A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase.

New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes-primarily those involved in macromolecular synthesis-are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α-ß-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

Translating slow-binding inhibition kinetics into cellular and in vivo effects.

Many drug candidates fail in clinical trials owing to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug-target complex has a longer half-life than off-target-drug complexes. However, though slow-binding inhibition kinetics are a key feature of many marketed drugs, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters, including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.

Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors.

The structure-activity and structure-kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11Å channel leading to the Zn(2+) catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.

Novel broad-spectrum inhibitors of bacterial methionine aminopeptidase.

With increasing emergence of multi-drug resistant infections, there is a dire need for new classes of compounds that act through unique mechanisms. In this work, we describe the discovery and optimization of a novel series of inhibitors of bacterial methionine aminopeptidase (MAP). Through a high-throughput screening campaign, one azepinone amide hit was found that resembled the native peptide substrate and possessed moderate biochemical potency against three bacterial isozymes. X-ray crystallography was used in combination with substrate-based design to direct the rational optimization of analogs with sub-micromolar potency. The novel compounds presented here represent potent broad-spectrum biochemical inhibitors of bacterial MAP and have the potential to lead to the development of new medicines to combat serious multi-drug resistant infections.

Avibactam is a covalent, reversible, non-ß-lactam ß-lactamase inhibitor.

Avibactam is a ß-lactamase inhibitor that is in clinical development, combined with ß-lactam partners, for the treatment of bacterial infections comprising gram-negative organisms. Avibactam is a structural class of inhibitor that does not contain a ß-lactam core but maintains the capacity to covalently acylate its ß-lactamase targets. Using the TEM-1 enzyme, we characterized avibactam inhibition by measuring the on-rate for acylation and the off-rate for deacylation. The deacylation off-rate was 0.045 min(-1), which allowed investigation of the deacylation route from TEM-1. Using NMR and MS, we showed that deacylation proceeds through regeneration of intact avibactam and not hydrolysis. Other than TEM-1, four additional clinically relevant ß-lactamases were shown to release intact avibactam after being acylated. We showed that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among ß-lactamase inhibitors.

Kinetics of avibactam inhibition against Class A, C, and D ß-lactamases.

Avibactam is a non-ß-lactam ß-lactamase inhibitor with a spectrum of activity that includes ß-lactamase enzymes of classes A, C, and selected D examples. In this work acylation and deacylation rates were measured against the clinically important enzymes CTX-M-15, KPC-2, Enterobacter cloacae AmpC, Pseudomonas aeruginosa AmpC, OXA-10, and OXA-48. The efficiency of acylation (k2/Ki) varied across the enzyme spectrum, from 1.1 × 10(1) m(-1)s(-1) for OXA-10 to 1.0 × 10(5) for CTX-M-15. Inhibition of OXA-10 was shown to follow the covalent reversible mechanism, and the acylated OXA-10 displayed the longest residence time for deacylation, with a half-life of greater than 5 days. Across multiple enzymes, acyl enzyme stability was assessed by mass spectrometry. These inhibited enzyme forms were stable to rearrangement or hydrolysis, with the exception of KPC-2. KPC-2 displayed a slow hydrolytic route that involved fragmentation of the acyl-avibactam complex. The identity of released degradation products was investigated, and a possible mechanism for the slow deacylation from KPC-2 is proposed.

Mechanistic insights into a heterobifunctional degrader-induced PTPN2/N1 complex.

PTPN2 (protein tyrosine phosphatase non-receptor type 2, or TC-PTP) and PTPN1 are attractive immuno-oncology targets, with the deletion of Ptpn1 and Ptpn2 improving response to immunotherapy in disease models. Targeted protein degradation has emerged as a promising approach to drug challenging targets including phosphatases. We developed potent PTPN2/N1 dual heterobifunctional degraders (Cmpd-1 and Cmpd-2) which facilitate efficient complex assembly with E3 ubiquitin ligase CRL4CRBN, and mediate potent PTPN2/N1 degradation in cells and mice. To provide mechanistic insights into the cooperative complex formation introduced by degraders, we employed a combination of structural approaches. Our crystal structure reveals how PTPN2 is recognized by the tri-substituted thiophene moiety of the degrader. We further determined a high-resolution structure of DDB1-CRBN/Cmpd-1/PTPN2 using single-particle cryo-electron microscopy (cryo-EM). This structure reveals that the degrader induces proximity between CRBN and PTPN2, albeit the large conformational heterogeneity of this ternary complex. The molecular dynamic (MD)-simulations constructed based on the cryo-EM structure exhibited a large rigid body movement of PTPN2 and illustrated the dynamic interactions between PTPN2 and CRBN. Together, our study demonstrates the development of PTPN2/N1 heterobifunctional degraders with potential applications in cancer immunotherapy. Furthermore, the developed structural workflow could help to understand the dynamic nature of degrader-induced cooperative ternary complexes.

Exploitation of structural and regulatory diversity in glutamate racemases.

Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we describe three distinct mechanisms of regulation for the family of glutamate racemases: allosteric activation by metabolic precursors, kinetic regulation through substrate inhibition, and D-glutamate recycling using a d-amino acid transaminase. In a search for selective inhibitors, we identified a series of uncompetitive inhibitors specifically targeting Helicobacter pylori glutamate racemase that bind to a cryptic allosteric site, and used these inhibitors to probe the mechanistic and dynamic features of the enzyme. These structural, kinetic and mutational studies provide insight into the physiological regulation of these essential enzymes and provide a basis for designing narrow-spectrum antimicrobial agents.

High-Throughput Quantitative Assay Technologies for Accelerating the Discovery and Optimization of Targeted Protein Degradation Therapeutics.

The aberrant regulation of protein expression and function can drastically alter cellular physiology and lead to numerous pathophysiological conditions such as cancer, inflammatory diseases, and neurodegeneration. The steady-state expression levels of endogenous proteins are controlled by a balance of de novo synthesis rates and degradation rates. Moreover, the levels of activated proteins in signaling cascades can be further modulated by a variety of posttranslational modifications and protein-protein interactions. The field of targeted protein degradation is an emerging area for drug discovery in which small molecules are used to recruit E3 ubiquitin ligases to catalyze the ubiquitination and subsequent degradation of disease-causing target proteins by the proteasome in both a dose- and time-dependent manner. Traditional approaches for quantifying protein level changes in cells, such as Western blots, are typically low throughput with limited quantification, making it hard to drive the rapid development of therapeutics that induce selective, rapid, and sustained protein degradation. In the last decade, a number of techniques and technologies have emerged that have helped to accelerate targeted protein degradation drug discovery efforts, including the use of fluorescent protein fusions and reporter tags, flow cytometry, time-resolved fluorescence energy transfer (TR-FRET), and split luciferase systems. Here we discuss the advantages and disadvantages associated with these technologies and their application to the development and optimization of degraders as therapeutics.

Evaluation of the Small-molecule BRD4 Degrader CFT-2718 in Small-cell Lung Cancer and Pancreatic Cancer Models.

Targeted, catalytic degradation of oncoproteins using heterobifunctional small molecules is an attractive modality, particularly for hematologic malignancies, which are often initiated by aberrant transcription factors and are challenging to drug with inhibitors. BRD4, a member of the bromodomain and extraterminal family, is a core transcriptional and epigenetic regulator that recruits the P-TEFb complex, which includes Cdk9 and cyclin T, to RNA polymerase II (pol II). Together, BRD4 and CDK9 phosphorylate serine 2 (pSer2) of heptad repeats in the C-terminal domain of RPB1, the large subunit of pol II, promote transcriptional elongation. Small-molecule degraders of BRD4 have shown encouraging efficacy in preclinical models for several tumor types but less efficacy in other cancers including small-cell lung cancer (SCLC) and pancreatic cancer. Here, we evaluated CFT-2718, a new BRD4-targeting degrader with enhanced catalytic activity and in vivo properties. In vivo, CFT-2718 has significantly greater efficacy than the CDK9 inhibitor dinaciclib in reducing growth of the LX-36 SCLC patient-derived xenograft (PDX) model and performed comparably to dinaciclib in limiting growth of the PNX-001 pancreatic PDX model. In vitro, CFT-2718 reduced cell viability in four SCLC and two pancreatic cancer models. In SCLC models, this activity significantly exceeded that of dinaciclib; furthermore, CFT-2718 selectively increased the expression of cleaved PARP, an indicator of apoptosis. CFT-2718 caused rapid BRD4 degradation and reduced levels of total and pSer2 RPB1 protein. These and other findings suggest that BRD-mediated transcriptional suppression merits further exploration in the setting of SCLC.

Structural Characterization of Degrader-Induced Ternary Complexes Using Hydrogen-Deuterium Exchange Mass Spectrometry and Computational Modeling: Implications for Structure-Based Design.

The field of targeted protein degradation (TPD) has grown exponentially over the past decade with the goal of developing therapies that mark proteins for destruction leveraging the ubiquitin-proteasome system. One common approach to achieve TPD is to employ a heterobifunctional molecule, termed as a degrader, to recruit the protein target of interest to the E3 ligase machinery. The resultant generation of an intermediary ternary complex (target-degrader-ligase) is pivotal in the degradation process. Understanding the ternary complex geometry offers valuable insight into selectivity, catalytic efficiency, linker chemistry, and rational degrader design. In this study, we utilize hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify degrader-induced protein-protein interfaces. We then use these data in conjunction with constrained protein docking to build three-dimensional models of the ternary complex. The approach was used to characterize complex formation between the E3 ligase CRBN and the first bromodomain of BRD4, a prominent oncology target. We show marked differences in the ternary complexes formed in solution based on distinct patterns of deuterium uptake for two degraders, CFT-1297 and dBET6. CFT-1297, which exhibited positive cooperativity, altered the deuterium uptake profile revealing the degrader-induced protein-protein interface of the ternary complex. For CFT-1297, the ternary complexes generated by the highest scoring HDX-constrained docking models differ markedly from those observed in the published crystal structures. These results highlight the potential utility of HDX-MS to provide rapidly accessible structural insights into degrader-induced protein-protein interfaces in solution. They further suggest that degrader ternary complexes exhibit significant conformation flexibility and that biologically relevant complexes may well not exhibit the largest interaction surfaces between proteins. Taken together, the results indicate that methods capable of incorporating linker conformation uncertainty may prove an important component in degrader design moving forward. In addition, the development of scoring functions modified to handle interfaces with no evolved complementarity, for example, through consideration of high levels of water infiltration, may prove valuable. Furthermore, the use of crystal structures as validation tools for novel degrader methods needs to be considered with caution.

A Method for Determining the Kinetics of Small-Molecule-Induced Ubiquitination.

Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin-proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.

Conservation of the structure and function of bacterial tryptophan synthases.

Tryptophan biosynthesis is one of the most characterized processes in bacteria, in which the enzymes from Salmonella typhimurium and Escherichia coli serve as model systems. Tryptophan synthase (TrpAB) catalyzes the final two steps of tryptophan biosynthesis in plants, fungi and bacteria. This pyridoxal 5'-phosphate (PLP)-dependent enzyme consists of two protein chains, α (TrpA) and ß (TrpB), functioning as a linear αßßα heterotetrameric complex containing two TrpAB units. The reaction has a complicated, multistep mechanism resulting in the ß-replacement of the hydroxyl group of l-serine with an indole moiety. Recent studies have shown that functional TrpAB is required for the survival of pathogenic bacteria in macrophages and for evading host defense. Therefore, TrpAB is a promising target for drug discovery, as its orthologs include enzymes from the important human pathogens Streptococcus pneumoniae, Legionella pneumophila and Francisella tularensis, the causative agents of pneumonia, legionnaires' disease and tularemia, respectively. However, specific biochemical and structural properties of the TrpABs from these organisms have not been investigated. To fill the important phylogenetic gaps in the understanding of TrpABs and to uncover unique features of TrpAB orthologs to spearhead future drug-discovery efforts, the TrpABs from L. pneumophila, F. tularensis and S. pneumoniae have been characterized. In addition to kinetic properties and inhibitor-sensitivity data, structural information gathered using X-ray crystallo-graphy is presented. The enzymes show remarkable structural conservation, but at the same time display local differences in both their catalytic and allosteric sites that may be responsible for the observed differences in catalysis and inhibitor binding. This functional dissimilarity may be exploited in the design of species-specific enzyme inhibitors.

Targeted protein degradation and the enzymology of degraders.

Targeted protein degradation is an emerging strategy for drug discovery that employs small molecules to catalyze the ubiquitination of target proteins, ultimately causing their degradation by the proteasome. Current degrader designs employ hetero-bivalent molecules to recruit E3 ubiquitin ligases such as VHL, Cereblon, and the IAPs to the target protein to be ubiquitinated. In this review, we describe some of the foundational studies underpinning the use of heterobivalent degraders for targeted protein degradation. We also present a framework for degraders as programmable essential activators of ubiquitin ligase enzymes, connecting their features as catalysts with established enzymology concepts.

A quantitative mechanistic PK/PD model directly connects Btk target engagement and in vivo efficacy.

Correlating target engagement with in vivo drug activity remains a central challenge in efforts to improve the efficiency of drug discovery. Previously we described a mechanistic pharmacokinetic-pharmacodynamic (PK/PD) model that used drug-target binding kinetics to successfully predict the in vivo efficacy of antibacterial compounds in models of Pseudomonas aeruginosa and Staphylococcus aureus infection. In the present work we extend this model to quantitatively correlate the engagement of Bruton's tyrosine kinase (Btk) by the covalent inhibitor CC-292 with the ability of this compound to reduce ankle swelling in an animal model of arthritis. The modeling studies include the rate of Btk turnover and reveal the vulnerability of Btk to engagement by CC-292.

Correlating Drug-Target Kinetics and In vivo Pharmacodynamics: Long Residence Time Inhibitors of the FabI Enoyl-ACP Reductase.

Drug-target kinetics enable time-dependent changes in target engagement to be quantified as a function of drug concentration. When coupled to drug pharmacokinetics (PK), drug-target kinetics can thus be used to predict in vivo pharmacodynamics (PD). Previously we described a mechanistic PK/PD model that successfully predicted the antibacterial activity of an LpxC inhibitor in a model of Pseudomonas aeruginosa infection. In the present work we demonstrate that the same approach can be used to predict the in vivo activity of an enoyl-ACP reductase (FabI) inhibitor in a model of methicillin-resistant Staphylococcus aureus (MRSA) infection. This is significant because the LpxC inhibitors are cidal, whereas the FabI inhibitors are static. In addition P. aeruginosa is a Gram-negative organism whereas MRSA is Gram-positive. Thus this study supports the general applicability of our modeling approach across antibacterial space.

An Isochemogenic Set of Inhibitors To Define the Therapeutic Potential of Histone Deacetylases in ß-Cell Protection.

Modulation of histone deacetylase (HDAC) activity has been implicated as a potential therapeutic strategy for multiple diseases. However, it has been difficult to dissect the role of individual HDACs due to a lack of selective small-molecule inhibitors. Here, we report the synthesis of a series of highly potent and isoform-selective class I HDAC inhibitors, rationally designed by exploiting minimal structural changes to the clinically experienced HDAC inhibitor CI-994. We used this toolkit of isochemogenic or chemically matched inhibitors to probe the role of class I HDACs in ß-cell pathobiology and demonstrate for the first time that selective inhibition of an individual HDAC isoform retains beneficial biological activity and mitigates mechanism-based toxicities. The highly selective HDAC3 inhibitor BRD3308 suppressed pancreatic ß-cell apoptosis induced by inflammatory cytokines, as expected, or now glucolipotoxic stress, and increased functional insulin release. In addition, BRD3308 had no effect on human megakaryocyte differentiation, while inhibitors of HDAC1 and 2 were toxic. Our findings demonstrate that the selective inhibition of HDAC3 represents a potential path forward as a therapy to protect pancreatic ß-cells from inflammatory cytokines and nutrient overload in diabetes.

Biochemical characterization of an inhibitor of Escherichia coli UDP-N-acetylmuramyl-l-alanine ligase.

UDP-N-acetylmuramyl-l-alanine ligase (MurC) is an essential bacterial enzyme involved in peptidoglycan biosynthesis and a target for the discovery of novel antibacterial agents. As a result of a high-throughput screen (HTS) against a chemical library for inhibitors of MurC, a series of benzofuran acyl-sulfonamides was identified as potential leads. One of these compounds, Compound A, inhibited Escherichia coli MurC with an IC(50) of 2.3 microM. Compound A exhibited time-dependent, partially reversible inhibition of E. coli MurC. Kinetic studies revealed a mode of inhibition consistent with the compound acting competitively with the MurC substrates ATP and UDP-N-acetyl-muramic acid (UNAM) with a K(i) of 4.5 microM against ATP and 6.3 microM against UNAM. Fluorescence binding experiments yielded a K(d) of 3.1 microM for the compound binding to MurC. Compound A also exhibited high-affinity binding to bovine serum albumin (BSA) as evidenced by a severe reduction in MurC inhibition upon addition of BSA. This finding is consistent with the high lipophilicity of the compound. Advancement of this compound series for further drug development will require reduction of albumin binding.

Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis.

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated.