Oxidative cyclization reagents reveal tryptophan cation-π interactions.

Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics1-3. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity3,4. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues that participate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.

Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors.

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly "druggable" or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.

An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4.

Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. However, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed cross-talk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. By identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.

Impact of Statins on Cardiovascular Outcomes Following Coronary Artery Calcium Scoring.

BACKGROUND: Compared with traditional risk factors, coronary artery calcium (CAC) scores improve prognostic accuracy for atherosclerotic cardiovascular disease (ASCVD) outcomes. However, the relative impact of statins on ASCVD outcomes stratified by CAC scores is unknown. OBJECTIVES: The authors sought to determine whether CAC can identify patients most likely to benefit from statin treatment. METHODS: The authors identified consecutive subjects without pre-existing ASCVD or malignancy who underwent CAC scoring from 2002 to 2009 at Walter Reed Army Medical Center. The primary outcome was first major adverse cardiovascular event (MACE), a composite of acute myocardial infarction, stroke, and cardiovascular death. The effect of statin therapy on outcomes was analyzed stratified by CAC presence and severity, after adjusting for baseline comorbidities with inverse probability of treatment weights based on propensity scores. RESULTS: A total of 13,644 patients (mean age 50 years; 71% men) were followed for a median of 9.4 years. Comparing patients with and without statin exposure, statin therapy was associated with reduced risk of MACE in patients with CAC (adjusted subhazard ratio: 0.76; 95% confidence interval: 0.60 to 0.95; p = 0.015), but not in patients without CAC (adjusted subhazard ratio: 1.00; 95% confidence interval: 0.79 to 1.27; p = 0.99). The effect of statin use on MACE was significantly related to the severity of CAC (p < 0.0001 for interaction), with the number needed to treat to prevent 1 initial MACE outcome over 10 years ranging from 100 (CAC 1 to 100) to 12 (CAC >100). CONCLUSIONS: In a largescale cohort without baseline ASCVD, the presence and severity of CAC identified patients most likely to benefit from statins for the primary prevention of cardiovascular diseases.

JP-8 jet fuel can promote auditory impairment resulting from subsequent noise exposure in rats.

We report on the transient and persistent effects of JP-8 jet fuel exposure on auditory function in rats. JP-8 has become the standard jet fuel utilized in the United States and North Atlantic Treaty Organization countries for military use and it is closely related to Jet A fuel, which is used in U.S. domestic aviation. Rats received JP-8 fuel (1000 mg/m(3)) by nose-only inhalation for 4 h and half of them were immediately subjected to an octave band of noise ranging between 97 and 105 dB in different experiments. The noise by itself produces a small, but permanent auditory impairment. The current permissible exposure level for JP-8 is 350 mg/m(3). Additionally, a positive control group received only noise exposure, and a fourth group consisted of untreated control subjects. Exposures occurred either on 1 day or repeatedly on 5 successive days. Impairments in auditory function were assessed using distortion product otoacoustic emissions and compound action potential testing. In other rats, tissues were harvested following JP-8 exposure for assessment of hydrocarbon levels or glutathione (GSH) levels. A single JP-8 exposure by itself at 1000 mg/m(3) did not disrupt auditory function. However, exposure to JP-8 and noise produced an additive disruption in outer hair cell function. Repeated 5-day JP-8 exposure at 1000 mg/m(3) for 4 h produced impairment of outer hair cell function that was most evident at the first postexposure assessment time. Partial though not complete recovery was observed over a 4-week postexposure period. The adverse effects of repeated JP-8 exposures on auditory function were inconsistent, but combined treatment with JP-8 + noise yielded greater impairment of auditory function, and hair cell loss than did noise by itself. Qualitative comparison of outer hair cell loss suggests an increase in outer hair cell death among rats treated with JP-8 + noise for 5 days as compared to noise alone. In most instances, hydrocarbon constituents of the fuel were largely eliminated in all tissues by 1-h postexposure with the exception of fat. Finally, JP-8 exposure did result in a significant depletion of total GSH that was observable in liver with a nonsignificant trend toward depletion in the brain and lung raising the possibility that the promotion of noise-induced hearing loss by JP-8 might have resulted from oxidative stress.

Promotion of noise-induced cochlear injury by toluene and ethylbenzene in the rat.

Ethylbenzene + toluene are known individually to have ototoxic potential at high exposure levels and with prolonged exposure times generally of 4-16 weeks. Both ethylbenzene + toluene are minor constituents of JP-8 jet fuel; this fuel has recently been determined to promote susceptibility to noise-induced hearing loss. Therefore, the current study evaluates the ototoxic potential of combined exposure to ethylbenzene + toluene exposure in a ratio calculated from the average found in three laboratories. Rats received ethylbenzene + toluene by inhalation and half of them were subjected simultaneously to an octave band of noise (OBN) of 93-95 dB. Another group received only the noise exposure which was designed to produce a small, but permanent auditory impairment while an unexposed control group was also included. In two separate experiments, exposures occurred either repeatedly on 5 successive days for 1 week or for 5 days on 2 successive weeks to 4000 mg/m(3) total hydrocarbons for 6 h based upon initial pilot studies. The concentration of toluene was 400 ppm and the concentration of ethylbenzene was 660 ppm. Impairments in auditory function were assessed using distortion product otoacoustic emissions and compound action potential testing. Following completion of these tests, the organs of Corti were dissected to permit evaluation of hair cell loss. The uptake and elimination of the solvents was assessed by harvesting key organs at two time points following ethylbenzene + toluene exposure from additional rats not used for auditory testing. Similarly, glutathione (GSH) levels were measured in light of suggestions that oxidative stress might result from solvent-noise exposures. Ethylbenzene + toluene exposure by itself at 4000 mg/m(3) for 6 h did not impair cochlear function or yield a loss of hair cells. However, when combined with a 93-dB OBN exposure combined solvent + noise did yield a loss in auditory function and a clear potentiation of outer hair cell death that exceeded the loss produced by noise alone. No evidence was found for a loss in total GSH in lung, liver, or brain as a consequence of ethylbenzene + toluene exposure.