Reactivity of [(PNP)Mn(CO)2] with Organophosphates.

Organophosphorus nerve agents (OPAs) are a toxic class of synthetic compounds that cause adverse effects with many biological systems. Development of methods for environmental remediation and passivation has been ongoing for years. However, little progress has been made in therapeutic development for exposure victims. Given the postexposure behavior of OPA materials in enzymes such as acetylcholinesterase (AChE), development of electrophilic compounds as therapeutics may be more beneficial than the currently employed nucleophilic countermeasures. In this report, we present our studies with an electrophilic, 16-electron manganese complex (iPrPNP)Mn(CO)2 (1) and the nucleophilic hydroxide derivative (iPrPNHP)Mn(CO)2(OH) (2). The reactivity of 1 with phosphorus acids and the reactivity of 2 with the P-F bond of diisopropylfluorophosphate (DIPF) were studied. The role of water in both nucleophilic and electrophilic reactivity was investigated with the use of 17O-labeled water. Promising results arising from reactions of both 1 and 2 with organophosphorus substrates are reported.

Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2.

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M-1 s-1, achieves sub-µM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

Label-free affinity screening, design and synthesis of inhibitors targeting the Mycobacterium tuberculosis L-alanine dehydrogenase.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structure-based design of lead compounds by targeting the hypoxia-associated enzyme L-alanine dehydrogenase (AlaDH). Biochemical and structural analyses of AlaDH confirmed binding of nucleoside derivatives and showed a site adjacent to the nucleoside binding pocket that can confer specificity to putative inhibitors. Using a combination of dye-ligand affinity chromatography, enzyme kinetics and protein crystallographic studies, we show the development and validation of drug prototypes. Crystal structures of AlaDH-inhibitor complexes with variations at the N6 position of the adenyl-moiety of the inhibitor provide insight into the molecular basis for the specificity of these compounds. We describe a drug-designing pipeline that aims to block Mtb to proliferate upon re-oxygenation by specifically blocking NAD accessibility to AlaDH. The collective approach to drug discovery was further evaluated through in silico analyses providing additional insight into an efficient drug development strategy that can be further assessed with the incorporation of in vivo studies.

PCP consensus protein/peptide alphavirus antigens stimulate broad spectrum neutralizing antibodies.

Vaccines based on proteins and peptides may be safer and if calculated based on many sequences, more broad-spectrum than those designed based on single strains. Physicochemical Property Consensus (PCPcon) alphavirus (AV) antigens from the B-domain of the E2 envelope protein were designed, synthesized recombinantly and shown to be immunogenic (i.e. sera after inoculation detected the antigen in dotspots and ELISA). Antibodies in sera after inoculation with B-region antigens based on individual AV species (eastern or Venezuelan equine encephalitis (EEEVcon, VEEVcon), or chikungunya (CHIKVcon) bound only their cognate protein, while those designed against multiple species (Mosaikcon and EVCcon) recognized all three serotype specific antigens. The VEEVcon and EEEVcon sera only showed antiviral activity against their related strains (in plaque reduction neutralization assays (PRNT50/80). Peptides designed to surface exposed areas of the E2-A-domain of CHIKVcon were added to CHIKVcon inocula to provide anti-CHIKV antibodies. EVCcon, based on three different alphavirus species, combined with E2-A-domain peptides from AllAVcon, a PCPcon of 24 diverse AV, generated broad spectrum, antiviral antibodies against VEEV, EEEV and CHIKV, AV with less than 35% amino acid identity to each other (>65% diversity). This is a promising start to a molecularly defined vaccine against all AV. Further study with these antigens can illuminate what areas are most important for a robust immune response, resistant to mutations in rapidly evolving viruses. The validated computational methods can also be used to design broad spectrum antigens against many other pathogen families.

Hepatitis C virus NS3/4A inhibitors and other drug-like compounds as covalent binders of SARS-CoV-2 main protease.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (Mpro also known as 3CLpro) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the Mpro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the Mpro as potential COVID-19 therapeutics for further testing and possibly clinical trials.

High-Throughput Virtual Screening and Validation of a SARS-CoV-2 Main Protease Noncovalent Inhibitor.

Despite the recent availability of vaccines against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the search for inhibitory therapeutic agents has assumed importance especially in the context of emerging new viral variants. In this paper, we describe the discovery of a novel noncovalent small-molecule inhibitor, MCULE-5948770040, that binds to and inhibits the SARS-Cov-2 main protease (Mpro) by employing a scalable high-throughput virtual screening (HTVS) framework and a targeted compound library of over 6.5 million molecules that could be readily ordered and purchased. Our HTVS framework leverages the U.S. supercomputing infrastructure achieving nearly 91% resource utilization and nearly 126 million docking calculations per hour. Downstream biochemical assays validate this Mpro inhibitor with an inhibition constant (Ki) of 2.9 µM (95% CI 2.2, 4.0). Furthermore, using room-temperature X-ray crystallography, we show that MCULE-5948770040 binds to a cleft in the primary binding site of Mpro forming stable hydrogen bond and hydrophobic interactions. We then used multiple µs-time scale molecular dynamics (MD) simulations and machine learning (ML) techniques to elucidate how the bound ligand alters the conformational states accessed by Mpro, involving motions both proximal and distal to the binding site. Together, our results demonstrate how MCULE-5948770040 inhibits Mpro and offers a springboard for further therapeutic design.

Conformational control via sequence for a heteropeptoid in water: coupled NMR and Rosetta modelling.

We report a critical advance in the generation and characterization of peptoid hetero-oligomers. A library of sub-monomers with amine and carboxylate side-chains are combined in different sequences using microwave-assisted synthesis. Their sequence-structure propensity is confirmed by circular dichroism, and conformer subtypes are enumerated by NMR. Biasing the ψ-angle backbone to trans (180°) in Monte Carlo modelling favors i to i + 3 naphthyl-naphthyl stacking, and matches experimental ensemble distributions. Taken together, high-yield synthesis of heterooligomers and NMR with structure prediction enables rapid determination of sequences that induce secondary structural propensities for predictive design of hydrophilic peptidomimetic foldamers and their future libraries.

Synthesis of stable isotope-labeled chloroquine and amodiaquine and their metabolites.

Anti-malaria drugs chloroquine and amodiaquine and their metabolites were synthesized to incorporate 13 C and 15 N starting from U-13 C-labeled benzene to give M + 7 isotopomers. Chloroquine and its metabolites were prepared from 7-chloro-1,2,3,4-tetrahydroquinolin-4-one through an aryl substitution with the corresponding amines; and the amodiaquine and its metabolites were prepared from 4,7-dichloroquinoline in a similar fashion.

Immunomagnetic separation and quantification of butyrylcholinesterase nerve agent adducts in human serum.

A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.

Detection of intact influenza viruses using biotinylated biantennary S-sialosides.

We report the modular synthesis of robust, biotinylated biantennary sialylglycoconjugates and their ability to differentiate between two type A influenza strains. This is the first demonstration of glycoconjugate-based discriminatory capture and detection of two strains of intact influenza virus, in the presence of the innate enzymatic activity of viral neuraminidases. We also demonstrate a "carboassay" using glycoconjugates as capture and reporter elements, which therefore, does not require antibodies. The capture of intact influenza viruses is of potential benefit for clinical diagnostics.

Functional PEG-modified thin films for biological detection.

We report a general procedure to prepare functional organic thin films for biological assays on oxide surfaces. Silica surfaces were functionalized by self-assembly of an amine-terminated silane film using both vapor- and solution-phase deposition of 3'-aminopropylmethyldiethoxysilane (APMDES). We found that vapor-phase deposition of APMDES under reduced pressure produced the highest quality monolayer films with uniform surface coverage, as determined by atomic force microscopy (AFM), ellipsometry, and contact angle measurements. The amine-terminated films were chemically modified with a mixture of carboxylic acid-terminated poly(ethylene glycol) (PEG) chains of varying functionality. A fraction of the PEG chains (0.1-10 mol %) terminated in biotin, which produced a surface with an affinity toward streptavidin. When used in pseudo-sandwich assays on waveguide platforms for the detection of Bacillus anthracis protective antigen (PA), these functional PEG surfaces significantly reduced nonspecific binding to the waveguide surface while allowing for highly specific binding. Detection of PA was used to validate these films for sensing applications in both buffer and complex media. Ultimately, these results represent a step toward the realization of a robust, reusable, and autonomous biosensor.

Botulinum neurotoxin detection and differentiation by mass spectrometry.

Botulinum neurotoxins (BoNTs) are proteases that cleave specific cellular proteins essential for neurotransmitter release. Seven BoNT serotypes (A-G) exist; 4 usually cause human botulism (A, B, E, and F). We developed a rapid, mass spectrometry-based method (Endopep-MS) to detect and differentiate active BoNTs A, B, E, and F. This method uses the highly specific protease activity of the toxins with target peptides specific for each toxin serotype. The product peptides derived from the endopeptidase activities of BoNTs are detected by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. In buffer, this method can detect toxin equivalents of as little as 0.01 mouse lethal dose (MLD)50 and concentrations as low as 0.62 MLD50/mL. A high-performance liquid chromatography-tandem mass spectrometry method for quantifying active toxin, where the amount of toxin can be correlated to the amount of product peptides, is also described.

From the mouse to the mass spectrometer: detection and differentiation of the endoproteinase activities of botulinum neurotoxins A-G by mass spectrometry.

We have developed an assay (Endopep-MS) that detects the specific endoproteinase activities of all seven BoNT types by mass spectrometry (MS). Each BoNT type cleaves a unique site on proteins involved in neuronal transmission. Target peptide substrates based on these proteins identify a BoNT type by its enzymatic action on the substrate and the production of two peptide products, which are then detected by matrix-assisted laser desorption/ionization time-of-flight MS or liquid chromatography electrospray ionization MS/MS. We showed the ability to detect all seven toxin types in a multiplexed assay format. The detection limits achieved range from 0.039 to 0.625 mouse LD(50)/mL for toxin types A, B, E, and F in a buffer system. The Endopep-MS assay is the first to differentiate all seven BoNT types, is sensitive, specific, and has the potential to quantify toxin activity.