TMPRSS2 Inhibitor Discovery Facilitated through an In Silico and Biochemical Screening Platform.

The COVID-19 pandemic has highlighted the need for new antiviral approaches because many of the currently approved drugs have proven ineffective against mitigating SARS-CoV-2 infections. The host transmembrane serine protease TMPRSS2 is a promising antiviral target because it plays a role in priming the spike protein before viral entry occurs for the most virulent variants. Further, TMPRSS2 has no established physiological role, thereby increasing its attractiveness as a target for antiviral agents. Here, we utilize virtual screening to curate large libraries into a focused collection of potential inhibitors. Optimization of a recombinant expression and purification protocol for the TMPRSS2 peptidase domain facilitates subsequent biochemical screening and characterization of selected compounds from the curated collection in a kinetic assay. In doing so, we identify new noncovalent TMPRSS2 inhibitors that block SARS-CoV-2 infectivity in a cellular model. One such inhibitor, debrisoquine, has high ligand efficiency, and an initial structure-activity relationship study demonstrates that debrisoquine is a tractable hit compound for TMPRSS2.

Allostery in the dynamic coactivator domain KIX occurs through minor conformational micro-states.

The coactivator KIX of CBP uses two binding surfaces to recognize multiple activators and exhibits allostery in ternary complex formation. Activator•coactivator interactions are central to transcriptional regulation, yet the microscopic origins of allostery in dynamic proteins like KIX are largely unknown. Here, we investigate the molecular recognition and allosteric manifestations involved in two KIX ternary systems c-Myb•KIX•MLL and pKID•KIX•MLL. Exploring the hypothesis that binary complex formation prepays an entropic cost for positive cooperativity, we utilize molecular dynamics simulations, side chain methyl order parameters, and differential scanning fluorimetry (DSF) to explore conformational entropy changes in KIX. The protein's configurational micro-states from structural clustering highlight the utility of protein plasticity in molecular recognition and allostery. We find that apo KIX occupies a wide distribution of lowly-populated configurational states. Each binding partner has its own suite of KIX states that it selects, building a model of molecular recognition fingerprints. Allostery is maximized with MLL pre-binding, which corresponds to the observation of a significant reduction in KIX micro-states observed when MLL binds. With all binding partners, the changes in KIX conformational entropy arise predominantly from changes in the most flexible loop. Likewise, we find that a small molecule and mutations allosterically inhibit/enhance activator binding by tuning loop dynamics, suggesting that loop-targeting chemical probes could be developed to alter KIX•activator interactions. Experimentally capturing KIX stabilization is challenging, particularly because of the disordered nature of particular activators. However, DSF melting curves allow for inference of relative entropic changes that occur across complexes, which we compare to our computed entropy changes using simulation methyl order parameters.

Norstictic Acid Is a Selective Allosteric Transcriptional Regulator.

Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways, and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (∼900 Å2) and have little topology, and thus, they do not present an array of attractive small-molecule binding sites for inhibitor discovery. Here we demonstrate that the depsidone natural product norstictic acid functions through an alternative binding site to block Med25-transcriptional activator PPIs in vitro and in cell culture. Norstictic acid targets a binding site comprising a highly dynamic loop flanking one canonical binding surface, and in doing so, it both orthosterically and allosterically alters Med25-driven transcription in a patient-derived model of triple-negative breast cancer. These results highlight the potential of Med25 as a therapeutic target as well as the inhibitor discovery opportunities presented by structurally dynamic loops within otherwise challenging proteins.

TMPRSS2 inhibitor discovery facilitated through an in silico and biochemical screening platform.

The COVID-19 pandemic has highlighted the need for new antiviral targets, as many of the currently approved drugs have proven ineffective against mitigating SARS-CoV-2 infections. The host transmembrane serine protease TMPRSS2 is a highly promising antiviral target, as it plays a direct role in priming the spike protein before viral entry occurs. Further, unlike other targets such as ACE2, TMPRSS2 has no known biological role. Here we utilize virtual screening to curate large libraries into a focused collection of potential inhibitors. Optimization of a recombinant expression and purification protocol for the TMPRSS2 peptidase domain facilitates subsequent biochemical screening and characterization of selected compounds from the curated collection in a kinetic assay. In doing so, we demonstrate that serine protease inhibitors camostat, nafamostat, and gabexate inhibit through a covalent mechanism. We further identify new non-covalent compounds as TMPRSS2 protease inhibitors, demonstrating the utility of a combined virtual and experimental screening campaign in rapid drug discovery efforts.

Selective Modulation of Dynamic Protein Complexes.

Dynamic proteins perform critical roles in cellular machines, including those that control proteostasis, transcription, translation, and signaling. Thus, dynamic proteins are prime candidates for chemical probe and drug discovery but difficult targets because they do not conform to classical rules of design and screening. Selectivity is pivotal for candidate probe molecules due to the extensive interaction network of these dynamic hubs. Recognition that the traditional rules of probe discovery are not necessarily applicable to dynamic proteins and their complexes, as well as technological advances in screening, have produced remarkable results in the last 2-4 years. Particularly notable are the improvements in target selectivity for small-molecule modulators of dynamic proteins, especially with techniques that increase the discovery likelihood of allosteric regulatory mechanisms. We focus on approaches to small-molecule screening that appear to be more suitable for highly dynamic targets and have the potential to streamline identification of selective modulators.

Bioorthogonal pro-metabolites for profiling short chain fatty acylation.

Short chain fatty acids (SCFAs) play a central role in health and disease. One function of these signaling molecules is to serve as precursors for short chain fatty acylation, a class of metabolically-derived posttranslational modifications (PTMs) that are established by lysine acetyltransferases (KATs) and lysine deacetylases (KDACs). Via this mechanism, short chain fatty acylation serves as an integrated reporter of metabolism as well as KAT and KDAC activity, and has the potential to illuminate the role of these processes in disease. However, few methods to study short chain fatty acylation exist. Here we report a bioorthogonal pro-metabolite strategy for profiling short chain fatty acylation in living cells. Inspired by the dietary component tributyrin, we synthesized a panel of ester-caged bioorthogonal short chain fatty acids. Cellular evaluation of these agents led to the discovery of an azido-ester that is metabolized to its cognate acyl-coenzyme A (CoA) and affords robust protein labeling profiles. We comprehensively characterize the metabolic dependence, toxicity, and histone deacetylase (HDAC) inhibitor sensitivity of these bioorthogonal pro-metabolites, and apply an optimized probe to identify novel candidate protein targets of short chain fatty acids in cells. Our studies showcase the utility of bioorthogonal pro-metabolites for unbiased profiling of cellular protein acylation, and suggest new approaches for studying the signaling functions of SCFAs in differentiation and disease.

Defining Metabolic and Nonmetabolic Regulation of Histone Acetylation by NSAID Chemotypes.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are well-known for their effects on inflammatory gene expression. Although NSAIDs are known to impact multiple cellular signaling mechanisms, a recent finding is that the NSAID salicylate can disrupt histone acetylation, in part through direct inhibition of the lysine acetyltransferase (KAT) p300/CBP. While salicylate is a relatively weak KAT inhibitor, its CoA-linked metabolite is more potent; however, the ability of NSAID metabolites to inhibit KAT enzymes biochemically and in cells remains relatively unexplored. Here we define the role of metabolic and nonmetabolic mechanisms in inhibition of KAT activity by NSAID chemotypes. First, we screen a small panel of NSAIDs for biochemical inhibition of the prototypical KAT p300, leading to the finding that many carboxylate-containing NSAIDs, including ibuprofen, are able to function as weak inhibitors. Assessing the inhibition of p300 by ibuprofen-CoA, a known NSAID metabolite, reveals that linkage of ibuprofen to CoA increases its biochemical potency toward p300 and other KAT enzymes. In cellular studies, we find that carboxylate-containing NSAIDs inhibit histone acetylation. Finally, we exploit the stereoselective metabolism of ibuprofen to assess the role of its acyl-CoA metabolite in regulation of histone acetylation. This unique strategy reveals that formation of ibuprofen-CoA and histone acetylation are poorly correlated, suggesting metabolism may not be required for ibuprofen to inhibit histone acetylation. Overall, these studies provide new insights into the ability of NSAIDs to alter histone acetylation, and illustrate how selective metabolism may be leveraged as a tool to explore the influence of metabolic acyl-CoAs on cellular enzyme activity.

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling.

Non-enzymatic protein modification driven by thioester reactivity is thought to play a major role in the establishment of cellular lysine acylation. However, the specific protein targets of this process are largely unknown. Here we report an experimental strategy to investigate non-enzymatic acylation in cells. Specifically, we develop a chemoproteomic method that separates thioester reactivity from enzymatic utilization, allowing selective enrichment of non-enzymatic acylation targets. Applying this method to cancer cell lines identifies numerous candidate targets of non-enzymatic acylation, including several enzymes in lower glycolysis. Functional studies highlight malonyl-CoA as a reactive thioester metabolite that can modify and inhibit glycolytic enzyme activity. Finally, we show that synthetic thioesters can be used as novel reagents to probe non-enzymatic acylation in living cells. Our studies provide new insights into the targets and drivers of non-enzymatic acylation, and demonstrate the utility of reactivity-based methods to experimentally investigate this phenomenon in biology and disease.

Co-opting a Bioorthogonal Reaction for Oncometabolite Detection.

Dysregulated metabolism is a hallmark of many diseases, including cancer. Methods to fluorescently detect metabolites have the potential to enable new approaches to cancer detection and imaging. However, fluorescent sensing methods for naturally occurring cellular metabolites are relatively unexplored. Here we report the development of a chemical approach to detect the oncometabolite fumarate. Our strategy exploits a known bioorthogonal reaction, the 1,3-dipolar cycloaddition of nitrileimines and electron-poor olefins, to detect fumarate via fluorescent pyrazoline cycloadduct formation. We demonstrate hydrazonyl chlorides serve as readily accessible nitrileimine precursors, whose reactivity and spectral properties can be tuned to enable detection of fumarate and other dipolarophile metabolites. Finally, we show this reaction can be used to detect enzyme activity changes caused by mutations in fumarate hydratase, which underlie the familial cancer predisposition syndrome hereditary leiomyomatosis and renal cell cancer. Our studies define a novel intersection of bioorthogonal chemistry and metabolite reactivity that may be harnessed to enable biological profiling, imaging, and diagnostic applications.

Global Profiling of Acetyltransferase Feedback Regulation.

Lysine acetyltransferases (KATs) are key mediators of cell signaling. Methods capable of providing new insights into their regulation thus constitute an important goal. Here we report an optimized platform for profiling KAT-ligand interactions in complex proteomes using inhibitor-functionalized capture resins. This approach greatly expands the scope of KATs, KAT complexes, and CoA-dependent enzymes accessible to chemoproteomic methods. This enhanced profiling platform is then applied in the most comprehensive analysis to date of KAT inhibition by the feedback metabolite CoA. Our studies reveal that members of the KAT superfamily possess a spectrum of sensitivity to CoA and highlight NAT10 as a novel KAT that may be susceptible to metabolic feedback inhibition. This platform provides a powerful tool to define the potency and selectivity of reversible stimuli, such as small molecules and metabolites, that regulate KAT-dependent signaling.

Characterizing the Covalent Targets of a Small Molecule Inhibitor of the Lysine Acetyltransferase P300.

C646 inhibits the lysine acetyltransferases (KATs) p300 and CBP and represents the most potent and selective small molecule KAT inhibitor identified to date. To gain insights into the cellular activity of this epigenetic probe, we applied chemoproteomics to identify covalent targets of the C646 chemotype. Modeling and synthetic derivatization was used to develop a clickable analogue (C646-yne) that inhibits p300 similarly to the parent compound and enables enrichment of bound proteins. LC-MS/MS identified the major covalent targets of C646-yne as highly abundant cysteine-containing proteins, and follow-up studies found that C646 can inhibit tubulin polymerization in vitro. Finally, we provide evidence that thiol reactivity of C646 may limit its ability to antagonize acetylation in cells. These findings should enable a more precise interpretation of studies utilizing C646 as a chemical probe of KAT activity and suggest that an underappreciated liability of electrophile-containing inhibitors is a reduction in their cellular potency due to consumption by abundant protein and metabolite thiol sinks.