Two Distinct Mechanisms of Inhibition of LpxA Acyltransferase Essential for Lipopolysaccharide Biosynthesis.

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.

Discovery and optimization of novel pyridines as highly potent and selective glycogen synthase kinase 3 inhibitors.

Glycogen synthase kinase-3 plays an essential role in multiple biochemical pathways in the cell, particularly in regards to energy regulation. As such, Glycogen synthase kinase-3 is an attractive target for pharmacological intervention in a variety of disease states, particularly non-insulin dependent diabetes mellitus. However, due to homology with other crucial kinases, such as the cyclin-dependent protein kinase CDC2, developing compounds that are both potent and selective is challenging. A novel series of derivatives of 5-nitro-N2-(2-(pyridine-2ylamino)ethyl)pyridine-2,6-diamine were synthesized and have been shown to potently inhibit glycogen synthase kinase-3 (GSK3). Potency in the low nanomolar range was obtained along with remarkable selectivity. The compounds activate glycogen synthase in insulin receptor-expressing CHO-IR cells and in primary rat hepatocytes, and have acceptable pharmacokinetics and pharmacodynamics to allow for oral dosing. The X-ray co-crystal structure of human GSK3-ß in complex with compound 2 is reported and provides insights into the structural determinants of the series responsible for its potency and selectivity.

Design and synthesis of potent RSK inhibitors.

Utilizing the already described 3,4-bi-aryl pyridine series as a starting point, incorporation of a second ring system with a hydrogen bond donor and additional hydrophobic contacts yielded the azaindole series which exhibited potent, picomolar RSK2 inhibition and the most potent in vitro target modulation seen thus far for a RSK inhibitor. In the context of the more potent core, several changes at the phenol moiety were assessed to potentially find a tool molecule appropriate for in vivo evaluation.

2-Amino-7-substituted benzoxazole analogs as potent RSK2 inhibitors.

2-Amino-7-substituted benzoxazole analogs were identified by HTS as inhibitors of RSK2. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus of improving in vitro and target modulation potency and physicochemical properties.

Design and synthesis of 6,6-fused heterocyclic amides as raf kinase inhibitors.

Compounds belonging to several scaffolds-quinazolines, quinolines and quinoxalines-were designed and synthesized as Raf kinase inhibitors. Scaffolds were assessed for in vitro Braf(V600E) inhibition, and overall kinase selectivity. Pharmacokinetic parameters for one of the scaffolds were also determined.

Design and synthesis of 5,6-fused heterocyclic amides as Raf kinase inhibitors.

Two scaffolds based on 5,6-fused heterocyclic backbones were designed and synthesized as Raf kinase inhibitors. The scaffolds were assessed for in vitro pan-Raf inhibition, activity in cell proliferation and target modulation assays, and pharmacokinetic parameters.

Design and Discovery of N-(3-(2-(2-Hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide, a Selective, Efficacious, and Well-Tolerated RAF Inhibitor Targeting RAS Mutant Cancers: The Path to the Clinic.

Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, 3 (RAF709; Nishiguchi, G. A.; et al. J. Med. Chem. 2017, 60, 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.

Synthesis, Binding Mode, and Antihyperglycemic Activity of Potent and Selective (5-Imidazol-2-yl-4-phenylpyrimidin-2-yl)[2-(2-pyridylamino)ethyl]amine Inhibitors of Glycogen Synthase Kinase 3.

In an effort to identify new antidiabetic agents, we have discovered a novel family of (5-imidazol-2-yl-4-phenylpyrimidin-2-yl)[2-(2-pyridylamino)ethyl]amine analogues which are inhibitors of human glycogen synthase kinase 3 (GSK3). We developed efficient synthetic routes to explore a wide variety of substitution patterns and convergently access a diverse array of analogues. Compound 1 (CHIR-911, CT-99021, or CHIR-73911) emerged from an exploration of heterocycles at the C-5 position, phenyl groups at C-4, and a variety of differently substituted linker and aminopyridine moieties attached at the C-2 position. These compounds exhibited GSK3 IC50s in the low nanomolar range and excellent selectivity. They activate glycogen synthase in insulin receptor-expressing CHO-IR cells and primary rat hepatocytes. Evaluation of lead compounds 1 and 2 (CHIR-611 or CT-98014) in rodent models of type 2 diabetes revealed that single oral doses lowered hyperglycemia within 60 min, enhanced insulin-stimulated glucose transport, and improved glucose disposal without increasing insulin levels.

Design and Discovery of N-(2-Methyl-5'-morpholino-6'-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A Potent, Selective, and Efficacious RAF Inhibitor Targeting RAS Mutant Cancers.

RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

Inhibition of prenylated KRAS in a lipid environment.

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.

Discovery of Potent and Selective RSK Inhibitors as Biological Probes.

While the p90 ribosomal S6 kinase (RSK) family has been implicated in multiple tumor cell functions, the full understanding of this kinase family has been restricted by the lack of highly selective inhibitors. A bis-phenol pyrazole was identified from high-throughput screening as an inhibitor of the N-terminal kinase of RSK2. Structure-based drug design using crystallography, conformational analysis, and scaffold morphing resulted in highly optimized difluorophenol pyridine inhibitors of the RSK kinase family as demonstrated cellularly by the inhibition of YB1 phosphorylation. These compounds provide for the first time in vitro tools with an improved selectivity and potency profile to examine the importance of RSK signaling in cancer cells and to fully evaluate RSK as a therapeutic target.

Structure-Based Drug Design of Novel, Potent, and Selective Azabenzimidazoles (ABI) as ATR Inhibitors.

Compound 13 was discovered through morphing of the ATR biochemical HTS hit 1. The ABI series was potent and selective for ATR. Incorporation of a 6-azaindole afforded a marked increase in cellular potency but was associated with poor PK and hERG ion channel inhibition. DMPK experiments established that CYP P450 and AO metabolism in conjunction with Pgp and BCRP efflux were major causative mechanisms for the observed PK. The series also harbored the CYP3A4 TDI liability driven by the presence of both a morpholine and an indole moiety. Incorporation of an adjacent fluorine or nitrogen into the 6-azaindole addressed many of the various medicinal chemistry issues encountered.

Discovery of RAF265: A Potent mut-B-RAF Inhibitor for the Treatment of Metastatic Melanoma.

Abrogation of errant signaling along the MAPK pathway through the inhibition of B-RAF kinase is a validated approach for the treatment of pathway-dependent cancers. We report the development of imidazo-benzimidazoles as potent B-RAF inhibitors. Robust in vivo efficacy coupled with correlating pharmacokinetic/pharmacodynamic (PKPD) and PD-efficacy relationships led to the identification of RAF265, 1, which has advanced into clinical trials.

Design and Synthesis of Orally Bioavailable Benzimidazole Reverse Amides as Pan RAF Kinase Inhibitors.

Benzimidazole reverse amides were designed and synthesized as Pan RAF kinase inhibitors. Investigation of the structure-activity relationship of the compounds revealed that they were potent in vitro and exhibited desirable in vivo properties.

Correction to Design and Synthesis of Orally Bioavailable Benzimidazole Reverse Amides as Pan RAF Kinase Inhibitors.

[This corrects the article DOI: 10.1021/ml5002272.].

Design and synthesis of orally bioavailable benzimidazoles as Raf kinase inhibitors.

A series of arylaminobenzimidazoles was designed and synthesized as Raf kinase inhibitors. Exploration of the structure-activity relationship resulted in compounds that are potent in vitro and show desirable in vivo properties.