Missense variants in phospholamban and cardiac myosin binding protein identified in patients with a family history and clinical diagnosis of dilated cardiomyopathy.

As the genetic landscape of cardiomyopathies continues to expand, the identification of missense variants in disease-associated genes frequently leads to a classification of variant of uncertain significance (VUS). For the proper reclassification of such variants, functional characterization is an important contributor to the proper assessment of pathogenic potential. Several missense variants in the calcium transport regulatory protein phospholamban have been associated with dilated cardiomyopathy. However, >40 missense variants in this transmembrane peptide are currently known and most remain classified as VUS with little clinical information. Similarly, missense variants in cardiac myosin binding protein have been associated with hypertrophic cardiomyopathy. However, hundreds of variants are known and many have low penetrance and are often found in control populations. Herein, we focused on novel missense variants in phospholamban, an Ala15-Thr variant found in a 4-year-old female and a Pro21-Thr variant found in a 60-year-old female, both with a family history and clinical diagnosis of dilated cardiomyopathy. The patients also harbored a Val896-Met variant in cardiac myosin binding protein. The phospholamban variants caused defects in the function, phosphorylation, and dephosphorylation of this calcium transport regulatory peptide, and we classified these variants as potentially pathogenic. The variant in cardiac myosin binding protein alters the structure of the protein. While this variant has been classified as benign, it has the potential to be a low-risk susceptibility variant because of the structural change in cardiac myosin binding protein. Our studies provide new biochemical evidence for missense variants previously classified as benign or VUS.

The Effect of Deuteration and Homologation of the Lactam Ring of Nirmatrelvir on Its Biochemical Properties and Oxidative Metabolism.

This study explores the relationship between structural alterations of nirmatrelvir, such as homologation and deuteration, and metabolic stability of newly synthesized derivatives. We developed a reliable synthetic protocol toward dideutero-nirmatrelvir and its homologated analogues with high isotopic incorporation. Deuteration of the primary metabolic site of nirmatrelvir provides a 3-fold improvement of its human microsomal stability but is accompanied by an increased metabolism rate at secondary sites. Homologation of the lactam ring allows the capping group modification to decrease and delocalize the molecule's lipophilicity, reducing the metabolic rate at secondary sites. The effect of deuteration was less pronounced for the 6-membered lactam than for its 5-membered analogue in human microsomes, but the trend is reversed in the case of mouse microsomes. X-ray data revealed that the homologation of the lactam ring favors the orientation of the drug's nitrile warhead for interaction with the catalytic sulfur of the SARS-CoV-2 Mpro, improving its binding. Comparable potency against SARS-CoV-2 Mpro from several variants of concern and selectivity over human cysteine proteases cathepsin B, L, and S was observed for the novel deuterated/homologated derivative and nirmatrelvir. Synthesized compounds displayed a large interspecies variability in hamster, rat, and human hepatocyte stability assays. Overall, we aimed to apply a rational approach in changing the physicochemical properties of the drug to refine its biochemical and biological parameters.

Deuteration for Metabolic Stabilization of SARS-CoV-2 Inhibitors GC373 and Nirmatrelvir.

Nirmatrelvir and GC373 inhibit the SARS-CoV-2 3CL protease and hinder viral replication in COVID-19. As nirmatrelvir in Paxlovid is oxidized by cytochrome P450 3A4, ritonavir is coadministered to block this. However, ritonavir undesirably alters the metabolism of other drugs. Hydrogens can be replaced with deuterium in nirmatrelvir and GC373 to slow oxidation. Results show that deuterium slows oxidation of nirmatrelvir adjacent to nitrogen by ∼40% and that the type of warhead can switch the site of oxidative metabolism.

Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin.

Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, development, structure, and applications of Lime, an improved SEP variant with higher fluorescence brightness and greater pH sensitivity. The X-ray crystal structure of Lime supports the mechanistic rationale that guided the introduction of beneficial mutations. Lime provides substantial improvements relative to SEP for imaging of endocytosis and exocytosis. Furthermore, Lime and its variants are advantageous for a broader range of applications including the detection of synaptic release and neuronal voltage changes.

SARS-CoV-2 Mpro Protease Variants of Concern Display Altered Viral Substrate and Cell Host Target Galectin-8 Processing but Retain Sensitivity toward Antivirals.

The main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 Mpro mutants provided structural insight into their altered functionality. Additionally, we show Mpro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mpro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

A sensitive and specific genetically-encoded potassium ion biosensor for in vivo applications across the tree of life.

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.

Acyl-CoA:diacylglycerol acyltransferase: Properties, physiological roles, metabolic engineering and intentional control.

Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the last reaction in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG). DGAT activity resides mainly in DGAT1 and DGAT2 in eukaryotes and bifunctional wax ester synthase-diacylglycerol acyltransferase (WSD) in bacteria, which are all membrane-bound proteins but exhibit no sequence homology to each other. Recent studies also identified other DGAT enzymes such as the soluble DGAT3 and diacylglycerol acetyltransferase (EaDAcT), as well as enzymes with DGAT activities including defective in cuticular ridges (DCR) and steryl and phytyl ester synthases (PESs). This review comprehensively discusses research advances on DGATs in prokaryotes and eukaryotes with a focus on their biochemical properties, physiological roles, and biotechnological and therapeutic applications. The review begins with a discussion of DGAT assay methods, followed by a systematic discussion of TAG biosynthesis and the properties and physiological role of DGATs. Thereafter, the review discusses the three-dimensional structure and insights into mechanism of action of human DGAT1, and the modeled DGAT1 from Brassica napus. The review then examines metabolic engineering strategies involving manipulation of DGAT, followed by a discussion of its therapeutic applications. DGAT in relation to improvement of traits of farmed animals is also discussed along with DGATs in various other eukaryotic organisms.

Primitive Phospholamban- and Sarcolipin-like Peptides Inhibit the Sarcoplasmic Reticulum Calcium Pump SERCA.

Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.

Crystallization of Feline Coronavirus Mpro With GC376 Reveals Mechanism of Inhibition.

Coronaviruses infect a variety of hosts in the animal kingdom, and while each virus is taxonomically different, they all infect their host via the same mechanism. The coronavirus main protease (Mpro, also called 3CLpro), is an attractive target for drug development due to its essential role in mediating viral replication and transcription. An Mpro inhibitor, GC376, has been shown to treat feline infectious peritonitis (FIP), a fatal infection in cats caused by internal mutations in the feline enteric coronavirus (FECV). Recently, our lab demonstrated that the feline drug, GC373, and prodrug, GC376, are potent inhibitors of SARS-CoV-2 Mpro and solved the structures in complex with the drugs; however, no crystal structures of the FIP virus (FIPV) Mpro with the feline drugs have been published so far. Here, we present crystal structures of FIPV Mpro-GC373/GC376 complexes, revealing the inhibitors covalently bound to Cys144 in the active site, similar to SARS-CoV-2 Mpro. Additionally, GC376 has a higher affinity for FIPV Mpro with lower nanomolar Ki values compared to SARS-CoV and SARS-CoV-2 Mpro. We also show that improved derivatives of GC376 have higher potency for FIPV Mpro. Since GC373 and GC376 represent strong starting points for structure-guided drug design, determining the crystal structures of FIPV Mpro with these inhibitors are important steps in drug optimization and structure-based broad-spectrum antiviral drug discovery.

Peptidomimetic α-Acyloxymethylketone Warheads with Six-Membered Lactam P1 Glutamine Mimic: SARS-CoV-2 3CL Protease Inhibition, Coronavirus Antiviral Activity, and in Vitro Biological Stability.

Recurring coronavirus outbreaks, such as the current COVID-19 pandemic, establish a necessity to develop direct-acting antivirals that can be readily administered and are active against a broad spectrum of coronaviruses. Described in this Article are novel α-acyloxymethylketone warhead peptidomimetic compounds with a six-membered lactam glutamine mimic in P1. Compounds with potent SARS-CoV-2 3CL protease and in vitro viral replication inhibition were identified with low cytotoxicity and good plasma and glutathione stability. Compounds 15e, 15h, and 15l displayed selectivity for SARS-CoV-2 3CL protease over CatB and CatS and superior in vitro SARS-CoV-2 antiviral replication inhibition compared with the reported peptidomimetic inhibitors with other warheads. The cocrystallization of 15l with SARS-CoV-2 3CL protease confirmed the formation of a covalent adduct. α-Acyloxymethylketone compounds also exhibited antiviral activity against an alphacoronavirus and non-SARS betacoronavirus strains with similar potency and a better selectivity index than remdesivir. These findings demonstrate the potential of the substituted heteroaromatic and aliphatic α-acyloxymethylketone warheads as coronavirus inhibitors, and the described results provide a basis for further optimization.

A genetically encoded fluorescent biosensor for extracellular L-lactate.

L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

Peptidomimetic nitrile warheads as SARS-CoV-2 3CL protease inhibitors.

Tragically, the death toll from the COVID-19 pandemic continues to rise, and with variants being observed around the globe new therapeutics, particularly direct-acting antivirals that are easily administered, are desperately needed. Studies targeting the SARS-CoV-2 3CL protease, which is critical for viral replication, with different peptidomimetics and warheads is an active area of research for development of potential drugs. To date, however, only a few publications have evaluated the nitrile warhead as a viral 3CL protease inhibitor, with only modest activity reported. This article describes our investigation of P3 4-methoxyindole peptidomimetic analogs with select P1 and P2 groups with a nitrile warhead that are potent inhibitors of SARS-CoV-2 3CL protease and demonstrate in vitro SARS-CoV-2 antiviral activity. A selectivity for SARS-CoV-2 3CL protease over human cathepsins B, S and L was also observed with the nitrile warhead, which was superior to that with the aldehyde warhead. A co-crystal structure with SARS-CoV-2 3CL protease and a reversibility study indicate that a reversible, thioimidate adduct is formed when the catalytic sulfur forms a covalent bond with the carbon of the nitrile. This effort also identified efflux as a property limiting antiviral activity of these compounds, and together with the positive attributes described these results provide insight for further drug development of novel nitrile peptidomimetics targeting SARS-CoV-2 3CL protease.

Accelerated discovery of novel glycoside hydrolases using targeted functional profiling and selective pressure on the rumen microbiome.

BACKGROUND: Carbohydrate-active enzymes (CAZymes) form the most widespread and structurally diverse set of enzymes involved in the breakdown, biosynthesis, or modification of lignocellulose that can be found in living organisms. However, the structural diversity of CAZymes has rendered the targeted discovery of novel enzymes extremely challenging, as these proteins catalyze many different chemical reactions and are sourced by a vast array of microbes. Consequently, many uncharacterized members of CAZyme families of interest have been overlooked by current methodologies (e.g., metagenomic screening) used to discover lignocellulolytic enzymes. RESULTS: In the present study, we combined phenotype-based selective pressure on the rumen microbiota with targeted functional profiling to guide the discovery of unknown CAZymes. In this study, we found 61 families of glycoside hydrolases (GH) (out of 182 CAZymes) from protein sequences deposited in the CAZy database-currently associated with more than 20,324 microbial genomes. Phenotype-based selective pressure on the rumen microbiome showed that lignocellulolytic bacteria (e.g., Fibrobacter succinogenes, Butyrivibrio proteoclasticus) and three GH families (e.g., GH11, GH13, GH45) exhibited an increased relative abundance in the rumen of feed efficient cattle when compared to their inefficient counterparts. These results paved the way for the application of targeted functional profiling to screen members of the GH11 and GH45 families against a de novo protein reference database comprised of 1184 uncharacterized enzymes, which led to the identification of 18 putative xylanases (GH11) and three putative endoglucanases (GH45). The biochemical proof of the xylanolytic activity of the newly discovered enzyme validated the computational simulations and demonstrated the stability of the most abundant xylanase. CONCLUSIONS: These findings contribute to the discovery of novel enzymes for the breakdown, biosynthesis, or modification of lignocellulose and demonstrate that the rumen microbiome is a source of promising enzyme candidates for the biotechnology industry. The combined approaches conceptualized in this study can be adapted to any microbial environment, provided that the targeted microbiome is easy to manipulate and facilitates enrichment for the microbes of interest. Video Abstract.

SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1.

SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them target the spike protein in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested SARS-CoV-2 spike RBD as a vaccine antigen and explored the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results show that RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralization of variants B.1.1.7 and B.1.351 and to a lesser extent, SARS-CoV-1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection.

Photocleavable proteins that undergo fast and efficient dissociation.

Photocleavable molecules can enable the light-dependent modulation of biomolecular activities with high spatiotemporal precision. We have previously reported a photocleavable protein (PhoCl1) that, uniquely, is a fully genetically encoded photocleavable molecule that can be introduced into cells in the form of its corresponding gene to enable optogenetic control of biomolecular activities. However, the first generation PhoCl1 exhibited a relatively slow rate of dissociation, potentially limiting its utility. Here, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Molecular dynamics (MD) simulations were performed to provide insight into the precise dissociation mechanism. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.

Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.

The sarco-plasmic reticulum calcium pump (SERCA) plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.

Improved SARS-CoV-2 Mpro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies.

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (Mpro) to cleave viral proteins. Consequently, Mpro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of Mpro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-Mpro complexes reveal that an alternative binding pocket in Mpro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na+ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for Mpro inhibitor design.

N-Terminal Finger Stabilizes the S1 Pocket for the Reversible Feline Drug GC376 in the SARS-CoV-2 Mpro Dimer.

The main protease (Mpro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drug's properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar Ki values with the Mpro from both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 Mpro. The comparison of a new X-ray crystal structure of Mpro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 Mpro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the Mpro, which facilitates coordination of the drug's P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials.

Insights into the catalytic properties of the mitochondrial rhomboid protease PARL.

The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac/Diablo, which have crucial roles in mitochondrial quality control and apoptosis. However, the catalytic properties of PARL, including the effect of lipids on the protease, have never been characterized in vitro. To address this, we isolated human PARL expressed in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show that PARL activity in detergent is enhanced by cardiolipin, a lipid enriched in the mitochondrial inner membrane. Significantly higher turnover rates were observed for PARL reconstituted in proteoliposomes, with Smac/Diablo being cleaved most rapidly at a rate of 1 min-1. In contrast, PGAM5 is cleaved with the highest efficiency (kcat/KM) compared with PINK1 and Smac/Diablo. In proteoliposomes, a truncated ß-cleavage form of PARL, a physiological form known to affect mitochondrial fragmentation, is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5, and Smac/Diablo. Multiplex profiling of 228 peptides reveals that PARL prefers substrates with a bulky side chain such as Phe in P1, which is distinct from the preference for small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences that enhance our understanding of its role in mitochondrial function and has implications for specific inhibitor design.

Structure and function of proteins in membranes and nanodiscs.

The field of membrane structural biology represents a fast-moving field with exciting developments including native nanodiscs that allow preparation of complexes of post-translationally modified proteins bound to biological lipids. This has led to conceptual advances including biological membrane:protein assemblies or "memteins" as the fundamental functional units of biological membranes. Tools including cryo-electron microscopy and X-ray crystallography are maturing such that it is becoming increasingly feasible to solve structures of large, multicomponent complexes, while complementary methods including nuclear magnetic resonance spectroscopy yield unique insights into interactions and dynamics. Challenges remain, including elucidating exactly how lipids and ligands are recognized at atomic resolution and transduce signals across asymmetric bilayers. In this special volume some of the latest thinking and methods are gathered through the analysis of a range of transmembrane targets. Ongoing work on areas including polymer design, protein labelling and microfluidic technologies will ensure continued progress on improving resolution and throughput, providing deeper understanding of this most important group of targets.