Targeting Enteropeptidase with Reversible Covalent Inhibitors To Achieve Metabolic Benefits.

Inhibition of the serine protease enteropeptidase (EP) opens a new avenue to the discovery of chemotherapeutics for the treatment of metabolic diseases. Camostat has been used clinically for treating chronic pancreatitis in Japan; however, the mechanistic basis of the observed clinical efficacy has not been fully elucidated. We demonstrate that camostat is a potent reversible covalent inhibitor of EP, with an inhibition potency (k inact/KI) of 1.5 × 104 M-1s-1 High-resolution liquid chromatography-mass spectrometry (LC-MS) showed addition of 161.6 Da to EP after the reaction with camostat, consistent with insertion of the carboxyphenylguanidine moiety of camostat. Covalent inhibition of EP by camostat is reversible, with an enzyme reactivation half-life of 14.3 hours. Formation of a covalent adduct was further supported by a crystal structure resolved to 2.19 Å, showing modification of the catalytic serine of EP by a close analog of camostat, leading to formation of the carboxyphenylguanidine acyl enzyme identical to that expected for the reaction with camostat. Of particular note, minor structural modifications of camostat led to changes in the mechanism of inhibition. We observed from other studies that sustained inhibition of EP is required to effect a reduction in cumulative food intake and body weight, with concomitant improved blood glucose levels in obese and diabetic leptin-deficient mice. Thus, the structure-activity relationship needs to be driven by not only the inhibition potency but also the mechanistic and kinetic characterization. Our findings support EP as a target for the treatment of metabolic diseases and demonstrate that reversible covalent EP inhibitors show clinically relevant efficacy. SIGNIFICANCE STATEMENT: Interest in targeted covalent drugs has expanded in recent years, particularly so for kinase targets, but also more broadly. This study demonstrates that reversible covalent inhibition of the serine protease enteropeptidase is a therapeutically viable approach to the treatment of metabolic diseases and that mechanistic details of inhibition are relevant to clinical efficacy. Our mechanistic and kinetic studies outline a framework for detailed inhibitor characterization that is proving essential in guiding discovery efforts in this area.

Discovery and optimization of new oxadiazole substituted thiazole RORγt inverse agonists through a bioisosteric amide replacement approach.

Starting from previously identified thiazole-2-carboxamides exemplified by compound 1/6, two new series of RORγt inverse agonists with significantly improved aqueous solubility, ADME parameters and oral PK properties were discovered. These scaffolds were identified from a bioisosteric amide replacement approach. Amongst the variety of heterocycles explored, a 1,3,4-oxadiazole led to compounds with the best overall profile for SAR development and in vivo exploration. In an ex vivo mouse PD model, concentration dependent efficacy was demonstrated and compounds 3/5 and 6/3 were profiled in a 5-day rat tolerability study.

Optimization and biological evaluation of thiazole-bis-amide inverse agonists of RORγt.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with small molecule therapeutics. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We recently described the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole scaffold. Herein we describe the successful optimization of this class by incorporation of an additional amide moiety at the 4-position of the thiazole core. In several optimization cycles, we have reduced human PXR activation, improved solubility, and increased potency while maintaining nuclear receptor selectivity. X-ray crystallographic analysis of compound 1g bound in the sterol binding site of the ligand binding domain of RORγt was largely consistent with an earlier structure, guiding further insight into the molecular mechanism for RORγt inhibition with this series. Compound 1g is orally bioavailable, potent in a human whole blood assay and proved to be efficacious in an ex-vivo IL-17A assay, and was selected for preclinical evaluation.

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.

3-Substituted Quinolines as RORγt Inverse Agonists.

We have previously reported the syntheses of a series of 3,6-disubstituted quinolines as modulators of the retinoic acid receptor-related orphan receptor gamma t (RORγt). These molecules are potent binders but are high molecular weight and they exhibited poor solubility at pH 2 and pH 7. This manuscript details our efforts at improving physical chemical properties for this series of compounds by increasing the diversity at the 3-position (i.e. introducing heteroatoms and lowering the molecular weight). These efforts have led to molecules which are potent binders with improved solubility.

Structure-Based Macrocycle Design in Small-Molecule Drug Discovery and Simple Metrics To Identify Opportunities for Macrocyclization of Small-Molecule Ligands.

Interest is growing in the use of macrocycles in pharmaceutical discovery. Macrocylization may provide a gateway to an expanded chemical space for small-molecule drug discovery, and this could be beneficial in prosecuting difficult targets, e.g., protein-protein interactions. Most, but not all, macrocycle drugs are derived from natural products. Studies on synthetic drug-like small-molecule macrocycles are limited, and our current understanding of macrocycle drugs is similarly limited. Following some background discussion, we review several examples of the structure-based design of synthetic macrocycles. Our opinion is that in conformationally suitable systems macrocycles are an analog class worthy of consideration. We then summarize an approach for the initial evaluation of molecules as candidates for macrocyclization.

Conformational Sampling of Macrocyclic Drugs in Different Environments: Can We Find the Relevant Conformations?

Conformational flexibility is a major determinant of the properties of macrocycles and other drugs in beyond rule of 5 (bRo5) space. Prediction of conformations is essential for design of drugs in this space, and we have evaluated three tools for conformational sampling of a set of 10 bRo5 drugs and clinical candidates in polar and apolar environments. The distance-geometry based OMEGA was found to yield ensembles spanning larger structure and property spaces than the ensembles obtained by MOE-LowModeMD (MOE) and MacroModel (MC). Both MC and OMEGA but not MOE generated different ensembles for polar and apolar environments. All three conformational search methods generated conformers similar to the crystal structure conformers for 9 of the 10 compounds, with OMEGA performing somewhat better than MOE and MC. MOE and OMEGA found all six conformers of roxithromycin that were identified by NMR in aqueous solutions, whereas only OMEGA sampled the three conformers observed in chloroform. We suggest that characterization of conformers using molecular descriptors, e.g., the radius of gyration and polar surface area, is preferred to energy- or root-mean-square deviation-based methods for selection of biologically relevant conformers in drug discovery in bRo5 space.

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.

Identification and biological evaluation of thiazole-based inverse agonists of RORγt.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with a small molecule. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We describe the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole core. Acid analog 1j demonstrated oral bioavailability in rats and was potent in a human whole blood assay, suggesting potential utility in treating autoimmune and inflammatory diseases such as psoriasis. X-ray crystallographic data helped to elucidate the molecular mechanism for RORγt inhibition with this series.

Protocols for the Design of Kinase-focused Compound Libraries.

Protocols for the design of kinase-focused compound libraries are presented. Kinase-focused compound libraries can be differentiated based on the design goal. Depending on whether the library should be a discovery library specific for one particular kinase, a general discovery library for multiple distinct kinase projects, or even phenotypic screening, there exists today a variety of in silico methods to design candidate compound libraries. We address the following scenarios: 1) Datamining of SAR databases and kinase focused vendor catalogues; 2) Predictions and virtual screening; 3) Structure-based design of combinatorial kinase inhibitors; 4) Design of covalent kinase inhibitors; 5) Design of macrocyclic kinase inhibitors; and 6) Design of allosteric kinase inhibitors and activators.

6-Substituted quinolines as RORγt inverse agonists.

We identified 6-substituted quinolines as modulators of the retinoic acid receptor-related orphan receptor gamma t (RORγt). The synthesis of this class of RORγt modulators is reported, and optimization of the substituents at the quinoline 6-position that produced compounds with high affinity for the receptor is detailed. This effort identified molecules that act as potent, full inverse agonists in a RORγt-driven cell-based reporter assay. The X-ray crystal structures of two full inverse agonists from this chemical series bound to the RORγt ligand binding domain are disclosed, and we highlight the interaction of a hydrogen-bond acceptor on the 6-position substituent of the inverse agonist with Glu379:NH as a conserved binding contact.

Identification and structure activity relationships of quinoline tertiary alcohol modulators of RORγt.

A high-throughput screen of the ligand binding domain of the nuclear receptor retinoic acid-related orphan receptor gamma t (RORγt) employing a thermal shift assay yielded a quinoline tertiary alcohol hit. Optimization of the 2-, 3- and 4-positions of the quinoline core using structure-activity relationships and structure-based drug design methods led to the discovery of a series of modulators with improved RORγt inhibitory potency and inverse agonism properties.

Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation.

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7ß, 27-dihydroxycholesterol (7ß, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7ß, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7ß, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

Discovery and development of simeprevir (TMC435), a HCV NS3/4A protease inhibitor.

Hepatitis C virus is a blood-borne infection and the leading cause of chronic liver disease (including cirrhosis and cancer) and liver transplantation. Since the identification of HCV in 1989, there has been an extensive effort to identify and improve treatment options. An important milestone was reached in 2011 with the approval of the first-generation HCV NS3/4A protease inhibitors. However, new therapies are needed to improve cure rates, shorten treatment duration, and improve tolerability. Here we summarize the extensive medicinal chemistry effort to develop novel P2 cyclopentane macrocyclic inhibitors guided by HCV NS3 protease assays, the cellular replicon system, structure-based design, and a panel of DMPK assays. The selection of compound 29 (simeprevir, TMC435) as clinical candidate was based on its excellent biological, PK, and safety pharmacology profile. Compound 29 has recently been approved for treatment of chronic HCV infection in combination with pegylated interferon-α and ribavirin in Japan, Canada, and USA.

Discovery and early development of TMC647055, a non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

Structure-based macrocyclization of a 6-carboxylic acid indole chemotype has yielded potent and selective finger-loop inhibitors of the hepatitis C virus (HCV) NS5B polymerase. Lead optimization in conjunction with in vivo evaluation in rats identified several compounds showing (i) nanomolar potency in HCV replicon cells, (ii) limited toxicity and off-target activities, and (iii) encouraging preclinical pharmacokinetic profiles characterized by high liver distribution. This effort culminated in the identification of TMC647055 (10a), a nonzwitterionic 17-membered-ring macrocycle characterized by high affinity, long polymerase residence time, and broad genotypic coverage. In vitro results of the combination of 10a with the HCV protease inhibitor TMC435 (simeprevir) supported an evaluation of this combination in patients with regard to virus suppression and resistance emergence. In a phase 1b trial with HCV genotype 1-infected patients, 10a was considered to be safe and well-tolerated and demonstrated potent antiviral activity, which was further enhanced in a combination study with TMC435.

Virologic response and characterisation of HCV genotype 2-6 in patients receiving TMC435 monotherapy (study TMC435-C202).

BACKGROUND & AIMS: TMC435 is a potent, once-daily, investigational hepatitis C virus (HCV) NS3/4A protease inhibitor in phase III clinical development. In the phase II trial TMC435-C202 (NCT00812331), TMC435 displayed potent activity in genotype 4, 5 and 6 patients and in 3/6 genotype 2 patients, whereas no activity was observed with genotype 3. METHODS: Thirty-seven patients received TMC435 monotherapy (200 mg once daily) for 7 days. HCV RNA, NS3 protease sequences and the corresponding phenotypes were evaluated. RESULTS: Genotype and isolate-specific baseline polymorphisms at NS3 positions known to affect HCV protease inhibitor activity were present in all genotypes. Consistent with the antiviral activity observed in genotypes 4 and 6, TMC435 was active in vitro against all genotype 4 isolates, and against most genotype 6 polymorphisms when tested as single or double mutants. In contrast, in genotype 3 where no HCV RNA decline was observed, isolates displayed >700-fold increases in EC(50) attributed to the D168Q polymorphism. In genotypes 2 and 5, HCV RNA changes from baseline to Day 3 ranged between -0.3 to -3.6 and -1.5 to -4.0 log(10)IU/ml, respectively, and isolates or site-directed mutants displayed intermediate in vitro susceptibility to TMC435 with fold changes in EC(50) between 15 and 78. Viral breakthrough in genotypes 4-6 was associated with emerging mutations including Q80R, R155K and/or D168E/V. CONCLUSIONS: Sequence and phenotypic analyses of baseline isolates identified polymorphisms which could explain the differences in antiviral activity between genotypes. Pathways of TMC435 resistance in genotypes 2-6 were similar to those identified in genotype 1.

TMC647055, a potent nonnucleoside hepatitis C virus NS5B polymerase inhibitor with cross-genotypic coverage.

Hepatitis C virus (HCV) infection is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. There remains an unmet medical need for efficacious and safe direct antivirals with complementary modes of action for combination in treatment regimens to deliver a high cure rate with a short duration of treatment for HCV patients. Here we report the in vitro inhibitory activity, mode of action, binding kinetics, and resistance profile of TMC647055, a novel and potent nonnucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase. In vitro combination studies with an HCV NS3/4A protease inhibitor demonstrated potent suppression of HCV RNA replication, confirming the potential for combination of these two classes in the treatment of chronic HCV infection. TMC647055 is a potent nonnucleoside NS5B polymerase inhibitor of HCV replication with a promising in vitro biochemical, kinetic, and virological profile that is currently undergoing clinical evaluation.

Finger loop inhibitors of the HCV NS5b polymerase. Part II. Optimization of tetracyclic indole-based macrocycle leading to the discovery of TMC647055.

Optimization of a novel series of macrocyclic indole-based inhibitors of the HCV NS5b polymerase targeting the finger loop domain led to the discovery of lead compounds exhibiting improved potency in cellular assays and superior pharmacokinetic profile. Further lead optimization performed on the most promising unsaturated-bridged subseries provided the clinical candidate 27-cyclohexyl-12,13,16,17-tetrahydro-22-methoxy-11,17-dimethyl-10,10-dioxide-2,19-methano-3,7:4,1-dimetheno-1H,11H-14,10,2,9,11,17-benzoxathiatetraazacyclo docosine-8,18(9H,15H)-dione, TMC647055 (compound 18a). This non-zwitterionic 17-membered ring macrocycle combines nanomolar cellular potency (EC(50) of 82 nM) with minimal associated cell toxicity (CC(50)>20 µM) and promising pharmacokinetic profiles in rats and dogs. TMC647055 is currently being evaluated in the clinic.

Finger-loop inhibitors of the HCV NS5b polymerase. Part 1: Discovery and optimization of novel 1,6- and 2,6-macrocyclic indole series.

Novel conformationaly constrained 1,6- and 2,6-macrocyclic HCV NS5b polymerase inhibitors, in which either the nitrogen or the phenyl ring in the C2 position of the central indole core is tethered to an acylsulfamide acid bioisostere, have been designed and tested for their anti-HCV potency. This transformational route toward non-zwitterionic finger loop-directed inhibitors led to the discovery of derivatives with improved cell potency and pharmacokinetic profile.