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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

Supplementary Figure from BCL10 Mutations Define Distinct Dependencies Guiding Precision Therapy for DLBCL

Supplementary Data from BCL10 Mutations Define Distinct Dependencies Guiding Precision Therapy for DLBCL

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

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.

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.

Background: Constitutive activation of the classical nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) pathway is a clear driver of B-cell non-Hodgkin lymphomas (NHL). Bruton's Tyrosine Kinase (BTK) and Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), which lies downstream of BTK, are key mediators of the classical NF-κB signaling pathway activated by BCR and TCR receptors. JNJ-67856633 is a first-in-class MALT1 protease inhibitor. JNJ-64264681 is a BTK inhibitor with improved selectivity against BTK. Blocking the BCR pathway at multiple points using these two orally bioavailable compounds could enhance clinical activity in B-cell lymphoma patients. Methods: JNJ-67856633 and JNJ-64264681 are currently being evaluated in phase 1 clinical trials designed to establish safety, PK, PD and the Recommended Phase 2 Dose (RP2D) of each agent. Results: JNJ-67856633 is a potent, selective, orally bioavailable, allosteric inhibitor of MALT1 protease activity. The compound inhibits proliferation of activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell lines bearing CD79b or CARD11 mutations as well as models mimicking resistance to covalent BTK inhibitors. JNJ-67856633 exhibits potent tumor growth inhibition in two human DLBCL xenograft models, OCI-Ly3 and OCI-Ly10, and mutation selected patient derived DLBCL xenografts. Furthermore, treatment with JNJ-67856633 leads to dose dependent inhibition of the generation of Tregs (CD4+CD25+FoxP3+) following CD3/28 stimulation in vitro, suggesting a potential immune modulatory role of MALT1 inhibition. JNJ-64264681 is an orally active small molecule that is a potent, selective, and irreversible covalent BTK inhibitor. JNJ-64264681 inhibits the growth of CD79b-mutant DLBCL cell lines in vitro and potently inhibits tumor growth in xenograft- or patient-derived DLBCL models in vivo. Treatment with JNJ-64264681 and JNJ-67856633 administered together demonstrated statistically significant tumor growth inhibition compared with vehicle control in two CD79b mutant mouse lymphoma models, one based on a DLBCL cell line (OCI-Ly10) and one based on a patient-derived DLBCL model (LY2298). In both models, the combination showed increased growth inhibition compared with single agents and tumor regression in the combination arm. Synergistic anti-proliferative activity was observed in three DLBCL cell lines carrying CD79b mutations and one MCL cell line. Conclusions: Taken together, the in vitro and in vivo data for JNJ-67856633 and JNJ-64264681 suggest that combination therapy can increase the anti-tumor effect of the monotherapies and provide a more sustained response, offering strong support for clinical investigation of the combination of these two novel agents. A phase 1b combination study is scheduled to initiate. Citation Format: Ulrike Philippar, Lorena Fontan, Ivo Cornelissen, Haopeng Rui, Sriram Balasubramanian, Marcello Gaudiano, Mariette Bekkers, Luc Van Nuffel, Tianbao Lu, Tianbao Lu, John Wiener, Mark Tichenor, Tony Greway, Kathryn Packman, Bie Verbist, Yusri Elsayed, Ricardo Attar, Jacqueline Bussolari, John Gerecitano. Combination therapy of JNJ-67856633, a novel, first-in-class MALT1 protease inhibitor, and JNJ-64264681, a novel BTK inhibitor, for the treatment of B-cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1267.

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.

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.

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 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.

// Yueyu Cao 1, 3 , Mengdan Wei 2, 3 , Bing Li 2, 3 , Yali Liu 2, 3 , Ying Lu 2, 3 , Zhipeng Tang 1, 3 , Tianbao Lu 3 , Yujiao Yin 1, 3 , Zhiqiang Qin 1, 2, 3, 4 , Zengguang Xu 2, 3 1 Department of Oncology, Shanghai East Hospital, Dalian Medical University, Shanghai 200120, China 2 Department of Oncology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China 3 Research Center for Translational Medicine and Key Laboratory of Arrhythmias, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China 4 Departments of Microbiology/Immunology/Parasitology, Louisiana State University Health Sciences Center, Louisiana Cancer Research Center, New Orleans, LA 70112, USA Correspondence to: Zhiqiang Qin, e-mail: zqin@lsuhsc.edu Zengguang Xu, e-mail: xuzg1998@163.com Keywords: EIF4G1, USP10, NSCLC, lung cancer Received: January 26, 2016 Accepted: March 02, 2016 Published: March 18, 2016 ABSTRACT 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.

Introduction: Constitutive activation of the classical nuclear factor kappa-light-chain-enhancer of activated B cells (NF κB) pathway is a clear driver of B-cell lymphomas, especially the aggressive activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a key mediator of the classical NF-κB signaling pathway downstream of B-cell receptor and T-cell receptor. MALT1 possesses two functions: a scaffolding function to recruit NF-κB signaling proteins and a protease function to cleave and inactivate inhibitors of the NF-κB signaling pathway. Methods: Using a high-throughput screen followed by iterative structure-activity relationship (SAR) analyses, the MALT1 inhibitor JNJ-67856633 was identified. JNJ-67856633 was evaluated using biochemical, cellular in vitro, in vivo tumor efficacy and safety models. Results: JNJ-67856633 is a potent, selective, allosteric inhibitor of MALT1 protease activity as measured by biochemical assays or downstream cellular cytokine readouts (IL6/10) or direct MALT1 substrate cleavage (RelB, BCL10). The compound inhibits proliferation of activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell lines bearing CD79b or CARD11 mutations as well as models mimicking resistance to covalent Bruton's tyrosine kinase (BTK) inhibitors. Furthermore, combination effects were observed in CD79b cellular ABC-DLBCL models when JNJ-67856633 was combined with a BTK inhibitor. JNJ-67856633 showed activity in organoid cultures derived from ABC-DLBCL patients. JNJ-67856633 leads to potent in vivo pharmacodynamic shutdown in CD79b- as well as CARD11-mutant ABC-DLBCL models as measured by serum IL10 or uncleaved BCL10 levels in tumors. JNJ-67856633 exhibits potent tumor growth inhibition in two human DLBCL xenograft models, OCI Ly3 and OCI Ly10. In addition, >5 patient-derived DLBCL xenografts were evaluated and activity in mutation selected models was observed. To address the role of MALT1 inhibition in T cells, primary human T cells derived from normal healthy volunteers were treated with JNJ-67856633 in vitro. Dose-dependent inhibition of the generation of Tregs (CD4+CD25+FoxP3+) following CD3/28 stimulation was observed upon treatment with JNJ-67856633, suggesting a potential immune-modulatory role of MALT1 inhibition. Conclusions: Phase 1 clinical trials assessing the safety and efficacy of JNJ-67856633 initiated in 2019. JNJ-67856633 is a combination partner for BTK inhibitors and a promising treatment option for BTKi-resistant tumors, with demonstrated preclinical activity in CARD11 mutant tumors. In addition to ABC-DLBCL, a MALT1 inhibitor is a promising treatment option for patients with CLL, MCL, WM, and FL whose tumors have been shown to be sensitive to inhibition of BTK. MALT lymphomas, characterized by MALT1 and BCL10 translocation, represent another attractive target for MALT1 inhibition. Citation Format: Ulrike Philippar, Tianbao Lu, Lorena Fontan, Nele Vloemans, Mariette Bekkers, Luc van Nuffel, Marcello Gaudiano, Katarzyna Wnuk-Lipinska, Bas-Jan Van Der Leede, Katie Amssoms, Kristof Kimpe, Bart Medaer, Tony Greway, Yann Abraham, Max Cummings, Emanuele Trella, Greet Vanhoof, Weimei Sun, Jan Willem Thuring, Peter Connolly, Haopeng Rui, Sriram Balasubramanian, John Gerecitano, Ari Melnick, Ricardo Attar. Discovery of JNJ-67856633: A novel, first-in-class MALT1 protease inhibitor for the treatment of B-cell lymphomas [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-49.

Introduction: Constitutive activation of the classical nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) pathway is a clear driver of B-cell lymphomas, especially the aggressive activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a key mediator of the classical NF-κB signaling pathway downstream of B-cell receptor and T-cell receptor. MALT1 possesses 2 functions: a scaffolding function to recruit NF-κB signaling proteins and a protease function to cleave and inactivate inhibitors of the NF-κB signaling pathway. Methods: Using a high-throughput screen followed by iterative structure-activity relationship (SAR) analyses, the MALT1 inhibitor JNJ-67856633 was identified. The lead compound was evaluated using biochemical, in vitro cellular and in vivo tumor efficacy and safety models. Results: JNJ-67856633 is a potent, selective, allosteric inhibitor of MALT1 protease activity as measured by biochemical assays or downstream cellular cytokine readouts (IL 6/10) or direct MALT1 substrate cleavage (RelB, BCL10). The compound inhibits proliferation of activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell lines bearing CD79b or CARD11 mutations as well as models mimicking resistance to covalent Bruton's Tyrosine Kinase (BTK) inhibitors. Furthermore, combination effects were observed in CD79b cellular ABC-DLBCL models when JNJ-67856633 was combined with a BTK inhibitor. JNJ-67856633 leads to potent in vivo pharmacodynamic shutdown in CD79b- as well as CARD11-mutant ABC-DLBCL models as measured by serum IL10 or uncleaved BCL10 levels in tumors. JNJ-67856633 exhibits potent tumor growth inhibition in two human DLBCL xenograft models, OCI Ly3 and OCI Ly10, and mutation selection patient derived DLBCL xenografts. To address the role of MALT1 inhibition in T cells, primary human T cells derived from normal healthy volunteers were treated with JNJ-67856633 in vitro. Dose dependent inhibition of the generation of Tregs (CD4+CD25+FoxP3+) following CD3/28 stimulation was observed upon treatment with JNJ-67856633 suggesting a potential immune modulatory role of MALT1 inhibition. Conclusions: Phase 1 clinical trials assessing the safety and efficacy of JNJ-67856633 initiated in 2019. JNJ-67856633 is a combination partner for BTK inhibitors and a promising treatment option for BTKi-resistant tumors, with demonstrated preclinical activity in CARD11 mutant tumors. In addition to ABC-DLBCL, a MALT1 inhibitor is a promising treatment option for patients with CLL, MCL, WM, and FL whose tumors have been shown to be sensitive to inhibition of BTK. MALT lymphomas, characterized by MALT1 and BCL10 translocation, represent another attractive target for MALT1 inhibition. Citation Format: Ulrike Philippar, Tianbao Lu, Nele Vloemans, Mariette Bekkers, Luc Van Nuffel, Marcello Gaudiano, Katarzyna Wnuk-Lipinska, Bas-jan Van Der Leede, Katie Amssoms, Kristof Kimpe, Bart Medaer, Tony Greway, Yann Abraham, Max Cummings, Emanuele Trella, Greet Vanhoof, Weimei Sun, Jan Willem Thuring, Peter Connolly, Jan Linders, Haopeng Rui, Sriram Balasubramanian, Amy Johnson, John Gerecitano, Jenna Goldberg, James P. Edwards, Yusri Elsayed, Jennifer Smit, Jaqueline Bussolari, Jaqueline Bussolari, Ricardo Attar. Discovery of JNJ-67856633: A novel, first-in-class MALT1 protease inhibitor for the treatment of B cell lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5690.

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.

Activation of the MET/HGF pathway has been linked to tumor initiation, metastasis, angiogenesis and resistance to therapeutic agents. Here we present pharmacological characterization of OMO-1 (formerly JNJ-38877618), a potent, highly selective, orally bioavailable MET kinase inhibitor with nM binding affinity (Kd=1.4 nM) and enzyme inhibitory activity against wt and M1268T mutant MET (2 and 3 nM IC50). MET inhibitory effects were assessed in proliferation, colony formation and motility assays. OMO-1 displayed nM potency against MET Ampl/mutant and therapy resistant models. In vivo, OMO-1 induced complete inhibition of tumor growth in 3 models: the SNU5 MET amp gastric, U87-MG HGF autocrine glioblastoma and Hs746T MET exon 14 skipping mutant gastric cancer. OMO-1 induced regression of large MET amplified EBC-1 SqNSCLC where OMO-1 led to dose- and time-dependent inhibition of MET kinase activation, with the duration of target shut down considerably exceeding plasma exposure times. Combination treatments were well tolerated and improved EGFR targeted therapy. Although single agent OMO-1 had no effect on NSCLC HCC827 EGFR, combination with Erlotinib led to delayed onset of tumor recurrence. The acquired EGFR inhibitor resistant model HCC827-ER1 was determined to be MET amplified. OMO-1 and erlotinib both inhibited tumor growth of this model whilst combination induced tumor regression. In an EGFR inhibitor resistant PDX having MET amplification, single agent OMO-1 caused tumour stasis whereas MetMab/erlotinib only led to tumor growth delay. The potent preclinical activity we have observed, supports ongoing clinical development of OMO-1 in patients with MET pathway-driven tumors. Citation Format: Marion Libouban, Eleonora Jovcehva, Desiree De Lange, Boudewijn Janssens, Tinne Verhulst, Souichi Ogata, Berthold Wroblowski, Laurence Mevellec, Tianbao Lu, Glen Clack, Timothy Perera. OMO-1, a potent, highly selective, orally bioavailable, MET kinase inhibitor with a favorable preclinical toxicity profile, shows both monotherapy activity, against MET pathway-driven tumors, and EGFR TKI combination activity in acquired resistance models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4791.

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.

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-1β 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.

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.

The activity and stability of the p53 tumor suppressor are regulated by the human homologue of the mouse double minute 2 (Hdm2) oncoprotein. It has been hypothesized that small molecules disrupting the Hdm2:p53 complex would allow for the activation of p53 and result in growth suppression. We have identified small-molecule inhibitors of the Hdm2:p53 interaction using our proprietary ThermoFluor microcalorimetry technology. Medicinal chemistry and structure-based drug design led to the development of an optimized series of benzodiazepinediones, including TDP521252 and TDP665759. Activities were dependent on the expression of wild-type (wt) p53 and Hdm2 as determined by lack of potency in mutant or null p53-expressing cell lines or cells engineered to no longer express Hdm2 and wt p53. TDP521252 and TDP665759 inhibited the proliferation of wt p53-expressing cell lines with average IC50s of 14 and 0.7 μmol/L, respectively. These results correlated with the direct cellular dissociation of Hdm2 from wt p53 observed within 15 minutes in JAR choriocarcinoma cells. Additional activities of these inhibitors in vitro include stabilization of p53 protein levels, up-regulation of p53 target genes in a DNA damage–independent manner, and induction of apoptosis in HepG2 cells. Administration of TDP665759 to mice led to an increase in p21waf1/cip1 levels in liver samples. Finally, TDP665759 synergizes with doxorubicin both in culture and in an A375 xenograft model to decrease tumor growth. Taken together, these data support the potential utility of small-molecule inhibitors of the Hdm2:p53 interaction for the treatment of wt p53-expressing tumors. [Mol Cancer Ther 2006;5(1):160–9]

A library of 1,4-benzodiazepine-2,5-diones was screened for binding to the p53-binding domain of HDM2 using Thermofluor ® , a miniaturized thermal denaturation assay. The hits obtained were shown to bind to HDM2 in the p53-binding pocket using a fluorescence polarization (FP) peptide displacement assay. The potency of the series was optimized, leading to sub-micromolar antagonists of the p53–HDM2 interaction.

HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.

Furan[3,2-b]pyrroles are important isosteres for the indole scaffold in which the benzene ring is replaced by the furan ring. A series of novel tetra-substituted furan[3,2-b]pyrroles was synthesized from a simple furaldehyde. The divergent synthesis allows for substitution on multiple positions on the scaffold, creating the potential for the formation of large libraries.

Although thrombin has been extensively researched with many examples of potent and selective inhibitors, the key characteristics of oral bioavailability and long half-life have been elusive. We report here a novel series non-peptidic phenyl-based, highly potent, highly selective and orally bioavailable thrombin inhibitors using oxyguanidines as guanidine-mimetics.

The structure–activity relationships of a novel series of non-amide-based thrombin inhibitors are described. Exploration of the P2 and the aryl binding region for this series has identified optimal groups for achieving nanomolar potency. The binding modes of these optimal groups have been confirmed by X-ray structural analysis.

We describe a new class of potent, non-amide-based small molecule thrombin inhibitors in which an amidinohydrazone is used as a guanidine bioisostere on a non-peptide scaffold. Compound 4 exhibits nM inhibition of thrombin, is selective for thrombin, and shows 60 and 23% bioavailability in rabbits and dogs, respectively. Crystallographic analysis of 4 bound to thrombin confirmed the amindinohydrazone binding mode.

We describe the in vitro evaluation and crystallographic analysis of a new class of potent and selective, non-amino acid-based, small-molecule thrombin inhibitors, exemplified by 14. This class of achiral inhibitors lacks an amide-based backbone, exhibits nM inhibition of thrombin, and is selective for thrombin. Compound 14 does not interact with the active-site catalytic apparatus and is anchored to the enzyme via a single network of hydrogen bonds to Asp189 of the S1 pocket.

The structure of the noncovalent complex of human alpha-thrombin with a nonpeptide inhibitor containing a central phenyl scaffold, N-[2-[5-methyl-3-(2-chlorophenylsulfonyloxy)phenoxy]ethyl]-N- methyl-4 -aminopyridine (1), has been determined to 2.20 A resolution. In addition, the thrombin-bound structures of two distinct amino acid-based inhibitors (3 and 4) containing different aminopyridine-derived guanidine mimetics have been determined. Each compound occupies the same region of the active site and projects an aminopyridine, a central hydrophobic group, and an aryl group, into the S1, S2, and aryl subsites on thrombin. Nonpeptide 1 forms only one direct intermolecular hydrogen bond to the thrombin active site and forms no hydrogen bonds to ordered molecules of solvent. Close contacts are observed between main-chain carbonyl groups on thrombin and the edges of the central phenyl and aminopyridine rings and the sulfonyl group of 1 such that atoms carrying opposite partial charges are juxtaposed. Aminopyridine groups in 3 and 4 also form close contacts with the edges of carbonyl groups on thrombin and are flexibly accommodated in the S1 subsite. Superposition of the bound conformations of 1 and D-Phe-Pro-amidobutylguanidine (2) revealed that the central phenyl scaffold of 1 substitutes for the peptide main chain of 2.

New drugs are needed for treatment of diseases caused by African trypanosomes. One possible target for chemotherapy is the biosynthesis of the glycosyl phosphatidyl-inositol (GPI) of this parasite's variant surface glycoprotein (VSG). Unlike mammalian GPIs, the diacylglycerol moiety of the VSG anchor contains only myristate (tetradecanoate), added in unique remodeling reactions. We previously found that 11-oxatetradecanoic acid [i.e., 10-(propoxy)decanoic acid] is selectively toxic to trypanosomes. We have now assayed 244 different fatty acid analogs, most with chain lengths comparable to that of myristate, for trypanocidal effects. In these assays we surveyed the effects on toxicity of systematic alterations in the analogs' steric, conformational, and hydrophobic properties. We also used three 3H-labeled oxatetradecanoic acids to explore the mechanism of analog action. Their incorporation into VSG correlated roughly with toxicity, although they also were incorporated into phospholipids and other proteins. Myristate analogs are useful for studying the mechanism of GPI myristolyation, and they are candidates for antitrypanosomal chemotherapy.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is a monomeric enzyme that is essential for vegetative growth. Nmt1p catalyzes the co-translational transfer of myristate from CoA to the amino-terminal Gly of cellular proteins in an ordered Bi Bi reaction mechanism that initially involves binding of myristoyl-CoA to the apoenzyme. Forty one fatty acid analogs were synthesized to define features in the acyl chain of myristoyl-CoA which are important determinants of its recognition by Nmt1p's acyl-CoA binding site as well as to help us deduce the structure of the binding site itself. These analogs included dicarboxylic acids, omega-nitrocarboxylic acids, analogs equivalent in length to C13:0-C15:0 which contain electronegative halogens at their omega-termini, hydroxytetradecanoic acids with hydrogen replaced by OH from C3 to C13, and azidophenyl-containing fatty acids with the linear azide unit attached either meta or para to phenyl and with variations in the length of their methylene chains. These compounds were converted to their CoA derivatives using Pseudomonas acyl-CoA synthetase and then surveyed as substrates for purified Nmt1p in an in vitro assay system that included an octapeptide derived from residues 1-8 of the human immunodeficiency virus Pr55gag polyprotein precursor. The results suggest that the myristoyl-CoA binding site contains a conical-shaped "receptor" that interacts with the omega-terminus of the bound acyl chain of acyl-CoAs. The acuteness of this cone determines the enzyme's capacity to accommodate steric bulk at the omega-terminus as well as Nmt1p's sensitivity to the distance between the eclipsed C5-C6 bond of a bound acyl chain and its omega-terminus. The activity profile of the various analog-CoAs also indicates that the enzyme's myristoyl-CoA binding site can accommodate fatty acid analogs with marked increases in polarity at their omega-terminus (compared to C14:0) as long as their chain length is equivalent to that of myristate.

3-Nitro-2,6-bis(4′-benzo-15-crown-5)aminopyridine and its rubidium picrate complex were synthesized, and the first known example of a bis(crown ether) rubidium picrate intramolecular sandwich complex was obtained. The structure is confirmed by solid state studies: C39H44N7O19Rb·CH3OH·H2O, triclinic, P1. a = 11.481(4), b = 14.519(4), c = 15.960(6) A, α = 63.28(3), β = 73.75(3), γ = 80.66(3)°, Dc = 1.53 g/cm3, Z = 2, and R = 0.091 for the 2350 observed reflections. The rubidium cation is intramolecularly sandwiched by ten oxygen atoms. The Rb[sbnd]O bond lengths range from 2.849(12) A to 3.02(2) A. The picrate anion is linked by ionic interactions to the large cation.

We report here general methodology for the synthesis of 1-substituted anthraquinone derivatives and two novel,catalytic processes for the replacement of a 1-chloro substituent

The direct nucleophilic aromatic substitution reactions of anthraquinones have permitted the syntheses of more than 30 novel podands, crown ethers and lariat ethers. Anthraquinones having (ethyleneoxy)n sidearms were obtained by direct displacement of chloride by the anion of CH3(OCH2CH2)nOH. The ethyleneoxy-substituted anthraquinones could, in turn, undergo direct replacement by nucleophiles that failed to displace chloride. This approach has been used for the preparation of two-armed podand derivatives and several novel crown derivatives of anthraquinone. Binding comparisons are presented for several of these new anthraquinones. Direct substitution did not prove successful in the preparation of anthraquinone- [2 · 2] -cryptand which was obtained by alkylation. The crystal structure of the latter reveals an orientation of ring and anthraquinone appropriate for cation binding, a fact confirmed by cation binding constant measurements.

We describe the in vitro evaluation and crystallographic analysis of a new class of potent and selective, non-amino acid-based, small-molecule thrombin inhibitors, exemplified by 14. This class of achiral inhibitors lacks an amide-based backbone, exhibits nM inhibition of thrombin, and is selective for thrombin. Compound 14 does not interact with the active-site catalytic apparatus and is anchored to the enzyme via a single network of hydrogen bonds to Asp189 of the S1 pocket.

The structure–activity relationships of a novel series of non-amide-based thrombin inhibitors are described. Exploration of the P2 and the aryl binding region for this series has identified optimal groups for achieving nanomolar potency. The binding modes of these optimal groups have been confirmed by X-ray structural analysis.

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.

Candida albicans and Cryptococcus neoformans are major causes of systemic fungal infections, particularly in patients with acquired immunodeficiency syndrome. Metabolic labeling studies revealed that these organisms synthesize a small number of N-myristoylproteins, the most prominent being 20-kDa ADP-ribosylation factors (Arfs). C. albicans Arf has approximately 80% identity with the essential Arf1 and Arf2 proteins of Saccharomyces cerevisiae. [3H]Myristic acid analogs with oxygen for -CH2- substitutions at C4, C6, C11, and C13 are incorporated into cellular N-myristoylproteins, phospholipids, and neutral lipids produced by these three yeasts during exponential growth at 30 degrees C in complex media. Analog- and organism-specific differences in the efficiency of labeling of proteins and lipid classes were observed. The effects of oxatetradecanoic acids with oxygen for -CH2- substitutions at C3-C13 on C. neoformans, C. albicans, and S. cerevisiae were assessed during mid-log phase growth at 30 degrees C. A single dose of 3-oxa-, 4-oxa-, 5-oxa- or 6-oxatetradecanoic acid (O3-O6, final concentration = 300 microM) was able to inhibit growth of C. neoformans in the order O4 greater than O5 greater than O3 approximately O6. The other compounds were inactive. 4-Oxatetradecanoic acid was fungicidal, producing a 10,000-fold reduction in viable cell number 1 h after administration and continued suppression of cell growth for 7 h. A clear dose response was observed over a concentration range of 100-300 microM. 4-Oxatridecanoic acid was 100-fold less potent in reducing cell viability than 4-oxatetradecanoic acid but more potent than 5-oxatridecanoic acid. O4 produced approximately 10-100-fold reductions in the viability of C. albicans and S. cerevisiae at 300-500 microM, respectively, whereas O5 and O6 were less active. Since N-myristoylation of the Pr55gag polyprotein precursor produced by human immunodeficiency virus I (HIV-I) is essential for its assembly, we also assessed the antiviral effects of 4-oxatetradecanoic acid. O4 is able to produce a 50% reduction in the replication of HIV-I in acutely infected human T-lymphocyte cell lines at a concentration of 18 microM. Together, these data suggest that (i) the position of the oxygen for methylene substitution is a critical determinant of the fungicidal activity of O4 and (ii) NMT may be an attractive therapeutic target for treating opportunistic fungal infections in patients infected with HIV-I.

The direct nucleophilic aromatic substitution reactions of anthraquinones have permitted the syntheses of more than 30 novel podands, crown ethers and lariat ethers. Anthraquinones having (ethyleneoxy)n sidearms were obtained by direct displacement of chloride by the anion of CH3(OCH2CH2)nOH. The ethyleneoxy-substituted anthraquinones could, in turn, undergo direct replacement by nucleophiles that failed to displace chloride. This approach has been used for the preparation of two-armed podand derivatives and several novel crown derivatives of anthraquinone. Binding comparisons are presented for several of these new anthraquinones. Direct substitution did not prove successful in the preparation of anthraquinone- [2 · 2] -cryptand which was obtained by alkylation. The crystal structure of the latter reveals an orientation of ring and anthraquinone appropriate for cation binding, a fact confirmed by cation binding constant measurements.

MyristoylCoA:protein N-myristoyl transferase (NMT) catalyzes the cotranslational covalent attachment of myristate (C14:0) to the ammo-terminal glycine residues of a variety of biologically important proteins. An extensive survey of myristic acid analogs has previously allowed us to infer the conformational constraints imposed upon the fatty acid when bound to the enzyme's acylCoA binding site. A series of myristic acid analogs has now been synthesized and studied in an in vitro assay to probe further the conformation of NMT's bound ligand and to assess the importance of the distance between the carboxyl and the proposed bend in the fatty acid.

We report here general methodology for the synthesis of 1-substituted anthraquinone derivatives and two novel,catalytic processes for the replacement of a 1-chloro substituent

To evaluate the association of single-nucleotide polymorphisms (SNPs) in PADI4 mRNA with rheumatoid arthritis (RA) in a Chinese population, we examined the distribution of four exonic SNPs of the PADI4 gene (padi4_89*G/A, padi4_90*T/C, padi4_92*G/C and padi4_104*T/C) and PADI4 gene expression in 70 RA patients and 81 controls. Increased RA susceptibility was associated with the minor alleles of padi4_89 (P = 0.012), padi4_90 (P = 0.002), padi4_104 (P = 0.001) and the functional haplotype carrying the four minor alleles (P = 0.008). Human leukocyte antigen (HLA)-DRB1 shared epitope (SE) alleles were also associated with increased RA susceptibility, and the individuals with minor alleles of four exonic SNPs and SE alleles showed more increased RA susceptibility. The PADI4 expression was significantly higher in RA patients than in controls (P < 0.001). HLA-DRB1 SE alleles and the genotypes carrying the minor alleles of four SNPs were associated with increased PADI4 expression. It is concluded that PADI4 SNPs, functional haplotype and PADI4 expression may contribute to an inherited predisposition to RA in a Chinese population.

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 (Ki = 1.2 nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.