HLA-DRB1 is associated with cefaclor-induced immediate hypersensitivity.

Background: Drug-induced hypersensitivity such as anaphylaxis is an important cause of drug-related morbidity and mortality. Cefaclor is a leading cause of drug induced type I hypersensitivity in Korea, but little is yet known about genetic biomarkers to predict this hypersensitivity reaction. We aimed to evaluate the possible involvement of genes in cefaclor induced type I hypersensitivity. Methods: Whole exome sequencing (WES) and HLA genotyping were performed in 43 patients with cefaclor induced type I hypersensitivity. In addition, homology modeling was performed to identify the binding forms of cefaclor to HLA site. Results: Anaphylaxis was the most common phenotype of cefaclor hypersensitivity (90.69%). WES results show that rs62242177 and rs62242178 located in LIMD1 region were genome-wide significant at the 5 × 10-8 significance level. Cefaclor induced type I hypersensitivity was significantly associated with HLA-DRB1∗04:03 (OR 4.61 [95% CI 1.51-14.09], P < 0.002) and HLA-DRB1∗14:54 (OR 3.86 [95% CI 1.09-13.67], P < 0.002). Conclusion: LIMD1, HLA-DRB1∗04:03 and HLA-DRB1∗14:54 may affect susceptibility to cefaclor induced type I hypersensitivity. Further confirmative studies with a larger patient population should be performed to ascertain the role of HLA-DRB1 and LIMD1 in the development of cefaclor induced hypersensitivity.

Sigma Receptor Ligands Prevent COVID Mortality In Vivo: Implications for Future Therapeutics.

The emergence of lethal coronaviruses follows a periodic pattern which suggests a recurring cycle of outbreaks. It remains uncertain as to when the next lethal coronavirus will emerge, though its eventual emergence appears to be inevitable. New mutations in evolving SARS-CoV-2 variants have provided resistance to current antiviral drugs, monoclonal antibodies, and vaccines, reducing their therapeutic efficacy. This underscores the urgent need to investigate alternative therapeutic approaches. Sigma receptors have been unexpectedly linked to the SARS-CoV-2 life cycle due to the direct antiviral effect of their ligands. Coronavirus-induced cell stress facilitates the formation of an ER-derived complex conducive to its replication. Sigma receptor ligands are believed to prevent the formation of this complex. Repurposing FDA-approved drugs for COVID-19 offers a timely and cost-efficient strategy to find treatments with established safety profiles. Notably, diphenhydramine, a sigma receptor ligand, is thought to counteract the virus by inhibiting the creation of ER-derived replication vesicles. Furthermore, lactoferrin, a well-characterized immunomodulatory protein, has shown antiviral efficacy against SARS-CoV-2 both in laboratory settings and in living organisms. In the present study, we aimed to explore the impact of sigma receptor ligands on SARS-CoV-2-induced mortality in ACE2-transgenic mice. We assessed the effects of an investigational antiviral drug combination comprising a sigma receptor ligand and an immunomodulatory protein. Mice treated with sigma-2 receptor ligands or diphenhydramine and lactoferrin exhibited improved survival rates and rapid rebound in mass following the SARS-CoV-2 challenge compared to mock-treated animals. Clinical translation of these findings may support the discovery of new treatment and research strategies for SARS-CoV-2.

Evidence of a Sjögren's disease-like phenotype following COVID-19 in mice and humans.

Sjögren's Disease (SjD) is a systemic autoimmune disease characterized by lymphocytic inflammation of the lacrimal and salivary glands (SG), dry eyes and mouth, and systemic symptoms. SARS-CoV-2 may trigger the development or progression of autoimmune diseases. To test this, we used a mouse model of SARS-CoV-2 infection and convalescent patients' blood and SG in order to understand the development of SjD-like autoimmunity after infection. First, SARS-CoV-2-infected human angiotensin-converting enzyme 2 (ACE2) transgenic mice exhibited decreased salivation, elevated antinuclear antibodies (ANA), and lymphocytic infiltration in the lacrimal and SG. The sera from patients with COVID-19 sera showed increased ANA (i.e., anti-SSA [Sjögren's-syndrome-related antigen A]/anti-Ro52 and anti-SSB [SS-antigen B]/anti-La). Male patients showed elevated anti-SSA compared with female patients, and female patients exhibited diverse ANA patterns. SG biopsies from convalescent COVID-19 patients were microscopically similar to SjD SG with focal lymphocytic infiltrates in 4 of 6 patients and 2 of 6 patients exhibiting focus scores of at least 2. Lastly, monoclonal antibodies produced in recovered patients blocked ACE2/spike interaction and cross-reacted with nuclear antigens. Our study shows a direct association between SARS-CoV-2 and SjD. Hallmark features of SjD-affected SGs were histologically indistinguishable from convalescent COVID-19 patients. The results implicate that SARS-CoV-2 could be an environmental trigger for SjD.

Molecular Mechanisms of Lupus Susceptibility Allele PBX1D.

Pre-B cell leukemia homeobox 1 (PBX1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, PBX1D, which lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus than healthy control subjects. PBX1D overexpression in CD4+ T cells impaired regulatory T cell homeostasis and expanded inflammatory CD4+ T cells. In this study, we showed that PBX1 message expression is downregulated by activation in CD4+ T cells as well as in B cells. PBX1D protein was less stable than the normal isoform, PBX1B, and it is degraded through the ubiquitin-proteasome-dependent pathway. The DNA binding domain lacking in PBX1D has two putative ubiquitin binding sites, K292 and K293, that are predicted to be in direct contact with DNA. Mutation of K292-293 reduced PBX1B stability to a level similar to PBX1D and abrogated DNA binding. In addition, contrary to PBX1B, PBX1D is retained in the cytoplasm without the help of the cofactors MEIS or PREP1, indicating a different requirement for nuclear translocation. Overall, these findings suggest that multiple post-transcriptional mechanisms are responsible for PBX1D loss of function and induction of CD4+ T cell inflammatory phenotypes in systemic lupus erythematosus.

Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.

Ancestral origins are associated with SARS-CoV-2 susceptibility and protection in a Florida patient population.

COVID-19 is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The severity of COVID-19 is highly variable and related to known (e.g., age, obesity, immune deficiency) and unknown risk factors. The widespread clinical symptoms encompass a large group of asymptomatic COVID-19 patients, raising a crucial question regarding genetic susceptibility, e.g., whether individual differences in immunity play a role in patient symptomatology and how much human leukocyte antigen (HLA) contributes to this. To reveal genetic determinants of susceptibility to COVID-19 severity in the population and further explore potential immune-related factors, we performed a genome-wide association study on 284 confirmed COVID-19 patients (cases) and 95 healthy individuals (controls). We compared cases and controls of European (EUR) ancestry and African American (AFR) ancestry separately. We identified two loci on chromosomes 5q32 and 11p12, which reach the significance threshold of suggestive association (p<1x10-5 threshold adjusted for multiple trait testing) and are associated with the COVID-19 susceptibility in the European ancestry (index rs17448496: odds ratio[OR] = 0.173; 95% confidence interval[CI], 0.08-0.36 for G allele; p = 5.15× 10-5 and index rs768632395: OR = 0.166; 95% CI, 0.07-0.35 for A allele; p = 4.25×10-6, respectively), which were associated with two genes, PPP2R2B at 5q32, and LRRC4C at 11p12, respectively. To explore the linkage between HLA and COVID-19 severity, we applied fine-mapping analysis to dissect the HLA association with mild and severe cases. Using In-silico binding predictions to map the binding of risk/protective HLA to the viral structural proteins, we found the differential presentation of viral peptides in both ancestries. Lastly, extrapolation of the identified HLA from the cohort to the worldwide population revealed notable correlations. The study uncovers possible differences in susceptibility to COVID-19 in different ancestral origins in the genetic background, which may provide new insights into the pathogenesis and clinical treatment of the disease.

Taste Receptors Function as Nutrient Sensors in Pancreatic Islets: A Potential Therapeutic Target for Diabetes.

Glucose, amino acids, and free fatty acids are critical nutrients participating in stimulating or regulating the hormone secretion of islets. These nutrients are believed to be metabolized by pancreatic endocrine cells to function. However, recent evidence suggests that taste receptors, which play key roles in the oral cavity to sense glucose (sweet taste), amino acids (umami taste), and free fatty acids (fatty taste), are expressed in pancreatic islet cells and may act to sense these nutrients to regulate pancreatic hormone secretion, including insulin and glucagon. Disorders in these taste receptor pathways in islets may contribute to the pathogenesis of diabetes, or it may influence hyperglycemia, disturbance in amino acid metabolism, or hyperlipidemia. In this review, we su mMarize the expression and hormone-regulating functions of sweet, umami, and fatty taste receptors acting as nutrient sensors in pancreatic islets in vitro and in vivo. We discuss the potential roles of these taste receptor-nutrient sensor pathways in islets targeted to develop therapeutic strategies for diabetes and related disease.

Evidence of a Sjögren's disease-like phenotype following COVID-19.

Objectives: Sjögren's Disease (SjD) is a chronic and systemic autoimmune disease characterized by lymphocytic infiltration and the development of dry eyes and dry mouth resulting from the secretory dysfunction of the exocrine glands. SARS-CoV-2 may trigger the development or progression of autoimmune diseases, as evidenced by increased autoantibodies in patients and the presentation of cardinal symptoms of SjD. The objective of the study was to determine whether SARS-CoV-2 induces the signature clinical symptoms of SjD. Methods: The ACE2-transgenic mice were infected with SARS-CoV-2. SJD profiling was conducted. COVID-19 patients' sera were examined for autoantibodies. Clinical evaluations of convalescent COVID-19 subjects, including minor salivary gland (MSG) biopsies, were collected. Lastly, monoclonal antibodies generated from single B cells of patients were interrogated for ACE2/spike inhibition and nuclear antigens. Results: Mice infected with the virus showed a decreased saliva flow rate, elevated antinuclear antibodies (ANAs) with anti-SSB/La, and lymphocyte infiltration in the lacrimal and salivary glands. Sera of COVID-19 patients showed an increase in ANA, anti-SSA/Ro52, and anti-SSB/La. The male patients showed elevated levels of anti-SSA/Ro52 compared to female patients, and female patients had more diverse ANA patterns. Minor salivary gland biopsies of convalescent COVID-19 subjects showed focal lymphocytic infiltrates in four of six subjects, and 2 of 6 subjects had focus scores >2. Lastly, we found monoclonal antibodies produced in recovered patients can both block ACE2/spike interaction and recognize nuclear antigens. Conclusion: Overall, our study shows a direct association between SARS-CoV-2 and SjD. Hallmark features of SjD salivary glands were histologically indistinguishable from convalescent COVID-19 subjects. The results potentially implicate that SARS-CoV-2 could be an environmental trigger for SjD. Key Messages: What is already known about this subject?SAR-CoV-2 has a tropism for the salivary glands. However, whether the virus can induce clinical phenotypes of Sjögren's disease is unknown.What does this study add?Mice infected with SAR-CoV-2 showed loss of secretory function, elevated autoantibodies, and lymphocyte infiltration in glands.COVID-19 patients showed an increase in autoantibodies. Monoclonal antibodies produced in recovered patients can block ACE2/spike interaction and recognize nuclear antigens.Minor salivary gland biopsies of some convalescent subjects showed focal lymphocytic infiltrates with focus scores.How might this impact on clinical practice or future developments?Our data provide strong evidence for the role of SARS-CoV-2 in inducing Sjögren's disease-like phenotypes.Our work has implications for how patients will be diagnosed and treated effectively.

2.1 Å crystal structure of the Mycobacterium tuberculosis serine hydrolase, Hip1, in its anhydro-form (Anhydrohip1).

The 2.6 Å crystal structure of the apo form of Hip1 (hydrolase important for pathogenesis) has been previously reported. However, very little is known about the active site architecture of this M. tuberculosis (Mtb), serine hydrolase drug target. To begin mapping the active site of Hip1, we cocrystallized Hip1 with the irreversible serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF). We chose AEBSF for cocrystallization with Hip1 since the similar inhibitor, phenylmethylsulfonyl fluoride (PMSF), interestingly exhibited no activity against Hip1. We obtained crystals that diffracted to 2.1 Å but to our bewilderment, we did not observe any electron density for the inhibitor in the omit map for the Hip1-AEBSF complex. Rather, in the active site, dehydroalanine (dAla) was found to occupy the expected position of the catalytic Ser228, thus yielding anhydrohip1. Here we present a comparative analysis of the crystal structures of anhydrohip1 and Hip1 and provide a mechanism for the conversion of the enzyme to the anhydro-form through reaction with AEBSF. With the aid of molecular docking, we propose an explanation for the differential inhibition of Hip1 by AEBSF and PMSF. We also present a preliminary definition of the S1 and S2 pockets of the protease's active site and propose a mechanism for a ligand-induced conformational change within the S2 pocket. Finally, we expand upon the previous demarcation of the putative lipid binding pocket in the α-domain of the enzyme. We believe that this detailed analysis of the structures of anhydrohip1 and Hip1 provides valuable information useful for the structure-based drug design of novel Hip1-directed Mtb therapeutics.

Alpha-1 antitrypsin: A novel biomarker and potential therapeutic approach for metabolic diseases.

It is well recognized that chronic low-grade systemic inflammation and autoimmunity contribute to the pathogenesis of metabolic syndrome, its associated diseases (e.g. type 2 diabetes, non-alcoholic fatty liver disease) and type 1 diabetes, respectively. Consequently, anti-inflammatory agents might play a role in managing these immune associated metabolic diseases. Alpha-1 antitrypsin (AAT), an endogenous acute phase protein being used for treatment of AAT deficiency (a rare genetic disease), has multiple functions including anti-inflammatory, immunomodulatory, anti-apoptosis and cytoprotective effects. In this review, we summarized basic and clinical studies that reported potential therapeutic role of AAT in metabolic syndrome associated diseases and type 1 diabetes. Studies that demonstrated AAT had the possibility to be used as a novel biomarker to predict these immune associated metabolic diseases were also included.

Epitope Mapping of Pathogenic Autoantigens on Sjögren's Syndrome-Susceptible Human Leukocyte Antigens Using In Silico Techniques.

Sjögren's syndrome (SjS) is characterized by lymphocytic infiltration and the dysfunction of the salivary and lacrimal glands. The autoimmune response is driven by the effector T cells and their cytokines. The activation of the effector helper T cells is mediated by autoantigen presentation by human leukocyte antigen (HLA) class II molecules of antigen-presenting cells. Studies using familial aggregation, animal models, and genome-wide association demonstrate a significant genetic correlation between specific risk HLAs and SjS. One of the key HLA alleles is HLA-DRB1*0301; it is one of the most influential associations with primary SjS, having the highest odds ratio and occurrence across different ethnic groups. The specific autoantigens attributed to SjS remain elusive, especially the specific antigenic epitopes presented by HLA-DRB1*0301. This study applied a high throughput in silico mapping technique to identify antigenic epitopes of known SjS autoantigens presented by high-risk HLAs. Furthermore, we identified specific binding HLA-DRB1*0301 epitopes using structural modeling tools such as Immune Epitope Database and Analysis Resource IEDB, AutoDock Vina, and COOT. By deciphering the critical epitopes of autoantigens presented by HLA-DRB1*0301, we gain a better understanding of the origin of the antigens, determine the T cell receptor function, learn the mechanism of disease progression, and develop therapeutic applications.

Low-frequency variants in mildly symptomatic vaccine breakthrough infections presents a doubled-edged sword.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) has raised questions regarding vaccine protection against SARS-CoV-2 infection, transmission, and ongoing virus evolution. Twenty-three mildly symptomatic "vaccination breakthrough" infections were identified as early as January 2021 in Alachua County, Florida, among individuals fully vaccinated with either the BNT162b2 (Pfizer) or the Ad26 (Janssen/J&J) vaccines. SARS-CoV-2 genomes were successfully generated for 11 of the vaccine breakthroughs, and 878 individuals in the surrounding area and were included for reference-based phylogenetic investigation. These 11 individuals were characterized by infection with VOCs, but also low-frequency variants present within the surrounding population. Low-frequency mutations were observed, which have been more recently identified as mutations of interest owing to their location within targeted immune epitopes (P812L) and association with increased replicative capacity (L18F). We present these results to posit the nature of the efficacy of vaccines in reducing symptoms as both a blessing and a curse-as vaccination becomes more widespread and self-motivated testing reduced owing to the absence of severe symptoms, we face the challenge of early recognition of novel mutations of potential concern. This case study highlights the critical need for continued testing and monitoring of infection and transmission among individuals regardless of vaccination status.

Emerging mutation patterns in SARS-CoV-2 variants.

There is an urgent need to understand the functional effects of mutations in emerging variants of SARS-CoV-2. Variants of concern (alpha, beta, gamma and delta) acquired four patterns of spike glycoprotein mutations that enhance transmissibility and immune evasion: 1) mutations in the N-terminal domain (NTD), 2) mutations in the Receptor Binding Domain (RBD), 3) mutations at interchain contacts of the spike trimer, and 4) furin cleavage site mutations. Most distinguishing mutations among variants of concern are exhibited in the NTD, localized to sites of high structural flexibility. Emerging variants of interest such as mu, lambda and C.1.2 exhibit the same patterns of mutations as variants of concern. There is a strong likelihood that SARS-CoV-2 variants will continue to emerge with mutations in these defined patterns, thus providing a basis for the development of next line antiviral drugs and vaccine candidates.

Blocking IAg7 class II major histocompatibility complex by drug-like small molecules alleviated Sjögren's syndrome in NOD mice.

BACKGROUND: Sjögren's syndrome (SjS) is an autoimmune disease with a strong genetic association. To date, no vaccine or therapeutic agent exists to cure SjS, and patients must rely on lifelong therapies to treat symptoms. Human leukocyte antigens (HLA) are primary susceptibility loci that form the genetic basis for many autoimmune diseases, including SjS. In this study, we sought to determine whether blocking MHC class II IAg7 antigen presentation in the NOD mouse would alleviate SjS by preventing the recognition of autoantigens by pathogenic T cells. METHODS: Mapping of the antigenic epitopes of Ro60 autoantigen to IAg7 of the NOD mice was performed using structural modeling and in-vitro stimulation. Tetraazatricyclo-dodecane (TATD) and 8-Azaguanine (8-Aza) were previously identified as potential binders to IAg7 of the NOD mice using in silico drug screening. Mice were treated with 20mgs/kg via IP every day five days/week for 23 weeks. Disease profiling was conducted. FINDINGS: Specific peptides of Ro60 autoantigen were identified to bind to IAg7 and stimulated splenocytes of the NOD mice. Treating NOD mice with TATD or 8-Azaguanine alleviated SjS symptoms by improving salivary and lacrimal gland secretory function, decreasing the levels of autoantibodies, and reducing the severity of lymphocytic infiltration in the salivary and lacrimal glands. INTERPRETATION: This study presents a novel therapeutic approach for SjS by identifying small molecules capable of inhibiting T cell response via antigen-specific presentation. FUNDING: CQN is supported financially in part by PHS grants AI130561, DE026450, and DE028544 from the National Institutes of Health.

The Elusive Coreceptors for the SARS-CoV-2 Spike Protein.

Evidence suggests that the N-terminal domain (NTD) of the SARS-CoV-2 spike protein interacts with host coreceptors that participate in viral entry. Resolving the identity of coreceptors has important clinical implications as it may provide the basis for the development of antiviral drugs and vaccine candidates. The majority of characteristic mutations in variants of concern (VOCs) have occurred in the NTD and receptor binding domain (RBD). Unlike the RBD, mutations in the NTD have clustered in the most flexible parts of the spike protein. Many possible coreceptors have been proposed, including various sugars such as gangliosides, sialosides, and heparan sulfate. Protein coreceptors, including neuropilin-1 and leucine-rich repeat containing 15 (LRRC15), are also proposed coreceptors that engage the NTD.

Molecular Docking-Based Screening for Novel Inhibitors of the Human Immunodeficiency Virus Type 1 Protease that Effectively Reduce the Viral Replication in Human Cells.

Therapeutic pressure by protease inhibitors (PIs) contributes to accumulation of mutations in the HIV type 1 (HIV-1) protease (PR) leading to development of drug resistance with subsequent therapy failure. Current PIs target the active site of PR in a competitive manner. Identification of molecules that exploit non-active site mechanisms of inhibition is essential to overcome resistance to current PIs. Potential non-active site HIV-1 protease (PR) inhibitors (PI) were identified by in silico screening of almost 140,000 molecules targeting the hinge region of PR. Inhibitory activity of best docking compounds was tested in an in vitro PR inhibition biochemical assay. Five compounds inhibited PR from multiple HIV-1 sub-types in vitro and reduced replicative capacity by PI-sensitive or multi-PI resistant HIV-1 variants in human cells ex vivo. Antiviral activity was boosted when combined with Ritonavir, potentially diminishing development of drug resistance, while providing effective treatment for drug resistant HIV-1 variants.

Highly Specific Sigma Receptor Ligands Exhibit Anti-Viral Properties in SARS-CoV-2 Infected Cells.

(1) Background: There is a strong need for prevention and treatment strategies for COVID-19 that are not impacted by SARS-CoV-2 mutations emerging in variants of concern. After virus infection, host ER resident sigma receptors form direct interactions with non-structural SARS-CoV-2 proteins present in the replication complex. (2) Methods: In this work, highly specific sigma receptor ligands were investigated for their ability to inhibit both SARS-CoV-2 genome replication and virus induced cellular toxicity. This study found antiviral activity associated with agonism of the sigma-1 receptor (e.g., SA4503), ligation of the sigma-2 receptor (e.g., CM398), and a combination of the two pathways (e.g., AZ66). (3) Results: Intermolecular contacts between these ligands and sigma receptors were identified by structural modeling. (4) Conclusions: Sigma receptor ligands and drugs with off-target sigma receptor binding characteristics were effective at inhibiting SARS-CoV-2 infection in primate and human cells, representing a potential therapeutic avenue for COVID-19 prevention and treatment.

Independent infections of porcine deltacoronavirus among Haitian children.

Coronaviruses have caused three major epidemics since 2003, including the ongoing SARS-CoV-2 pandemic. In each case, the emergence of coronavirus in our species has been associated with zoonotic transmissions from animal reservoirs1,2, underscoring how prone such pathogens are to spill over and adapt to new species. Among the four recognized genera of the family Coronaviridae, human infections reported so far have been limited to alphacoronaviruses and betacoronaviruses3-5. Here we identify porcine deltacoronavirus strains in plasma samples of three Haitian children with acute undifferentiated febrile illness. Genomic and evolutionary analyses reveal that human infections were the result of at least two independent zoonoses of distinct viral lineages that acquired the same mutational signature in the genes encoding Nsp15 and the spike glycoprotein. In particular, structural analysis predicts that one of the changes in the spike S1 subunit, which contains the receptor-binding domain, may affect the flexibility of the protein and its binding to the host cell receptor. Our findings highlight the potential for evolutionary change and adaptation leading to human infections by coronaviruses outside of the previously recognized human-associated coronavirus groups, particularly in settings where there may be close human-animal contact.

Generation of a Novel SARS-CoV-2 Sub-genomic RNA Due to the R203K/G204R Variant in Nucleocapsid: Homologous Recombination has Potential to Change SARS-CoV-2 at Both Protein and RNA Level.

BACKGROUND: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host's anti-viral immune response, in turn affecting the frequency of variants over time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. METHODS: Deep-sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. Results: Sequence analysis suggests that the 3 adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep-sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. CONCLUSIONS: The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans, resulting in both coding changes and novel sub-genomic RNA transcripts, suggests this as a mechanism for diversification and adaptation within its new host.

Identification and Characterization of Two Structurally Related Dipeptides that Enhance Catalytic Efficiency of Neurolysin.

Neurolysin (Nln) is a recently recognized endogenous mechanism functioning to preserve the brain from ischemic injury. To further understand the pathophysiological function of this peptidase in stroke and other neurologic disorders, the present study was designed to identify small molecule activators of Nln. Using a computational approach, the structure of Nln was explored, which was followed by docking and in silico screening of ∼140,000 molecules from the National Cancer Institute Developmental Therapeutics Program database. Top ranking compounds were evaluated in an Nln enzymatic assay, and two hit histidine-dipeptides were further studied in detail. The identified dipeptides enhanced the rate of synthetic substrate hydrolysis by recombinant (human and rat) and mouse brain-purified Nln in a concentration-dependent manner (micromolar A50 and Amax ≥ 300%) but had negligible effect on activity of closely related peptidases. Both dipeptides also enhanced hydrolysis of Nln endogenous substrates neurotensin, angiotensin I, and bradykinin and increased efficiency of the synthetic substrate hydrolysis (Vmax/Km ratio) in a concentration-dependent manner. The dipeptides and competitive inhibitor dynorphin A (1-13) did not affect each other's affinity for Nln, suggesting differing nature of their respective binding sites. Lastly, drug affinity responsive target stability (DARTS) and differential scanning fluorimetry (DSF) assays confirmed concentration-dependent interaction of Nln with the activator molecule. This is the first study demonstrating that Nln activity can be enhanced by small molecules, although the peptidic nature and low potency of the activators limit their application. The identified dipeptides provide a chemical scaffold to develop high-potency, drug-like molecules as research tools and potential drug leads. SIGNIFICANCE STATEMENT: This study describes discovery of two molecules that selectively enhance activity of peptidase Nln-a newly recognized cerebroprotective mechanism in the poststroke brain. The identified molecules will serve as a chemical scaffold for development of drug-like molecules to further study Nln and may become lead structures for a new class of drugs. In addition, our conceptual and methodological framework and research findings might be used for other peptidases and enzymes, the activation of which bears therapeutic potential.