Snapshots and ensembles of BTK and cIAP1 protein degrader ternary complexes.

Heterobifunctional chimeric degraders are a class of ligands that recruit target proteins to E3 ubiquitin ligases to drive compound-dependent protein degradation. Advancing from initial chemical tools, protein degraders represent a mechanism of growing interest in drug discovery. Critical to the mechanism of action is the formation of a ternary complex between the target, degrader and E3 ligase to promote ubiquitination and subsequent degradation. However, limited insights into ternary complex structures exist, including a near absence of studies on one of the most widely co-opted E3s, cellular inhibitor of apoptosis 1 (cIAP1). In this work, we use a combination of biochemical, biophysical and structural studies to characterize degrader-mediated ternary complexes of Bruton's tyrosine kinase and cIAP1. Our results reveal new insights from unique ternary complex structures and show that increased ternary complex stability or rigidity need not always correlate with increased degradation efficiency.

Delineating the role of cooperativity in the design of potent PROTACs for BTK.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.

Inducing protein-protein interactions with molecular glues.

The drugable proteome is limited by the number of functional binding sites that can bind small molecules and respond with a therapeutic effect. Orthosteric and allosteric modulators of enzyme function or receptor signaling are well-established mechanisms of drug action. Drugs that perturb protein-protein interactions have only recently been launched. This approach is more difficult due to the extensive contact surfaces that must be perturbed antagonistically. Compounds that promote novel protein-protein interactions promise to dramatically expand opportunities for therapeutic intervention. This approach is precedented with natural products (rapamycin, FK506, sanglifehrin A), synthetic small molecules (thalidomide and IMiD derivatives) and indisulam analogues.

Heterocyclic methylsulfone hydroxamic acid LpxC inhibitors as Gram-negative antibacterial agents.

The synthesis and antibacterial activity of heterocyclic methylsulfone hydroxamates is presented. Compounds in this series are potent inhibitors of the LpxC enzyme, a key enzyme involved in the production of lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria. SAR evaluation of compounds in this series revealed analogs with potent antibacterial activity against challenging Gram-negative species such as Pseudomonas aeruginosa and Klebsiella pneumoniae.

Novel monobactams utilizing a siderophore uptake mechanism for the treatment of gram-negative infections.

Novel siderophore-linked monobactams with in vitro and in vivo anti-microbial activity against MDR Gram-negative pathogens are described.

An asymmetric synthesis of (2S,5S)-5-substituted azepane-2-carboxylate derivatives.

To facilitate a drug discovery project, we needed to develop a robust asymmetric synthesis of (2S,5S)-5-substituted-azepane-2-carboxylate derivatives. Two key requirements for the synthesis were flexibility for elaboration at C5 and suitability for large scale preparation. To this end we have successfully developed a scalable asymmetric synthesis of these derivatives that starts with known hydroxy-ketone 8. The key step features an oxidative cleavage of aza-bicyclo[3.2.2]nonene 14, which simultaneously generates the C2 and C5 substituents in a stereoselective manner.

Ultra-High-Throughput Acoustic Droplet Ejection-Open Port Interface-Mass Spectrometry for Parallel Medicinal Chemistry.

High-throughput experimentation (HTE) has emerged as an important tool in drug discovery, providing a platform for preparing large compound libraries and enabling swift reaction screening over wide-ranging conditions. Recent advances in automated high-density, material-sparing HTE have necessitated the development of rapid analytics with sensitivity and resolution sufficient to identify products and/or assess reaction performance in a timely and data-rich manner. Combination of an ultrathroughput (UT) reader platform with Acoustic Droplet Ejection-Open Port Interface-Mass Spectrometry (ADE-OPI-MS) provides the requisite speed and sensitivity. Herein, we report the application of ADE-OPI-MS to HTE in the areas of parallel medicinal chemistry and reaction screening.

Potent, selective spiropyrrolidine pyrimidinetrione inhibitors of MMP-13.

Explorations in the pyrimidinetrione series of MMP-13 inhibitors led to the discovery of a series of spiro-fused compounds that are potent and selective inhibitors of MMP-13. While other spiro-fused motifs are hydrolytically unstable, presumably due to electronic destabilization of the pyrimidinetrione ring, the spiropyrrolidine series does not share this liability. Greater than 100-fold selectivity versus other MMP family members was achieved by incorporation of an extended aryl-heteroaryl P1'group. When dosed as the sodium salt, these compounds displayed excellent oral absorption and pharmacokinetic properties. Despite the selectivity, a representative of this series produced fibroplasia in a 14 day rat study.

Pharmacological characterization of CP-547,632, a novel vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for cancer therapy.

Signaling through vascular endothelial growth factor (VEGF) receptors (VEGFRs) is a key pathway initiating endothelial cell proliferation and migration resulting in angiogenesis, a requirement for human tumor growth and metastasis. Abrogation of signaling through VEGFR by a variety of approaches has been demonstrated to inhibit angiogenesis and tumor growth. Small molecule inhibitors of VEGFR tyrosine kinase have been shown to inhibit angiogenesis, inhibit tumor growth, and prevent metastases. Our goal was to discover and characterize an p.o. active VEGFR-2 small molecule inhibitor. A novel isothiazole, CP-547,632, was identified as a potent inhibitor of the VEGFR-2 and basic fibroblast growth factor (FGF) kinases (IC(50) = 11 and 9 nM, respectively). It is selective relative to epidermal growth factor receptor, platelet-derived growth factor beta, and other related TKs. It also inhibits VEGF-stimulated autophosphorylation of VEGFR-2 in a whole cell assay with an IC(50) value of 6 nM. After oral administration of CP-547,632 to mice bearing NIH3T3/H-ras tumors, VEGFR-2 phosphorylation in tumors was inhibited in a dose-dependent fashion (EC(50) = 590 ng/ml). These plasma concentrations correlated well with the observed concentrations of the compound necessary to inhibit VEGF-induced corneal angiogenesis in BALB/c mice. A sponge angiogenesis assay was used to directly compare the inhibitory activities of CP-547,632 against FGF receptor 2 or VEGFR-2; this compound potently inhibits both basic FGF and VEGF-induced angiogenesis in vivo. The antitumor efficacy of this agent was evaluated after once daily p.o. administration to athymic mice bearing human xenografts and resulted in as much as 85% tumor growth inhibition. CP-547,632 is a well-tolerated, orally-bioavailable inhibitor presently under clinical investigation for the treatment of human malignancies.

Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors.

Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymatic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry additional risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biologically relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.

Pyridone-conjugated monobactam antibiotics with gram-negative activity.

Herein we describe the structure-aided design and synthesis of a series of pyridone-conjugated monobactam analogues with in vitro antibacterial activity against clinically relevant Gram-negative species including Pseudomonas aeruginosa , Klebsiella pneumoniae , and Escherichia coli . Rat pharmacokinetic studies with compound 17 demonstrate low clearance and low plasma protein binding. In addition, evidence is provided for a number of analogues suggesting that the siderophore receptors PiuA and PirA play a role in drug uptake in P. aeruginosa strain PAO1.

Discovery of Dap-3 polymyxin analogues for the treatment of multidrug-resistant Gram-negative nosocomial infections.

We report novel polymyxin analogues with improved antibacterial in vitro potency against polymyxin resistant recent clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa . In addition, a human renal cell in vitro assay (hRPTEC) was used to inform structure-toxicity relationships and further differentiate analogues. Replacement of the Dab-3 residue with a Dap-3 in combination with a relatively polar 6-oxo-1-phenyl-1,6-dihydropyridine-3-carbonyl side chain as a fatty acyl replacement yielded analogue 5x, which demonstrated an improved in vitro antimicrobial and renal cytotoxicity profiles relative to polymyxin B (PMB). However, in vivo PK/PD comparison of 5x and PMB in a murine neutropenic thigh model against P. aeruginosa strains with matched MICs showed that 5x was inferior to PMB in vivo, suggesting a lack of improved therapeutic index in spite of apparent in vitro advantages.

Potent inhibitors of LpxC for the treatment of Gram-negative infections.

In this paper, we present the synthesis and SAR as well as selectivity, pharmacokinetic, and infection model data for representative analogues of a novel series of potent antibacterial LpxC inhibitors represented by hydroxamic acid.

Pyridone methylsulfone hydroxamate LpxC inhibitors for the treatment of serious gram-negative infections.

The synthesis and biological activity of a new series of LpxC inhibitors represented by pyridone methylsulfone hydroxamate 2a is presented. Members of this series have improved solubility and free fraction when compared to compounds in the previously described biphenyl methylsulfone hydroxamate series, and they maintain superior Gram-negative antibacterial activity to comparator agents.

Linezolid, the first oxazolidinone antibacterial agent.

Linezolid (Zyvox) is the first member of an entirely new class of antibiotics to reach the market in over 35 years; it was approved for use in 2000. A member of the oxazolidinone class of antibiotics, linezolid is highly effective for the treatment of serious Gram-positive infections and has activity that compares favorably with vancomycin for most clinically relevant pathogens. Zyvox is approved for use against serious Gram-positive infections, including those caused by Streptococcus pneumoniae, and the very challenging methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium organisms. Zyvox inhibits bacterial protein synthesis by binding to 23S rRNA in the catalytic site of the 50S ribosome. It can be administered both orally and intravenously and has good tissue distribution. Recent results have demonstrated that oxazolidinone analogs related to linezolid are effective in treating pulmonary tuberculosis caused by resistant Mycobacterium tuberculosis in animal infection models and suggest additional new therapeutic applications for these antibiotics.

Preparation, gram-negative antibacterial activity, and hydrolytic stability of novel siderophore-conjugated monocarbam diols.

A novel series of monocarbam compounds exhibiting promising antibacterial activity against multidrug resistant Gram-negative microorganisms is reported, along with the synthesis of one such molecule MC-1 (1). Also reported are structure-activity relationships associated with the in vitro and in vivo efficacy of 1 and related analogues in addition to the hydrolytic stability of such compounds and possible implications thereof.

Discovery of azetidinyl ketolides for the treatment of susceptible and multidrug resistant community-acquired respiratory tract infections.

Respiratory tract bacterial strains are becoming increasingly resistant to currently marketed macrolide antibiotics. The current alternative telithromycin (1) from the newer ketolide class of macrolides addresses resistance but is hampered by serious safety concerns, hepatotoxicity in particular. We have discovered a novel series of azetidinyl ketolides that focus on mitigation of hepatotoxicity by minimizing hepatic turnover and time-dependent inactivation of CYP3A isoforms in the liver without compromising the potency and efficacy of 1.

A short enantioselective synthesis of N-Boc-(2R,3R)-3-methyl-3-hydroxypipecolic acid from geraniol.

The asymmetric synthesis of (2R,3R)-3-methyl-3-hydroxypipecolic acid, a key intermediate in the synthesis of dual MMP-13/aggrecanase inhibitors, is described. The title compound is prepared in seven steps with an overall yield of 41% starting from geraniol. Key steps in the synthesis include Sharpless asymmetric epoxidation, which establishes the chiral centers, and a one-pot oxidative olefin cleavage/reductive amination sequence that closes the piperidine ring.

Discovery of novel isothiazole inhibitors of the TrkA kinase: structure-activity relationship, computer modeling, optimization, and identification of highly potent antagonists.

The design, synthesis, and biological evaluation of potent inhibitors of the TrkA kinase is presented. A homology model is created to aid in the enhancement of potency and selectivity of isothiazole inhibitors found during a high-throughput screen. Three different syntheses are utilized to make diverse analogs within this series. Aminoheterocycles are found to be good urea surrogates, whereas bicyclic substituents on the C3 thio group were found to be extremely potent TrkA inhibitors in kinase and cell assays.

Discovery of 3-OH-3-methylpipecolic hydroxamates: potent orally active inhibitors of aggrecanase and MMP-13.

A series of 3-hydroxy-3-methylpipecolic hydroxamate inhibitors of MMP-13 and aggrecanase was designed based on the observation of increased aggrecanase activity with substitution at the 3-position of the piperidine ring. Potency versus aggrecanase was optimized by modification of the benzyloxyarylsulfonamide group that binds in the S1' pocket. These compounds also possess markedly improved bioavailability and lower metabolic clearance compared to analogous 3,3-dimethyl-5-hydroxypipecolic hydroxamates. These improvements are attributed to lowered lipophilicity proximal to the metabolically labile hydroxamic acid. Synthesis, structure activity relationships, and in vivo efficacy data are described.