement assay identified active compounds. These activities were confirmed in several cell-based assays including induction of the p53 regulated gene (PIG-3) and caspase activity.