Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu.

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins.

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu.

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins.

What's the defect? Using mass defects to study oligomerization of membrane proteins and peptides in nanodiscs with native mass spectrometry.

Many membrane proteins form functional complexes that are either homo- or hetero-oligomeric. However, it is challenging to characterize membrane protein oligomerization in intact lipid bilayers, especially for polydisperse mixtures. Native mass spectrometry of membrane proteins and peptides inserted in lipid nanodiscs provides a unique method to study the oligomeric state distribution and lipid preferences of oligomeric assemblies.

A unique class of Zn-binding serine-based PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile.

Treatment with β-lactam antibiotics, particularly cephalosporins, is a major risk factor for Clostridioides difficile infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), which are serine-based enzymes that assemble the bacterial cell wall. However, C.

Discovery of Di- and Trihaloacetamides as Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity.

The main protease (M) is a validated antiviral drug target of SARS-CoV-2. A number of M inhibitors have now advanced to animal model study and human clinical trials. However, one issue yet to be addressed is the target selectivity over host proteases such as cathepsin L.

Influenza AM2 Channel Oligomerization Is Sensitive to Its Chemical Environment.

Viroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best-characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry and other techniques to characterize the oligomeric state of both the full-length and transmembrane (TM) domain of AM2 in a variety of different pH and detergent conditions.

Protein Modification via Mild Photochemical Isomerization of Triazenes to Release Aryl Diazonium Ions.

This work describes the development of phenyl diazenyl piperidine triazene derivatives that can be activated to release aryl diazonium ions for labeling of proteins using light. These probes show marked bench stability at room temperature and can be photoisomerized via low-intensity UVA irradiation at physiological pH. Upon isomerization, the triazenes are rendered more basic and readily protonate to release reactive aryl diazonium ions.

Discovery of SARS-CoV-2 Papain-like Protease Inhibitors through a Combination of High-Throughput Screening and a FlipGFP-Based Reporter Assay.

The papain-like protease (PL) of SARS-CoV-2 is a validated antiviral drug target. Through a fluorescence resonance energy transfer-based high-throughput screening and subsequent lead optimization, we identified several PL inhibitors including and with improved enzymatic inhibition and antiviral activity compared to , which was reported as a SARS-CoV PL inhibitor. Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular antiviral activity of PL inhibitors in the BSL-2 setting.

Expedited Approach toward the Rational Design of Noncovalent SARS-CoV-2 Main Protease Inhibitors.

The main protease (M) of SARS-CoV-2 is a validated antiviral drug target. Several M inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including , , , and , with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, () that is structurally distinct from the canonical M inhibitor .

Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay.

The papain-like protease (PL ) of SARS-CoV-2 is a validated antiviral drug target. PL is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PL inhibitors with IC values at the single-digit micromolar range.

Suzuki Coupling of Protected Aryl Diazonium Ions: Expanding the Knowledge of Triazabutadiene Compatible Reactions.

Aryl diazonium ions are important in synthesis and chemical biology, and the acid-labile triazabutadiene can protect this handle for future use. We report a Suzuki coupling strategy that is compatible with the triazabutadiene scaffold, expanding the scope of synthetically available triazabutadienes. Shown herein, the triazabutadiene scaffold remains intact and reactive after coupling, as demonstrated by releasing the aryl diazonium ion to label a tyrosine-rich model protein.

Copper-Free Click Enabled Triazabutadiene for Bioorthogonal Protein Functionalization.

Aryl diazonium ions have long been used in bioconjugation due to their reactivity toward electron-rich aryl residues, such as tyrosine. However, their utility in biological systems has been restricted due to the requirement of harsh conditions for their generation , as well as limited hydrolytic stability. Previous work describing a scaffold known as triazabutadiene (TBD) has shown the ability to protect aryl diazonium ions allowing for increased synthetic utility, as well as triggered release under biologically relevant conditions.

Ebselen, Disulfiram, Carmofur, PX-12, Tideglusib, and Shikonin Are Nonspecific Promiscuous SARS-CoV-2 Main Protease Inhibitors.

Among the drug targets being investigated for SARS-CoV-2, the viral main protease (M) is one of the most extensively studied. M is a cysteine protease that hydrolyzes the viral polyprotein at more than 11 sites. It is highly conserved and has a unique substrate preference for glutamine in the P1 position.

Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M and cathepsin L.

The main protease (M) of SARS-CoV-2 is a key antiviral drug target. While most M inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently found that several M inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II and XII, which are also active against human cathepsin L, a host protease that is important for viral entry. In this study, we solved x-ray crystal structures of M in complex with calpain inhibitors II and XII and three analogs of The structure of M with calpain inhibitor II confirmed that the S1 pocket can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position.

Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are non-specific promiscuous SARS-CoV-2 main protease inhibitors.

There is an urgent need for vaccines and antiviral drugs to combat the COVID-19 pandemic. Encouraging progress has been made in developing antivirals targeting SARS-CoV-2, the etiological agent of COVID-19. Among the drug targets being investigated, the viral main protease (M ) is one of the most extensively studied drug targets.

Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against M and cathepsin L.

The main protease (M) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 M inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently discovered several M inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of M in complex with calpain inhibitors II and XII, and three analogues of , one of the most potent M inhibitors .

Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease.

A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020.

Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease.

A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.96% as of May 4, 2020.

Imidazole Derivatives Improve Charge Reduction and Stabilization for Native Mass Spectrometry.

Noncovalent interactions between biomolecules are critical to their activity. Native mass spectrometry (MS) has enabled characterization of these interactions by preserving noncovalent assemblies for mass analysis, including protein-ligand and protein-protein complexes for a wide range of soluble and membrane proteins. Recent advances in native MS of lipoprotein nanodiscs have also allowed characterization of antimicrobial peptides and membrane proteins embedded in intact lipid bilayers.

Native Mass Spectrometry of Antimicrobial Peptides in Lipid Nanodiscs Elucidates Complex Assembly.

Antimicrobial peptides (AMPs) are generally cationic and amphipathic peptides that show potential applications to combat the growing threat of antibiotic resistant infections. AMPs are known to interact with bacterial membranes, but their mechanisms of toxicity and selectivity are poorly understood, in part because it is challenging to characterize AMP oligomeric complexes within lipid bilayers. Here, we used native mass spectrometry to measure the stoichiometry of AMPs inserted into lipoprotein nanodiscs with different lipid components.