Discovery of 4-aminopyrimidine analogs as highly potent dual P70S6K/Akt inhibitors.

Activation of the PI3K/Akt/mTOR kinase pathway is associated with human cancers. A dual p70S6K/Akt inhibitor is sufficient to inhibit strong tumor growth and to block negative impact of the compensatory Akt feedback loop activation. A scaffold docking strategy based on an existing quinazoline carboxamide series identified 4-aminopyrimidine analog 6, which showed a single-digit nanomolar and a micromolar potencies in p70S6K and Akt enzymatic assays. SAR optimization improved Akt enzymatic and p70S6K cellular potencies, reduced hERG liability, and ultimately discovered the promising candidate 37, which exhibited with a single digit nanomolar value in both p70S6K and Akt biochemical assays, and hERG activities (IC50 = 17.4 µM). This agent demonstrated dose-dependent efficacy in inhibiting mice breast cancer tumor growth and covered more than 90% pS6 inhibition up to 24 h at a dose of 200 mg/kg po.

Large-Scale Assessment of Binding Free Energy Calculations in Active Drug Discovery Projects.

Accurate ranking of compounds with regards to their binding affinity to a protein using computational methods is of great interest to pharmaceutical research. Physics-based free energy calculations are regarded as the most rigorous way to estimate binding affinity. In recent years, many retrospective studies carried out both in academia and industry have demonstrated its potential. Here, we present the results of large-scale prospective application of the FEP+ method in active drug discovery projects in an industry setting at Merck KGaA, Darmstadt, Germany. We compare these prospective data to results obtained on a new diverse, public benchmark of eight pharmaceutically relevant targets. Our results offer insights into the challenges faced when using free energy calculations in real-life drug discovery projects and identify limitations that could be tackled by future method development. The new public data set we provide to the community can support further method development and comparative benchmarking of free energy calculations.

Discovery of a novel series of pyridine and pyrimidine carboxamides as potent and selective covalent inhibitors of Btk.

Btk is an attractive target for the treatment of a range of Bcell malignancies as well as several autoimmune diseases such as murine lupus and rheumatoid arthritis. Several covalent irreversible inhibitors of Btk are currently in development including ibrutinib which was approved for treatment of B-cell malignancies. Herein, we describe our efforts using X-ray guided structure based design (SBD) to identify a novel chemical series of covalent Btk inhibitors. The resulting pyridine carboxamides were potent and selective inhibitors of Btk having excellent enzymatic and cellular inhibitory activity.

Discovery of potent, highly selective covalent irreversible BTK inhibitors from a fragment hit.

Bruton's Tyrosine Kinase (BTK) is a member of the TEC kinase family that is expressed in cells of hematopoietic lineage (e.g., in B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible BTK inhibitor targeting Cys481 within the ATP-binding pocket, for example ibrutinib, has been applied in the treatment of B-cell malignancies. Starting from a fragment hit, we discovered a novel series of potent covalent irreversible BTK inhibitors that occupy selectivity pocket of the active site of the BTK kinase domain. Guided by X-ray structures and a fragment-based drug design (FBDD) approach, we generated molecules showing comparable cellular potency to ibrutinib and higher kinome selectivity against undesirable off-targets like EGFR.

Optimization of the efflux ratio and permeability of covalent irreversible BTK inhibitors.

Bruton's tyrosine kinase (Btk) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage (e.g. B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible Btk inhibitors targeting Cys481 within the ATP-binding pocket have been applied in the treatment of B-cell malignancies. Starting from a fragment, we discovered a novel series of potent covalent irreversible Btk inhibitors that bear N-linked groups occupying the solvent accessible pocket (SAP) of the active site of the Btk kinase domain. The hit molecules, however, displayed high P-gp mediated efflux ratio (ER) and poor A-B permeability in Caco-2 assay. By decreasing tPSA, installing steric hindrance and adjusting clogP, one top molecule 9 was discovered, which showed a 99% decrease in efflux ratio and a 90-fold increase in A-B permeability compared to hit molecule 1.

Recent progress towards clinically relevant ATP-competitive Akt inhibitors.

The frequency of PI3K/Akt/mTOR (PAM) Pathway mutations in human cancers sparked interest to determine if the pathway is druggable. The modest clinical benefit observed with mTOR rapalogs (temsirolimus and everolimus) provided further motivation to identify additional nodes of pathway inhibition that lead to improved clinical benefit. Akt is a central signaling node of the PAM pathway and could be an ideal target for improved pathway inhibition. Furthermore, inhibitors of Akt may be especially beneficial in tumors with Akt1 mutations. Recently, multiple ATP-competitive Akt inhibitors have been identified and are currently in clinical development. This review details the medicinal chemistry efforts towards identification of these molecules, highlights relevant preclinical data supporting clinical evaluation, and summarizes current clinical development plans.

Structure based design of an in vivo active hydroxamic acid inhibitor of P. aeruginosa LpxC.

Lipid A is an essential component of the Gram negative outer membrane, which protects the bacterium from attack of many antibiotics. The Lipid A biosynthesis pathway is essential for Gram negative bacterial growth and is unique to these bacteria. The first committed step in Lipid A biosynthesis is catalysis by LpxC, a zinc dependent deacetylase. We show the design of an LpxC inhibitor utilizing a robust model which directed efficient design of picomolar inhibitors. Analysis of physiochemical properties drove design to focus on an optimal lipophilicity profile. Further structure based design took advantage of a conserved water network over the active site, and with the optimal lipophilicity profile, led to an improved LpxC inhibitor with in vivo activity against wild type Pseudomonas aeruginosa.

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore.

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

Identification of Clinical Candidate M2698, a Dual p70S6K and Akt Inhibitor, for Treatment of PAM Pathway-Altered Cancers.

Herein, we report the discovery of a novel class of quinazoline carboxamides as dual p70S6k/Akt inhibitors for the treatment of tumors driven by alterations to the PI3K/Akt/mTOR (PAM) pathway. Through the screening of in-house proprietary kinase library, 4-benzylamino-quinazoline-8-carboxylic acid amide 1 stood out, with sub-micromolar p70S6k biochemical activity, as the starting point for a structurally enabled p70S6K/Akt dual inhibitor program that led to the discovery of M2698, a dual p70S6k/Akt inhibitor. M2698 is kinase selective, possesses favorable physical, chemical, and DMPK profiles, is orally available and well tolerated, and displayed tumor control in multiple in vivo studies of PAM pathway-driven tumors.

Identification of annotated bioactive molecules that impair motility of the blood fluke Schistosoma mansoni.

Neglected tropical diseases are of growing worldwide concern and schistosomiasis, caused by parasitic flatworms, continues to be a major threat with more than 200 million people requiring preventive treatment. As praziquantel (PZQ) remains the treatment of choice, an urgent need for alternative treatments motivates research to identify new lead compounds that would complement PZQ by filling the therapeutic gaps associated with this treatment. Because impairing parasite neurotransmission remains a core strategy for control of parasitic helminths, we screened a library of 708 compounds with validated biological activity in humans on the blood fluke Schistosoma mansoni, measuring their effect on the motility on schistosomulae and adult worms. The primary phenotypic screen performed on schistosomulae identified 70 compounds that induced changes in viability and/or motility. Screening different concentrations and incubation times identified molecules with fast onset of activity on both life stages at low concentration (1 µM). To complement this study, similar assays were performed with chemical analogs of the cholinomimetic drug arecoline and the calcilytic molecule NPS-2143, two compounds that rapidly inhibited schistosome motility; 17 arecoline and 302 NPS-2143 analogs were tested to enlarge the pool of schistosomicidal molecules. Finally, validated hit compounds were tested on three functionally-validated neuroregulatory S. mansoni G-protein coupled receptors (GPCRs): Sm5HTR (serotonin-sensitive), SmGPR2 (histamine) and SmD2 (dopamine), revealing NPS-2143 and analogs as potent inhibitors of dopamine/epinine responses on both human and S. mansoni GPCRs. This study highlights the potential for repurposing known human therapeutic agents for potential schistosomicidal effects and expands the list of hits for further progression.

Exploration of 4,4-disubstituted pyrrolidine-1,2-dicarboxamides as potent, orally active Factor Xa inhibitors with extended duration of action.

Aiming to improve upon previously disclosed Factor Xa inhibitors, a series of 4,4-disubstituted pyrrolidine-1,2-dicarboxamides were explored with the intent of increasing the projected human half-life versus 5 (projected human t(1/2)=6 h). A stereospecific route to compounds containing a 4-aryl-4-hydroxypyrrolidine scaffold was developed, resulting in several compounds that demonstrated an increase in the half-life as well as an increase in the in vitro potency compared to 5. Reported herein is the discovery of 26, containing a (2R,4S)-4-hydroxy-4-(2,4-difluorophenyl)-pyrrolidine scaffold, which is a selective, orally bioavailable, efficacious Factor Xa inhibitor that appears suitable for a once-daily dosing (projected human t(1/2)=23 h).

Discovery of Affinity-Based Probes for Btk Occupancy Assays.

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.

Discovery of Evobrutinib: An Oral, Potent, and Highly Selective, Covalent Bruton's Tyrosine Kinase (BTK) Inhibitor for the Treatment of Immunological Diseases.

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Crystal structure of LpxC from Pseudomonas aeruginosa complexed with the potent BB-78485 inhibitor.

The cell wall in Gram-negative bacteria is surrounded by an outer membrane comprised of charged lipopolysaccharide (LPS) molecules that prevent entry of hydrophobic agents into the cell and protect the bacterium from many antibiotics. The hydrophobic anchor of LPS is lipid A, the biosynthesis of which is essential for bacterial growth and viability. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is an essential zinc-dependant enzyme that catalyzes the conversion of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)glucosamine and acetate in the biosynthesis of lipid A, and for this reason, LpxC is an attractive target for antibacterial drug discovery. Here we disclose a 1.9 A resolution crystal structure of LpxC from Pseudomonas aeruginosa (paLpxC) in a complex with the potent BB-78485 inhibitor. To our knowledge, this is the first crystal structure of LpxC with a small-molecule inhibitor that shows antibacterial activity against a wide range of Gram-negative pathogens. Accordingly, this structure can provide important information for lead optimization and rational design of the effective small-molecule LpxC inhibitors for successful treatment of Gram-negative infections.

Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site.

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram-negative and Gram-positive bacteria. Here we disclose a 1.9 A resolution crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high-throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC(50) approximately 18 microM in a racemic mixture) against hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc-1-P substrate-binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure-guided design of a new class of mechanism-based inhibitors of GlmU.

Characterization of substrate binding and catalysis in the potential antibacterial target N-acetylglucosamine-1-phosphate uridyltransferase (GlmU).

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the first step in peptidoglycan biosynthesis in both Gram-positive and Gram-negative bacteria. The products of the GlmU reaction are essential for bacterial survival, making this enzyme an attractive target for antibiotic drug discovery. A series of Haemophilus influenzae GlmU (hiGlmU) structures were determined by X-ray crystallography in order to provide structural and functional insights into GlmU activity and inhibition. The information derived from these structures was combined with biochemical characterization of the K25A, Q76A, D105A, Y103A, V223A, and E224A hiGlmU mutants in order to map these active-site residues to catalytic activity of the enzyme and refine the mechanistic model of the GlmU uridyltransferase reaction. These studies suggest that GlmU activity follows a sequential substrate-binding order that begins with UTP binding noncovalently to the GlmU enzyme. The uridyltransferase active site then remains in an open apo-like conformation until N-acetylglucosamine-1-phosphate (GlcNAc-1-P) binds and induces a conformational change at the GlcNAc-binding subsite. Following the binding of GlcNAc-1-P to the UTP-charged uridyltransferase active site, the non-esterified oxygen of GlcNAc-1-P performs a nucleophilic attack on the alpha-phosphate group of UTP. The new data strongly suggest that the mechanism of phosphotransfer in the uridyltransferase reaction in GlmU is primarily through an associative mechanism with a pentavalent phosphate intermediate and an inversion of stereochemistry. Finally, the structural and biochemical characterization of the uridyltransferase active site and catalytic mechanism described herein provides a basis for the structure-guided design of novel antibacterial agents targeting GlmU activity.

Oncogenic BRAF Deletions That Function as Homodimers and Are Sensitive to Inhibition by RAF Dimer Inhibitor LY3009120.

UNLABELLED: We have identified previously undiscovered BRAF in-frame deletions near the αC-helix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers. These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the ß3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors dimer formation. Expression of L485-P490-deleted BRAF is able to transform NIH/3T3 cells in a BRAF dimer-dependent manner. BRAF homodimer is confirmed to be the dominant RAF dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions, LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive. SIGNIFICANCE: This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer.

Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers.

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

Discovery of antibacterial biotin carboxylase inhibitors by virtual screening and fragment-based approaches.

As part of our effort to inhibit bacterial fatty acid biosynthesis through the recently validated target biotin carboxylase, we employed a unique combination of two emergent lead discovery strategies. We used both de novo fragment-based drug discovery and virtual screening, which employs 3D shape and electrostatic property similarity searching. We screened a collection of unbiased low-molecular-weight molecules and identified a structurally diverse collection of weak-binding but ligand-efficient fragments as potential building blocks for biotin carboxylase ATP-competitive inhibitors. Through iterative cycles of structure-based drug design relying on successive fragment costructures, we improved the potency of the initial hits by up to 3000-fold while maintaining their ligand-efficiency and desirable physicochemical properties. In one example, hit-expansion efforts resulted in a series of amino-oxazoles with antibacterial activity. These results successfully demonstrate that virtual screening approaches can substantially augment fragment-based screening approaches to identify novel antibacterial agents.