Targeted protein degradation has emerged from the chemical biology toolbox as one of the most exciting areas for novel therapeutic development across the pharmaceutical industry. The ability to ...induce the degradation, and not just inhibition, of target proteins of interest (POIs) with high potency and selectivity is a particularly attractive property for a protein degrader therapeutic. However, the physicochemical properties and mechanism of action for protein degraders can lead to unique pharmacokinetic (PK) and pharmacodynamic (PD) properties relative to traditional small molecule drugs, requiring a shift in perspective for translational pharmacology. In this review, we provide practical insights for building the PK-PD understanding of protein degraders in the context of translational drug development through the use of quantitative mathematical frameworks and standard experimental assays. Published datasets describing protein degrader pharmacology are used to illustrate the applicability of these insights. The learnings are consolidated into a translational PK-PD roadmap for targeted protein degradation that can enable a systematic, rational design workflow for protein degrader therapeutics.
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.
Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in ...kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.
Endoplasmic reticulum (ER) stress is activated in nonalcoholic fatty liver disease (NAFLD), raising the possibility that ER stress‐dependent metabolic dysfunction, inflammation, and cell death ...underlie the transition from steatosis to steatohepatitis (nonalcoholic steatohepatitis; NASH). B‐cell lymphoma 2 (BCL2)‐associated X protein (Bax) inhibitor‐1 (BI‐1), a negative regulator of the ER stress sensor, inositol‐requiring enzyme 1 alpha (IRE1α), has yet to be explored in NAFLD as a hepatoprotective agent. We hypothesized that the genetic ablation of BI‐1 would render the liver vulnerable to NASH because of unrestrained IRE1α signaling. ER stress was induced in wild‐type and BI‐1–/– mice acutely by tunicamycin (TM) injection (1 mg/kg) or chronically by high‐fat diet (HFD) feeding to determine NAFLD phenotype. Livers of TM‐treated BI‐1–/– mice showed IRE1α‐dependent NOD‐like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation, hepatocyte death, fibrosis, and dysregulated lipid homeostasis that led to liver failure within a week. The analysis of human NAFLD liver biopsies revealed BI‐1 down‐regulation parallel to the up‐regulation of IRE1α endoribonuclease (RNase) signaling. In HFD‐fed BI‐1–/– mice that presented NASH and type 2 diabetes, exaggerated hepatic IRE1α, X‐box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP) expression was linked to activated NLRP3 inflammasome and caspase‐1/‐11. Rises in interleukin (IL)‐1β, IL‐6, monocyte chemoattractant protein 1 (MCP1), chemokine (C‐X‐C motif) ligand 1 (CXCL1), and alanine transaminase (ALT)/aspartate transaminase (AST) levels revealed significant inflammation and injury, respectively. Pharmacological inhibition of IRE1α RNase activity with the small molecules, STF‐083010 or 4μ8c, was evaluated in HFD‐induced NAFLD. In BI‐1–/– mice, either treatment effectively counteracted IRE1α RNase activity, improving glucose tolerance and rescuing from NASH. The hepatocyte‐specific role of IRE1α RNase activity in mediating NLRP3 inflammasome activation and cell death was confirmed in primary mouse hepatocytes by IRE1α axis knockdown or its inhibition with STF‐083010 or 4μ8c. Conclusion: Targeting IRE1α‐dependent NLRP3 inflammasome signaling with pharmacological agents or by BI‐1 may represent a tangible therapeutic strategy for NASH. (Hepatology 2018).
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•Molecular glues promote the unnatural association of proteins to produce a therapeutic effect.•Molecular glues alter the surface of proteins to promote their association.•Natural ...products that promote protein interactions have inspired synthetic ligands with diverse pharmacology.•Targeted protein association offers a new method for modulating cellular physiology and expands opportunity for drug discovery.
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.
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.
Bispecific protein degraders (BPDs) engage the ubiquitin–proteasome system (UPS) to catalytically degrade intracellular proteins through the formation of ternary complexes with the target protein and ...E3 ubiquitin ligases. Here, we describe the development of a mechanistic modeling framework for BPDs that includes the reaction network governing ternary complex formation and degradation via the UPS. A critical element of the model framework is a multi-step process that results in a time delay between ternary complex formation and protein degradation, thereby balancing ternary complex stability against UPS degradation rates akin to the kinetic proofreading concept that has been proposed to explain the accuracy and specificity of biological processes including protein translation and T cell receptor signal transduction. Kinetic proofreading likely plays a central role in the cell’s ability to regulate substrate recognition and degradation by the UPS, and the model presented here applies this concept in the context of a quantitative pharmacokinetic (PK)-pharmacodynamic (PD) framework to inform the design of potent and selective BPDs.
Know your target, know your molecule Bunnage, Mark E; Gilbert, Adam M; Jones, Lyn H ...
Nature chemical biology,
06/2015, Letnik:
11, Številka:
6
Journal Article
Recenzirano
The pharmaceutical industry continues to experience signicant attrition of drug candidates during phase 2 proof-of-concept clinical studies. We describe some questions about the characteristics of ...protein targets and small-molecule drugs that may be important to consider in drug-discovery projects and could improve prospects for future clinical success.
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