Jay Chauhan

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Significance Excess Toll-like receptor 2 (TLR2) signaling has been implicated in numerous inflammatory diseases, yet there is no TLR2 inhibitor licensed for human use. Using computer-aided drug design (CADD), we identified a compound, C16H15NO4 (C29), and a derivative, ortho-vanillin, that inhibit TLR2 signaling in vitro and in vivo. Our findings also revealed unexpected differences between TLR2/1 and TLR2/6 signaling in mice vs. humans. Importantly, our data provide proof of principle that the CADD-targeted BB loop pocket residues are critical for TLR2 signaling and may be targeted therapeutically. Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein–protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors.

Small-molecule inhibitors of dimeric transcription factors: Antagonism of protein–protein and protein–DNA interactions

Transcription factors are DNA-binding proteins that – usually in combination with other proteins to form the pre-initiation complex (PIC) – regulate the transcription of specific DNA sequences (genes) into mRNA by controlling the recruitment of RNA polymerase II. Constitutive activation of transcription factors can lead to a variety of cancers, and are, therefore, important therapeutic targets. However, in stark contrast to targeting enzyme active sites, disruption of protein–protein or protein–DNA interactions involved in the transcriptional machinery is particularly challenging owing to the large interfacial areas involved, a lack of obvious binding sites and often non-contiguous contact points. Especially problematic for the development of small-molecules is the need by such agents to overcome the large free energy of association between protein–protein and, to a lesser extent, protein–DNA interfaces. Nevertheless, recent years have seen considerable success in this area of medicinal chemistry, cementing the notion that disruption of such interactions is feasible with small-molecule, drug-like compounds. We discuss, in particular, the disruption of dimeric transcription factors, such as STAT3–STAT3, c-Myc–Max and c-Jun–c-Fos (AP-1), with small-molecules that block their protein–protein interactions or their interactions with DNA.

Structural modifications of (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione that improve selectivity for inhibiting the proliferation of melanoma cells containing active ERK signaling

We herein report on the pharmacophore determination of the ERK docking domain inhibitor (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione, which has led to the discovery of compounds with greater selectivities for inhibiting the proliferation of melanoma cells containing active ERK signaling.

Structure-based design of N-substituted 1-hydroxy-4-sulfamoyl-2-naphthoates as selective inhibitors of the Mcl-1 oncoprotein.

Structure-based drug design was utilized to develop novel, 1-hydroxy-2-naphthoate-based small-molecule inhibitors of Mcl-1. Ligand design was driven by exploiting a salt bridge with R263 and interactions with the p2 pocket of the protein. Significantly, target molecules were accessed in just two synthetic steps, suggesting further optimization will require minimal synthetic effort. Molecular modeling using the Site-Identification by Ligand Competitive Saturation (SILCS) approach was used to qualitatively direct ligand design as well as develop quantitative models for inhibitor binding affinity to Mcl-1 and the Bcl-2 relative Bcl-xL as well as for the specificity of binding to the two proteins. Results indicated hydrophobic interactions in the p2 pocket dominated affinity of the most favourable binding ligand (3bl: Ki = 31 nM). Compounds were up to 19-fold selective for Mcl-1 over Bcl-xL. Selectivity of the inhibitors was driven by interactions with the deeper p2 pocket in Mcl-1 versus Bcl-xL. The SILCS-based SAR of the present compounds represents the foundation for the development of Mcl-1 specific inhibitors with the potential to treat a wide range of solid tumours and hematological cancers, including acute myeloid leukemia.

Discovery of methyl 4'-methyl-5-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)-[1,1'-biphenyl]-3-carboxylate, an improved small-molecule inhibitor of c-Myc-max dimerization.

c‐Myc is a basic helix‐loop‐helix‐leucine zipper (bHLH‐ZIP) transcription factor that is responsible for the transcription of a wide range of target genes involved in many cancer‐related cellular processes. Over‐expression of c‐Myc has been observed in, and directly contributes to, a variety of human cancers including those of the hematopoietic system, lung, prostate and colon. To become transcriptionally active, c‐Myc must first dimerize with Myc‐associated factor X (Max) via its own bHLH‐ZIP domain. A proven strategy towards the inhibition of c‐Myc oncogenic activity is to interfere with the structural integrity of the c‐Myc–Max heterodimer. The small molecule 10074‐G5 is an inhibitor of c‐Myc–Max dimerization (IC50=146 μM) that operates by binding and stabilizing c‐Myc in its monomeric form. We have identified a congener of 10074‐G5, termed 3jc48‐3 (methyl 4′‐methyl‐5‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)‐[1,1′‐biphenyl]‐3‐carboxylate), that is about five times as potent (IC50=34 μM) at inhibiting c‐Myc–Max dimerization as the parent compound. 3jc48‐3 exhibited an approximate twofold selectivity for c‐Myc–Max heterodimers over Max–Max homodimers, suggesting that its mode of action is through binding c‐Myc. 3jc48‐3 inhibited the proliferation of c‐Myc‐over‐expressing HL60 and Daudi cells with single‐digit micromolar IC50 values by causing growth arrest at the G0/G1 phase. Co‐immunoprecipitation studies indicated that 3jc48‐3 inhibits c‐Myc–Max dimerization in cells, which was further substantiated by the specific silencing of a c‐Myc‐driven luciferase reporter gene. Finally, 3jc48‐3′s intracellular half‐life was >17 h. Collectively, these data demonstrate 3jc48‐3 to be one of the most potent, cellularly active and stable c‐Myc inhibitors reported to date.

Structure–activity exploration of a small-molecule Lipid II inhibitor

We have recently identified low-molecular weight compounds that act as inhibitors of Lipid II, an essential precursor of bacterial cell wall biosynthesis. Lipid II comprises specialized lipid (bactoprenol) linked to a hydrophilic head group consisting of a peptidoglycan subunit (N-acetyl glucosamine [GlcNAc]–N-acetyl muramic acid [MurNAc] disaccharide coupled to a short pentapeptide moiety) via a pyrophosphate. One of our lead compounds, a diphenyl-trimethyl indolene pyrylium, termed BAS00127538, interacts with the MurNAc moiety and the isoprenyl tail of Lipid II. Here, we report on the structure–activity relationship of BAS00127538 derivatives obtained by in silico analyses and de novo chemical synthesis. Our results indicate that Lipid II binding and bacterial killing are related to three features: the diphenyl moiety, the indolene moiety, and the positive charge of the pyrylium. Replacement of the pyrylium moiety with an N-methyl pyridinium, which may have importance in stability of the molecule, did not alter Lipid II binding or antibacterial potency.

Discovery of Mcl-1 inhibitors based on a thiazolidine-2,4-dione scaffold

Inspired by a rhodanine-based dual inhibitor of Bcl-xL and Mcl-1, a focused library of analogues was prepared wherein the rhodanine core was replaced with a less promiscuous thiazolidine-2,4-dione scaffold. Compounds were initially evaluated for their abilities to inhibit Mcl-1. The most potent compound 12b inhibited Mcl-1 with a Ki of 155 nM. Further investigation revealed comparable inhibition of Bcl-xL (Ki = 90 nM), indicating that the dual inhibitory profile of the initial rhodanine lead had been retained upon switching the heterocycle core.

Synthetic, structural mimetics of the β-hairpin flap of HIV-1 protease inhibit enzyme function

Small-molecule mimetics of the β-hairpin flap of HIV-1 protease (HIV-1 PR) were designed based on a 1,4-benzodiazepine scaffold as a strategy to interfere with the flap-flap protein-protein interaction, which functions as a gated mechanism to control access to the active site. Michaelis-Menten kinetics suggested our small-molecules are competitive inhibitors, which indicates the mode of inhibition is through binding the active site or sterically blocking access to the active site and preventing flap closure, as designed. More generally, a new bioactive scaffold for HIV-1PR inhibition has been discovered, with the most potent compound inhibiting the protease with a modest K(i) of 11 μM.

Abstract 5062: Development of a first in class inhibitor of BET bromodomains and dopamine receptor 2

ConverGene has developed a first-in-class dual-active small molecule inhibitor that i) inhibits BET family of bromodomain-containing proteins, and ii) antagonizes dopamine receptor D2 (DRD2). BET protein family includes BRD4, an epigenetic reader protein that mediates expression of MYC oncogene. Thus, BRD4 is considered as a cancer therapeutic target to indirectly suppress MYC expression. In addition to being a therapeutic target for psychiatric diseases, DRD2 is emerging as a potential therapeutic target in neuroendocrine tumors, subsets of pancreatic ductal adenocarcinoma and small cell lung cancer. Our lead compound showed high activity in a binding test against BRD4 (Ki = 34 nM); exhibited high bioavailability upon oral administration; profoundly suppressed MYC expression both in vitro and in vivo; inhibited growth of AML and solid tumor cells in xenograft models; potently inhibited both isoforms of DRD2 (IC50 0.1 µM); and interfered with DRD2/β-arrestin/Akt pathway in vitro. Therefore, our BRD4/DRD2 dual-active compounds may hold promise as a novel class of therapeutics that interferes with both cancer growth and maintenance by simultaneously interfering with MYC and DRD2 pathways. We currently are investigating these dual-active compounds in multiple in vitro and in vivo models and expect to report the outcomes at the AACR Annual Meeting in 2017. Citation Format: Makoto Yoshioka, Jay Chauhan, Steven Fletcher, Jeffrey W. Strovel. Development of a first in class inhibitor of BET bromodomains and dopamine receptor 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5062. doi:10.1158/1538-7445.AM2017-5062