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.

Imidazo[1,2-a]pyridin-6-yl-benzamide analogs as potent RAF inhibitors.

A series of imidazo[1,2-a]pyridin-6-yl-benzamide analogs was designed as inhibitors of B-RAFV600E. Medicinal chemistry techniques were employed to explore the SAR for this series and improve selectivity versus P38 and VEGFR2.

Discovery of the 1,7-diazacarbazole class of inhibitors of checkpoint kinase 1.

Checkpoint kinase 1 (ChK1) is activated in response to DNA damage, acting to temporarily block cell cycle progression and allow for DNA repair. It is envisaged that inhibition of ChK1 will sensitize tumor cells to treatment with DNA-damaging therapies, and may enhance the therapeutic window. High throughput screening identified carboxylate-containing diarylpyrazines as a prominent hit series, but with limited biochemical potency and no cellular activity. Through a series of SAR investigations and X-ray crystallographic analysis the critical role of polar contacts with conserved waters in the kinase back pocket was established. Structure-based design, guided by in silico modeling, transformed the series to better satisfy these contacts and the novel 1,7-diazacarbazole class of inhibitors was discovered. Here we present the genesis of this novel series and the identification of GNE-783, a potent, selective and orally bioavailable inhibitor of ChK1.

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.

Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding.

Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.

Inhibition of Wnt signaling by Dishevelled PDZ peptides.

Dishevelled proteins are key regulators of Wnt signaling pathways that have been implicated in the progression of human cancers. We found that the binding cleft of the Dishevelled PDZ domain is more flexible than those of canonical PDZ domains and enables recognition of both C-terminal and internal peptides. These peptide ligands inhibit Wnt/beta-catenin signaling in cells, showing that Dishevelled PDZ domains are potential targets for small-molecule cancer therapeutics.

A specificity map for the PDZ domain family.

PDZ domains are protein-protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes in multicellular organisms. By scanning billions of random peptides, we accurately map binding specificity for approximately half of the over 330 PDZ domains in the human and Caenorhabditis elegans proteomes. The domains recognize features of the last seven ligand positions, and we find 16 distinct specificity classes conserved from worm to human, significantly extending the canonical two-class system based on position -2. Thus, most PDZ domains are not promiscuous, but rather are fine-tuned for specific interactions. Specificity profiling of 91 point mutants of a model PDZ domain reveals that the binding site is highly robust, as all mutants were able to recognize C-terminal peptides. However, many mutations altered specificity for ligand positions both close and far from the mutated position, suggesting that binding specificity can evolve rapidly under mutational pressure. Our specificity map enables the prediction and prioritization of natural protein interactions, which can be used to guide PDZ domain cell biology experiments. Using this approach, we predicted and validated several viral ligands for the PDZ domains of the SCRIB polarity protein. These findings indicate that many viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, and that highly pathogenic strains target PDZ domains involved in cell polarity and growth.

GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis.

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here, we show that during the conjugation of ATG8 to single membranes (CASM), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal capacity. GABARAP directly binds to a previously unidentified LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex and mediates sequestration to GABARAP-conjugated membrane compartments. This disrupts FLCN/FNIP GAP function toward RagC/D, resulting in impaired substrate-specific mTOR-dependent phosphorylation of TFEB. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

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.

The crystal structure of murine coronin-1: a regulator of actin cytoskeletal dynamics in lymphocytes.

Mammalian coronin-1 is preferentially expressed in hematopoietic cells and plays a poorly understood role in the dynamic reorganization of the actin cytoskeleton. Sequence analysis of coronin-1 revealed five WD40 repeats that were predicted to form a beta propeller. They are followed by a 130 residue extension and a 30 residue leucine zipper domain that is responsible for multimerization of the protein. Here, we present the crystal structure of murine coronin-1 without the leucine zipper at 1.75 A resolution. Coronin-1 forms a seven-bladed beta propeller composed of the five predicted WD40 repeats and two additional blades that lack any homology to the canonical WD40 motif. The C-terminal extension adopts an extended conformation, packs tightly against the bottom surface of the propeller, and is likely to be required for the structural stability of the propeller. Analysis of charged and conserved surface residues delineate possible binding sites for F-actin on the beta propeller.

Mathematical and Structural Characterization of Strong Nonadditive Structure-Activity Relationship Caused by Protein Conformational Changes.

In medicinal chemistry, accurate prediction of additivity-based structure-activity relationship (SAR) analysis rests on three assumptions: (1) a consistent binding pose of the central scaffold, (2) no interaction between the R group substituents, and (3) a relatively rigid binding pocket in which the R group substituents act independently. Previously, examples of nonadditive SAR have been documented in systems that deviate from the first two assumptions. Local protein structural change upon ligand binding, through induced fit or conformational selection, although a well-known phenomenon that invalidates the third assumption, has not been linked to nonadditive SAR conclusively. Here, for the first time, we present clear structural evidence that the formation of a hydrophobic pocket upon ligand binding in PDE2 catalytic site reduces the size of another distinct subpocket and contributes to strong nonadditive SAR between two otherwise distant R groups.

Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine Adenylyltransferase from Gram-Negative Bacteria.

The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli Δ tolC mutant strain.

Discovery and Optimization of Phosphopantetheine Adenylyltransferase Inhibitors with Gram-Negative Antibacterial Activity.

In the preceding manuscript [ Moreau et al. 2018 , 10.1021/acs.jmedchem.7b01691 ] we described a successful fragment-based lead discovery (FBLD) strategy for discovery of bacterial phosphopantetheine adenylyltransferase inhibitors (PPAT, CoaD). Following several rounds of optimization two promising lead compounds were identified: triazolopyrimidinone 3 and 4-azabenzimidazole 4. Here we disclose our efforts to further optimize these two leads for on-target potency and Gram-negative cellular activity. Enabled by a robust X-ray crystallography system, our structure-based inhibitor design approach delivered compounds with biochemical potencies 4-5 orders of magnitude greater than their respective fragment starting points. Additional optimization was guided by observations on bacterial permeability and physicochemical properties, which ultimately led to the identification of PPAT inhibitors with cellular activity against wild-type E. coli.

Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ domain.

The mitochondrial serine protease HtrA2/Omi helps to maintain mitochondrial function by handling misfolded proteins in the intermembrane space. In addition, HtrA2/Omi has been implicated as a proapoptotic factor upon release into the cytoplasm during the cell death cascade. The protein contains a C-terminal PDZ domain that packs against the protease active site and inhibits proteolytic activity. Engagement of the PDZ domain by peptide ligands has been shown to activate the protease and also has been proposed to mediate substrate recognition. We report a detailed structural and functional analysis of the human HtrA2/Omi PDZ domain using peptide libraries and affinity assays to define specificity, X-ray crystallography to view molecular details of PDZ-ligand interactions, and alanine-scanning mutagenesis to probe the peptide-binding groove. We show that the HtrA2/Omi PDZ domain recognizes both C-terminal and internal stretches of extended, hydrophobic polypeptides. High-affinity ligand recognition requires contacts with up to five hydrophobic side chains by distinct sites on the PDZ domain. However, no particular residue type is absolutely required at any position, and thus, the HtrA2/Omi PDZ domain appears to be a promiscuous module adapted to recognize unstructured, hydrophobic polypeptides. This type of specificity is consistent with the biological role of HtrA2/Omi in mitochondria, which requires the recognition of diverse, exposed stretches of hydrophobic sequences in misfolded proteins. The findings are less consistent with, but do not exclude, a role for the PDZ domain in targeting the protease to specific substrates during apoptosis.

Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3.

High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.

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.

Discovery of a Selective and Potent Inhibitor of Mitogen-Activated Protein Kinase-Interacting Kinases 1 and 2 (MNK1/2) Utilizing Structure-Based Drug Design.

The discovery of a highly potent and selective small molecule inhibitor 9 for in vitro target validation of MNK1/2 kinases is described. The aminopyrazine benzimidazole series was derived from an HTS hit and optimized by utilization of a docking model, conformation analysis, and binding pocket comparison against antitargets.

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.

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.

Perception of noxious and innocuous heat stimulation among healthy women and women with fibromyalgia: association with mood, somatic focus, and catastrophizing.

Recent studies have demonstrated that persons with fibromyalgia display abnormal processing of different types of painful stimulation, suggesting the disorder is characterized by a central pain-processing deficit not limited specifically to muscle pain. In the present study, 20 women with fibromyalgia and 20 normal, healthy women were compared on measures of pressure pain stimulation and response to contact thermal heat at both noxious and innocuous intensities. Women with fibromyalgia displayed significantly lower pressure pain thresholds at 18 tender point locations as defined by the American College of Rheumatology criteria, as well as lower pressure pain thresholds at five control sites. Women with fibromyalgia had significantly lower heat pain thresholds and tolerances when stimulated on the volar surface of the left forearm. When examining visual analog ratings of intensity and unpleasantness to constant stimuli, a multivariate analysis of variance performed on these ratings indicated that there were significant main effects of level of stimulation and group. Individual analysis of variances at each temperature revealed significant differences between the groups in pain intensity and unpleasantness ratings at both noxious and innocuous temperatures. Multiple regression analyses indicated that greater pain catastrophizing and diagnosis of fibromyalgia were associated with decreased pain thresholds and tolerances in the entire sample, whereas, self-report of depressive symptoms was associated with increased thresholds and tolerances. Self-report of somatic symptoms was not associated with these measures. These findings indicate that persons with fibromyalgia display altered perception of both pressure and thermal stimulation, even at innocuous levels. They also suggest that catastrophic thoughts about pain may play a role in increased pain perception in this population.