pre-clinical assessment of pharmacological and molecular properties

SARS-CoV-2, the cause of the COVID-19 pandemic, exploits host cell proteins for viral entry into human lung cells. One of them, the protease TMPRSS2, is required to activate the viral spike protein (S). Even though two inhibitors, camostat and nafamostat, are known to inhibit TMPRSS2 and block cell entry of SARS-CoV-2, finding further potent therapeutic options is still an important task. In this study, we report that a late-stage drug candidate, otamixaban, inhibits SARS-CoV-2 cell entry. We show that otamixaban suppresses TMPRSS2 activity and SARS-CoV-2 infection of a human lung cell line, although with lower potency than camostat or nafamostat. In contrast, otamixaban inhibits SARS-CoV-2 infection of precision cut lung slices with the same potency as camostat. Furthermore, we report that otamixaban's potency can be significantly enhanced by (sub-) nanomolar nafamostat or camostat supplementation. Dominant molecular TMPRSS2-otamixaban interactions are assessed by extensive 109 μs of atomistic molecular dynamics simulations. Our findings suggest that combinations of otamixaban with supplemental camostat or nafamostat are a promising option for the treatment of COVID-19

Chemical Control of a CRISPR-Cas9 Acetyltransferase

Lysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Toward this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First, we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype and reveal a role for p300s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tunable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci

Chemical Control of a CRISPR-Cas9 Acetyltransferase

Lysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Toward this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First, we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype and reveal a role for p300s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tunable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci

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

Assay interference and off-target liabilities of reported histone acetyltransferase inhibitors

A substantial obstacle in basic research is the use of poorly validated tool compounds with purported useful biological functions. Here, the authors systematically profile widely used histone acetyltransferase inhibitors and find that the majority are nonselective interference compounds

Revealing CD38 Cellular Localization Using a Cell Permeable, Mechanism-Based Fluorescent Small-Molecule Probe

Nicotinamide adenine dinucleotide (NAD) is increasingly recognized as an important signaling molecule that affects numerous biological pathways. Thus, enzymes that metabolize NAD can have important biological functions. One NAD-metabolizing enzyme in mammals is CD38, a type II transmembrane protein that converts NAD primarily to adenosine diphosphate ribose (ADPR) and a small amount of cyclic adenosine diphosphate ribose (cADPR). Localization of CD38 was originally thought to be only on the plasma membrane, but later reports showed either significant or solely, intracellular CD38. With the efficient NAD-hydrolysis activity, the intracellular CD38 may lead to depletion of cellular NAD, thus producing harmful effects. Therefore, the intracellular localization of CD38 needs to be carefully validated. Here, we report the synthesis and application of a cell permeable, fluorescent small molecule (SR101–F-araNMN) that can covalently label enzymatically active CD38 with minimal perturbation of live cells. Using this fluorescent probe, we revealed that CD38 is predominately on the plasma membrane of Raji and retinoic acid (RA)-treated HL-60 cells. Additionally, the probe revealed no CD38 expression in K562 cells, which was previously reported to have solely intracellular CD38. The finding that very little intracellular CD38 exists in these cell lines suggests that the major enzymatic function of CD38 is to hydrolyze extracellular rather than intracellular NAD. The fluorescent activity-based probes that we developed allow the localization of CD38 in different cells to be determined, thus enabling a better understanding of the physiological function

Mitoribosome sensitivity to HSP70 inhibition uncovers metabolic liabilities of castration-resistant prostate cancer.

The androgen receptor is a key regulator of prostate cancer and the principal target of current prostate cancer therapies collectively termed androgen deprivation therapies. Insensitivity to these drugs is a hallmark of progression to a terminal disease state termed castration-resistant prostate cancer. Therefore, novel therapeutic options that slow progression of castration-resistant prostate cancer and combine effectively with existing agents are in urgent need. We show that JG-98, an allosteric inhibitor of HSP70, re-sensitizes castration-resistant prostate cancer to androgen deprivation drugs by targeting mitochondrial HSP70 (HSPA9) to suppress aerobic respiration. Rather than impacting androgen receptor stability as previously described, JG-98's primary effect is inhibition of mitochondrial translation, leading to disruption of electron transport chain activity. Although functionally distinct from HSPA9 inhibition, direct inhibition of the electron transport chain with a complex I or II inhibitor creates a similar physiological state capable of re-sensitizing castration-resistant prostate cancer to androgen deprivation therapies. These data identify a significant role for HspA9 in mitochondrial ribosome function and highlight an actionable metabolic vulnerability of castration-resistant prostate cancer

Microfluidic Mobility Shift Profiling of Lysine Acetyltransferases Enables Screening and Mechanistic Analysis of Cellular Acetylation Inhibitors

Lysine acetyltransferases (KATs) are critical regulators of signaling in many diseases, including cancer. A major challenge in establishing the targetable functions of KATs in disease is a lack of well-characterized, cell-active KAT inhibitors. To confront this challenge, here we report a microfluidic mobility shift platform for the discovery and characterization of small molecule KAT inhibitors. Novel fluorescent peptide substrates were developed for four well-known KAT enzymes (p300, Crebbp, Morf, and Gcn5). Enzyme-catalyzed acetylation alters the electrophoretic mobility of these peptides in a microfluidic chip, allowing facile and direct monitoring of KAT activity. A pilot screen was used to demonstrate the utility of microfluidic mobility shift profiling to identify known and novel modulators of KAT activity. Real-time kinetic monitoring of KAT activity revealed that garcinol, a natural product KAT inhibitor used in cellular studies, exhibits time-dependent and detergent-sensitive inhibition, consistent with an aggregation-based mechanism. In contrast, the cell-permeable bisubstrate inhibitor Tat-CoA exhibited potent and time-independent KAT inhibition, highlighting its potential utility as a cellular inhibitor of KAT activity. These studies define microfluidic mobility shift profiling as a powerful platform for the discovery and characterization of small molecule inhibitors of KAT activity, and provide mechanistic insights potentially important for the application of KAT inhibitors in cellular contexts