Discovery of Clinical Candidate BMS-823778 as an Inhibitor of Human 11ß-Hydroxysteroid Dehydrogenase Type 1 (11ß-HSD-1).

BMS-823778 (2), a 1,2,4-triazolopyridinyl-methanol derived analog, was identified as a potent and selective inhibitor of human 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD-1) enzyme (IC50 = 2.3 nM) with >10,000-fold selectivity over 11ß-HSD-2. Compound 2 exhibits robust acute pharmacodynamic effects in cynomolgus monkeys (ED50 = 0.6 mg/kg) and in diet-induced obese (DIO) mice (ED50 = 34 mg/kg). Compound 2 also showed excellent inhibition in an ex vivo adipose DIO mouse model (ED50 = 5.2 mg/kg). Oral bioavailability ranges from 44% to 100% in preclinical species. Its favorable development properties, pharmacokinetics, high adipose-to-plasma concentration ratio, and preclinical pharmacology profile have prompted the evaluation of 2 for the treatment of type 2 diabetes and metabolic syndrome in phase 2 clinical trials.

Radiosynthesis and preclinical PET evaluation of 89Zr-nivolumab (BMS-936558) in healthy non-human primates.

Cancer immunotherapy, unlike traditional cytotoxic chemotherapeutic treatments, engages the immune system to identify cancer cells and stimulate immune responses. The Programmed Death-1 (PD-1) protein is an immunoinhibitory receptor expressed by activated cytotoxic T-lymphocytes (CTL) that seek out and destroy cancer cells. Multiple cancer types express and upregulate the Programmed Death-Ligand 1 (PD-L1) and 2 (PD-L2) which bind to PD-1 as an immune escape mechanism. Nivolumab is a fully human IgG4 anti-PD-1 monoclonal antibody (mAb) approved for treatment of multiple cancer types. This study reports the preparation and in vivo evaluation of 89Zr labeled nivolumab in healthy non-human primates (NHP) as a preliminary study of biodistribution and clearance. The radiochemical and in vivo stabilities of the 89Zr complex were shown to be acceptable for imaging. Three naïve NHPs were intravenously injected with tracer only or tracer co-injected with nivolumab followed by co-registered by positron emission tomography (PET) and magnetic resonance imaging (MRI), acquired for eight days following injection. Image-derived standardized uptake values (SUV) were quantified by region of interest (ROI) analysis. Radioactivity in the spleen was significantly reduced by addition of excess nivolumab compared to the tracer only study at all imaging time points. Liver uptake of the radiotracer was consistent as a clearance organ with minimal signal from other tissues: lung, muscle, brain, heart, and kidney. The results indicate specific biodistribution to the spleen, which can be blocked by co-administration of excess nivolumab. Distribution to other organs is consistent with elimination pathways of antibodies, with primary clearance through the liver.

Discovery of Clinical Candidate 2-((2S,6S)-2-Phenyl-6-hydroxyadamantan-2-yl)-1-(3'-hydroxyazetidin-1-yl)ethanone [BMS-816336], an Orally Active Novel Selective 11ß-Hydroxysteroid Dehydrogenase Type 1 Inhibitor.

BMS-816336 (6n-2), a hydroxy-substituted adamantyl acetamide, has been identified as a novel, potent inhibitor against human 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) enzyme (IC50 3.0 nM) with >10000-fold selectivity over human 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). 6n-2 exhibits a robust acute pharmacodynamic effect in cynomolgus monkeys (ED50 0.12 mg/kg) and in DIO mice. It is orally bioavailable (%F ranges from 20 to 72% in preclinical species) and has a predicted pharmacokinetic profile of a high peak to trough ratio and short half-life in humans. This ADME profile met our selection criteria for once daily administration, targeting robust inhibition of 11ß-HSD1 enzyme for the first 12 h period after dosing followed by an "inhibition holiday" so that the potential for hypothalamic-pituitary-adrenal (HPA) axis activation might be mitigated. 6n-2 was found to be well-tolerated in phase 1 clinical studies and represents a potential new treatment for type 2 diabetes, metabolic syndrome, and other human diseases modulated by glucocorticoid control.

Novel phenylalanine derived diamides as Factor XIa inhibitors.

The synthesis, structural activity relationships (SAR), and selectivity profile of a potent series of phenylalanine diamide FXIa inhibitors will be discussed. Exploration of P1 prime and P2 prime groups led to the discovery of compounds with high FXIa affinity, good potency in our clotting assay (aPPT), and high selectivity against a panel of relevant serine proteases as exemplified by compound 21. Compound 21 demonstrated good in vivo efficacy (EC50=2.8µM) in the rabbit electrically induced carotid arterial thrombosis model (ECAT).

Structure-based design of inhibitors of coagulation factor XIa with novel P1 moieties.

Compound 2 was previously identified as a potent inhibitor of factor XIa lacking oral bioavailability. A structure-based approach was used to design analogs of 2 with novel P1 moieties with good selectivity profiles and oral bioavailability. Further optimization of the P1 group led to the identification of a 4-chlorophenyltetrazole P1 analog, which when combined with further modifications to the linker and P2' group provided compound 32 with FXIa Ki=6.7 nM and modest oral exposure in dogs.

Discovery of novel P1 groups for coagulation factor VIIa inhibition using fragment-based screening.

A multidisciplinary, fragment-based screening approach involving protein ensemble docking and biochemical and NMR assays is described. This approach led to the discovery of several structurally diverse, neutral surrogates for cationic factor VIIa P1 groups, which are generally associated with poor pharmacokinetic (PK) properties. Among the novel factor VIIa inhibitory fragments identified were aryl halides, lactams, and heterocycles. Crystallographic structures for several bound fragments were obtained, leading to the successful design of a potent factor VIIa inhibitor with a neutral lactam P1 and improved permeability.

Hydrogen/deuterium exchange mass spectrometry applied to IL-23 interaction characteristics: potential impact for therapeutics.

IL-23 is an important therapeutic target for the treatment of inflammatory diseases. Adnectins are targeted protein therapeutics that are derived from domain III of human fibronectin and have a similar protein scaffold to antibodies. Adnectin 2 was found to bind to IL-23 and compete with the IL-23/IL-23R interaction, posing a potential protein therapeutic. Hydrogen/deuterium exchange mass spectrometry and computational methods were applied to probe the binding interactions between IL-23 and Adnectin 2 and to determine the correlation between the two orthogonal methods. This review summarizes the current structural knowledge about IL-23 and focuses on the applicability of hydrogen/deuterium exchange mass spectrometry to investigate the higher order structure of proteins, which plays an important role in the discovery of new and improved biotherapeutics.

Pyridine and pyridinone-based factor XIa inhibitors.

The structure-activity relationships (SAR) of six-membered ring replacements for the imidazole ring scaffold is described. This work led to the discovery of the potent and selective pyridine (S)-23 and pyridinone (±)-24 factor XIa inhibitors. SAR and X-ray crystal structure data highlight the key differences between imidazole and six-membered ring analogs.

Phenylimidazoles as potent and selective inhibitors of coagulation factor XIa with in vivo antithrombotic activity.

Novel inhibitors of FXIa containing an (S)-2-phenyl-1-(4-phenyl-1H-imidazol-2-yl)ethanamine core have been optimized to provide compound 16b, a potent, reversible inhibitor of FXIa (Ki = 0.3 nM) having in vivo antithrombotic efficacy in the rabbit AV-shunt thrombosis model (ID50 = 0.6 mg/kg + 1 mg kg(-1) h(-1)). Initial analog selection was informed by molecular modeling using compounds 11a and 11h overlaid onto the X-ray crystal structure of tetrahydroquinoline 3 complexed to FXIa. Further optimization was achieved by specific modifications derived from careful analysis of the X-ray crystal structure of the FXIa/11h complex. Compound 16b was well tolerated and enabled extensive pharmacologic evaluation of the FXIa mechanism up to the ID90 for thrombus inhibition.

The influence of adnectin binding on the extracellular domain of epidermal growth factor receptor.

The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.ᅟ

Optimization of 1,2,4-Triazolopyridines as Inhibitors of Human 11ß-Hydroxysteroid Dehydrogenase Type 1 (11ß-HSD-1).

Small alkyl groups and spirocyclic-aromatic rings directly attached to the left side and right side of the 1,2,4-triazolopyridines (TZP), respectively, were found to be potent and selective inhibitors of human 11ß-hydroxysteroid dehydrogenase-type 1 (11ß-HSD-1) enzyme. 3-(1-(4-Chlorophenyl)cyclopropyl)-8-cyclopropyl-[1,2,4]triazolo[4,3-a]pyridine (9f) was identified as a potent inhibitor of the 11ß-HSD-1 enzyme with reduced Pregnane-X receptor (PXR) transactivation activity. The binding orientation of this TZP series was revealed by X-ray crystallography structure studies.

Discovery and preclinical characterization of the cyclopropylindolobenzazepine BMS-791325, a potent allosteric inhibitor of the hepatitis C virus NS5B polymerase.

Described herein are structure-activity relationship studies that resulted in the optimization of the activity of members of a class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors. Subsequent iterations of analogue design and syntheses successfully addressed off-target activities, most notably human pregnane X receptor (hPXR) transactivation, and led to significant improvements in the physicochemical properties of lead compounds. Those analogues exhibiting improved solubility and membrane permeability were shown to have notably enhanced pharmacokinetic profiles. Additionally, a series of alkyl bridged piperazine carboxamides was identified as being of particular interest, and from which the compound BMS-791325 (2) was found to have distinguishing antiviral, safety, and pharmacokinetic properties that resulted in its selection for clinical evaluation.

Tetrahydroquinoline derivatives as potent and selective factor XIa inhibitors.

Antithrombotic agents that are inhibitors of factor XIa (FXIa) have the potential to demonstrate robust efficacy with a low bleeding risk profile. Herein, we describe a series of tetrahydroquinoline (THQ) derivatives as FXIa inhibitors. Compound 1 was identified as a potent and selective tool compound for proof of concept studies. It exhibited excellent antithrombotic efficacy in rabbit thrombosis models and did not prolong bleeding times. This demonstrates proof of concept for the FXIa mechanism in animal models with a reversible, small molecule inhibitor.

Discovery of 3-hydroxy-4-cyano-isoquinolines as novel, potent, and selective inhibitors of human 11ß-hydroxydehydrogenase 1 (11ß-HSD1).

Derived from the HTS hit 1, a series of hydroxyisoquinolines was discovered as potent and selective 11ß-HSD1 inhibitors with good cross species activity. Optimization of substituents at the 1 and 4 positions of the isoquinoline group in addition to the core modifications, with a special focus on enhancing metabolic stability and aqueous solubility, resulted in the identification of several compounds as potent advanced leads.

Synthesis and SAR of indole-and 7-azaindole-1,3-dicarboxamide hydroxyethylamine inhibitors of BACE-1.

Heterocyclic replacement of the isophthalamide phenyl ring in hydroxyethylamine (HEA) BACE-1 inhibitors was explored. A variety of indole-1,3-dicarboxamide HEAs exhibited potent BACE-1 enzyme inhibition, but displayed poor cellular activity. Improvements in cellular activity and aspartic protease selectivity were observed for 7-azaindole-1,3-dicarboxamide HEAs. A methylprolinol-bearing derivative (10n) demonstrated robust reductions in rat plasma Aß levels, but did not lower rat brain Aß due to poor central exposure. The same analog exhibited a high efflux ratio in a bidirectional Caco-2 assay and was likely a substrate of the efflux transporter P-glycoprotein. X-ray crystal structures are reported for two indole HEAs in complex with BACE-1.

Pyridine amides as potent and selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1.

Several series of pyridine amides were identified as selective and potent 11beta-HSD1 inhibitors. The most potent inhibitors feature 2,6- or 3,5-disubstitution on the pyridine core. Various linkers (CH(2)SO(2), CH(2)S, CH(2)O, S, O, N, bond) between the distal aryl and central pyridyl groups are tolerated, and lipophilic amide groups are generally favored. On the distal aryl group, a number of substitutions are well tolerated. A crystal structure was obtained for a complex between 11beta-HSD1 and the most potent inhibitor in this series.

Structure-activity relationships of anthranilamide-based factor Xa inhibitors containing piperidinone and pyridinone P4 moieties.

Introduction of the phenyl piperidinone and phenyl pyridinone P4 moieties in the anthranilamide scaffold led to potent, selective, and orally bioavailable inhibitors of factor Xa. Anthranilamide 28 displayed comparable efficacy to apixaban in the rabbit arteriovenous-shunt (AV) thrombosis model.

SAR and X-ray structures of enantiopure 1,2-cis-(1R,2S)-cyclopentyldiamine and cyclohexyldiamine derivatives as inhibitors of coagulation Factor Xa.

In the search of Factor Xa (FXa) inhibitors structurally different from the pyrazole-based series, we identified a viable series of enantiopure cis-(1R,2S)-cycloalkyldiamine derivatives as potent and selective inhibitors of FXa. Among them, cyclohexyldiamide 7 and cyclopentyldiamide 9 were the most potent neutral compounds, and had good anticoagulant activity comparable to the pyrazole-based analogs. Crystal structures of 7-FXa and 9-FXa illustrate binding similarities and differences between the five- and the six-membered core systems, and provide rationales for the observed SAR of P1 and linker moieties.

Comparative studies of active site-ligand interactions among various recombinant constructs of human beta-amyloid precursor protein cleaving enzyme.

Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimer's disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.