All Publications

  • Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2. Nature communications Sanders, B. C., Pokhrel, S., Labbe, A. D., Mathews, I. I., Cooper, C. J., Davidson, R. B., Phillips, G., Weiss, K. L., Zhang, Q., O'Neill, H., Kaur, M., Schmidt, J. G., Reichard, W., Surendranathan, S., Parvathareddy, J., Phillips, L., Rainville, C., Sterner, D. E., Kumaran, D., Andi, B., Babnigg, G., Moriarty, N. W., Adams, P. D., Joachimiak, A., Hurst, B. L., Kumar, S., Butt, T. R., Jonsson, C. B., Ferrins, L., Wakatsuki, S., Galanie, S., Head, M. S., Parks, J. M. 2023; 14 (1): 1733


    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.

    View details for DOI 10.1038/s41467-023-37254-w

    View details for PubMedID 36977673

    View details for PubMedCentralID PMC10044120

  • SGLT2 inhibitor ameliorates endothelial dysfunction associated with the common ALDH2 alcohol flushing variant. Science translational medicine Guo, H., Yu, X., Liu, Y., Paik, D. T., Justesen, J. M., Chandy, M., Jahng, J. W., Zhang, T., Wu, W., Rwere, F., Zhao, S. R., Pokhrel, S., Shivnaraine, R. V., Mukherjee, S., Simon, D. J., Manhas, A., Zhang, A., Chen, C. H., Rivas, M. A., Gross, E. R., Mochly-Rosen, D., Wu, J. C. 2023; 15 (680): eabp9952


    The common aldehyde dehydrogenase 2 (ALDH2) alcohol flushing variant known as ALDH2*2 affects ∼8% of the world's population. Even in heterozygous carriers, this missense variant leads to a severe loss of ALDH2 enzymatic activity and has been linked to an increased risk of coronary artery disease (CAD). Endothelial cell (EC) dysfunction plays a determining role in all stages of CAD pathogenesis, including early-onset CAD. However, the contribution of ALDH2*2 to EC dysfunction and its relation to CAD are not fully understood. In a large genome-wide association study (GWAS) from Biobank Japan, ALDH2*2 was found to be one of the strongest single-nucleotide polymorphisms associated with CAD. Clinical assessment of endothelial function showed that human participants carrying ALDH2*2 exhibited impaired vasodilation after light alcohol drinking. Using human induced pluripotent stem cell-derived ECs (iPSC-ECs) and CRISPR-Cas9-corrected ALDH2*2 iPSC-ECs, we modeled ALDH2*2-induced EC dysfunction in vitro, demonstrating an increase in oxidative stress and inflammatory markers and a decrease in nitric oxide (NO) production and tube formation capacity, which was further exacerbated by ethanol exposure. We subsequently found that sodium-glucose cotransporter 2 inhibitors (SGLT2i) such as empagliflozin mitigated ALDH2*2-associated EC dysfunction. Studies in ALDH2*2 knock-in mice further demonstrated that empagliflozin attenuated ALDH2*2-mediated vascular dysfunction in vivo. Mechanistically, empagliflozin inhibited Na+/H+-exchanger 1 (NHE-1) and activated AKT kinase and endothelial NO synthase (eNOS) pathways to ameliorate ALDH2*2-induced EC dysfunction. Together, our results suggest that ALDH2*2 induces EC dysfunction and that SGLT2i may potentially be used as a preventative measure against CAD for ALDH2*2 carriers.

    View details for DOI 10.1126/scitranslmed.abp9952

    View details for PubMedID 36696485

  • Egg-Derived Anti-SARS-CoV-2 Immunoglobulin Y (IgY) With Broad Variant Activity as Intranasal Prophylaxis Against COVID-19. Frontiers in immunology Frumkin, L. R., Lucas, M., Scribner, C. L., Ortega-Heinly, N., Rogers, J., Yin, G., Hallam, T. J., Yam, A., Bedard, K., Begley, R., Cohen, C. A., Badger, C. V., Abbasi, S. A., Dye, J. M., McMillan, B., Wallach, M., Bricker, T. L., Joshi, A., Boon, A. C., Pokhrel, S., Kraemer, B. R., Lee, L., Kargotich, S., Agochiya, M., John, T. S., Mochly-Rosen, D. 2022; 13: 899617


    COVID-19 emergency use authorizations and approvals for vaccines were achieved in record time. However, there remains a need to develop additional safe, effective, easy-to-produce, and inexpensive prevention to reduce the risk of acquiring SARS-CoV-2 infection. This need is due to difficulties in vaccine manufacturing and distribution, vaccine hesitancy, and, critically, the increased prevalence of SARS-CoV-2 variants with greater contagiousness or reduced sensitivity to immunity. Antibodies from eggs of hens (immunoglobulin Y; IgY) that were administered the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were developed for use as nasal drops to capture the virus on the nasal mucosa. Although initially raised against the 2019 novel coronavirus index strain (2019-nCoV), these anti-SARS-CoV-2 RBD IgY surprisingly had indistinguishable enzyme-linked immunosorbent assay binding against variants of concern that have emerged, including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529). This is different from sera of immunized or convalescent patients. Culture neutralization titers against available Alpha, Beta, and Delta were also indistinguishable from the index SARS-CoV-2 strain. Efforts to develop these IgY for clinical use demonstrated that the intranasal anti-SARS-CoV-2 RBD IgY preparation showed no binding (cross-reactivity) to a variety of human tissues and had an excellent safety profile in rats following 28-day intranasal delivery of the formulated IgY. A double-blind, randomized, placebo-controlled phase 1 study evaluating single-ascending and multiple doses of anti-SARS-CoV-2 RBD IgY administered intranasally for 14 days in 48 healthy adults also demonstrated an excellent safety and tolerability profile, and no evidence of systemic absorption. As these antiviral IgY have broad selectivity against many variants of concern, are fast to produce, and are a low-cost product, their use as prophylaxis to reduce SARS-CoV-2 viral transmission warrants further evaluation.Clinical Trial Registration:, identifier NCT04567810.

    View details for DOI 10.3389/fimmu.2022.899617

    View details for PubMedID 35720389

  • Natural variants in SARS-CoV-2 Spike protein pinpoint structural and functional hotspots with implications for prophylaxis and therapeutic strategies. Scientific reports Pokhrel, S., Kraemer, B. R., Burkholz, S., Mochly-Rosen, D. 2021; 11 (1): 13120


    In December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.

    View details for DOI 10.1038/s41598-021-92641-x

    View details for PubMedID 34162970

  • Paired SARS-CoV-2 spike protein mutations observed during ongoing SARS-CoV-2 viral transfer from humans to minks and Back to humans. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases Burkholz, S., Pokhrel, S., Kraemer, B. R., Mochly-Rosen, D., Carback, R. T., Hodge, T., Harris, P., Ciotlos, S., Wang, L., Herst, C. V., Rubsamen, R. 2021: 104897


    A mutation analysis of SARS-CoV-2 genomes collected around the world sorted by sequence, date, geographic location, and species has revealed a large number of variants from the initial reference sequence in Wuhan. This analysis also reveals that humans infected with SARS-CoV-2 have infected mink populations in the Netherlands, Denmark, United States, and Canada. In these animals, a small set of mutations in the spike protein receptor binding domain (RBD), often occurring in specific combinations, has transferred back into humans. The viral genomic mutations in minks observed in the Netherlands and Denmark show the potential for new mutations on the SARS-CoV-2 spike protein RBD to be introduced into humans by zoonotic transfer. Our data suggests that close attention to viral transfer from humans to farm animals and pets will be required to prevent build-up of a viral reservoir for potential future zoonotic transfer.

    View details for DOI 10.1016/j.meegid.2021.104897

    View details for PubMedID 33971305

  • Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Grebert, T., Nguyen, A. A., Pokhrel, S., Joseph, K., Ratin, M., Dufour, L., Chen, B., Haney, A. M., Karty, J. A., Trinidad, J. C., Garczarek, L., Schluchter, W. M., Kehoe, D. M., Partensky, F. 2021; 118 (9)


    Marine Synechococcus cyanobacteria owe their ubiquity in part to the wide pigment diversity of their light-harvesting complexes. In open ocean waters, cells predominantly possess sophisticated antennae with rods composed of phycocyanin and two types of phycoerythrins (PEI and PEII). Some strains are specialized for harvesting either green or blue light, while others can dynamically modify their light absorption spectrum to match the dominant ambient color. This process, called type IV chromatic acclimation (CA4), has been linked to the presence of a small genomic island occurring in two configurations (CA4-A and CA4-B). While the CA4-A process has been partially characterized, the CA4-B process has remained an enigma. Here we characterize the function of two members of the phycobilin lyase E/F clan, MpeW and MpeQ, in Synechococcus sp. strain A15-62 and demonstrate their critical role in CA4-B. While MpeW, encoded in the CA4-B island and up-regulated in green light, attaches the green light-absorbing chromophore phycoerythrobilin to cysteine-83 of the PEII α-subunit in green light, MpeQ binds phycoerythrobilin and isomerizes it into the blue light-absorbing phycourobilin at the same site in blue light, reversing the relationship of MpeZ and MpeY in the CA4-A strain RS9916. Our data thus reveal key molecular differences between the two types of chromatic acclimaters, both highly abundant but occupying distinct complementary ecological niches in the ocean. They also support an evolutionary scenario whereby CA4-B island acquisition allowed former blue light specialists to become chromatic acclimaters, while former green light specialists would have acquired this capacity by gaining a CA4-A island.

    View details for DOI 10.1073/pnas.2019715118

    View details for Web of Science ID 000625304300045

    View details for PubMedID 33627406

    View details for PubMedCentralID PMC7936332

  • Increased elastase sensitivity and decreased intramolecular interactions in the more transmissible 501Y.V1 and 501Y.V2 SARS-CoV-2 variants' spike protein-an in silico analysis. PloS one Pokhrel, S., Kraemer, B. R., Lee, L., Samardzic, K., Mochly-Rosen, D. 2021; 16 (5): e0251426


    Two SARS-CoV-2 variants of concern showing increased transmissibility relative to the Wuhan virus have recently been identified. Although neither variant appears to cause more severe illness nor increased risk of death, the faster spread of the virus is a major threat. Using computational tools, we found that the new SARS-CoV-2 variants may acquire an increased transmissibility by increasing the propensity of its spike protein to expose the receptor binding domain via proteolysis, perhaps by neutrophil elastase and/or via reduced intramolecular interactions that contribute to the stability of the closed conformation of spike protein. This information leads to the identification of potential treatments to avert the imminent threat of these more transmittable SARS-CoV-2 variants.

    View details for DOI 10.1371/journal.pone.0251426

    View details for PubMedID 34038453

  • CpeT is the phycoerythrobilin lyase for Cys-165 on beta-phycoerythrin from Fremyella diplosiphon and the chaperone-like protein CpeZ greatly improves its activity BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS Nguyen, A. A., Joseph, K., Bussell, A. N., Pokhrel, S., Karty, J. A., Kronfel, C. M., Kehoe, D. M., Schluchter, W. M. 2020; 1861 (12): 148284


    Bilin lyases are enzymes which ligate linear tetrapyrrole chromophores to specific cysteine residues on light harvesting proteins present in cyanobacteria and red algae. The lyases responsible for chromophorylating the light harvesting protein phycoerythrin (PE) have not been fully characterized. In this study, we explore the role of CpeT, a putative bilin lyase, in the biosynthesis of PE in the cyanobacterium Fremyella diplosiphon. Recombinant protein studies show that CpeT alone can bind phycoerythrobilin (PEB), but CpeZ, a chaperone-like protein, is needed in order to correctly and efficiently attach PEB to the β-subunit of PE. MS analyses of the recombinant β-subunit of PE coexpressed with CpeT and CpeZ show that PEB is attached at Cys-165. Purified phycobilisomes from a cpeT knockout mutant and wild type (WT) samples from F. diplosiphon were analyzed and compared. The cpeT mutant contained much less PE and more phycocyanin than WT cells grown under green light, conditions which should maximize the production of PE. In addition, Northern blot analyses showed that the cpeCDESTR operon mRNAs were upregulated while the cpeBcpeA mRNAs were downregulated in the cpeT mutant strain when compared with WT, suggesting that CpeT may also play a direct or indirect regulatory role in transcription of these operons or their mRNA stability, in addition to its role as a PEB lyase for Cys-165 on β-PE.

    View details for DOI 10.1016/j.bbabio.2020.148284

    View details for Web of Science ID 000572681900002

    View details for PubMedID 32777305

  • Interplay between differentially expressed enzymes contributes to light color acclimation in marine Synechococcus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sanfilippo, J. E., Nguyen, A. A., Garczarek, L., Karty, J. A., Pokhrel, S., Strnat, J. A., Partensky, F., Schluchter, W. M., Kehoe, D. M. 2019; 116 (13): 6457-6462


    Marine Synechococcus, a globally important group of cyanobacteria, thrives in various light niches in part due to its varied photosynthetic light-harvesting pigments. Many Synechococcus strains use a process known as chromatic acclimation to optimize the ratio of two chromophores, green-light-absorbing phycoerythrobilin (PEB) and blue-light-absorbing phycourobilin (PUB), within their light-harvesting complexes. A full mechanistic understanding of how Synechococcus cells tune their PEB to PUB ratio during chromatic acclimation has not yet been obtained. Here, we show that interplay between two enzymes named MpeY and MpeZ controls differential PEB and PUB covalent attachment to the same cysteine residue. MpeY attaches PEB to the light-harvesting protein MpeA in green light, while MpeZ attaches PUB to MpeA in blue light. We demonstrate that the ratio of mpeY to mpeZ mRNA determines if PEB or PUB is attached. Additionally, strains encoding only MpeY or MpeZ do not acclimate. Examination of strains of Synechococcus isolated from across the globe indicates that the interplay between MpeY and MpeZ uncovered here is a critical feature of chromatic acclimation for marine Synechococcus worldwide.

    View details for DOI 10.1073/pnas.1810491116

    View details for Web of Science ID 000462382800093

    View details for PubMedID 30846551

    View details for PubMedCentralID PMC6442610