Expression of IL-4 in Tumors: A Safety Surrogate to Predict Cancer Survival Associated With Biologic Therapies.

Interleukin (IL)-4-targeted therapies have revolutionized management of inflammatory dermatoses.

Integrating High-Resolution Mass Spectral Data, Bioassays and Computational Models to Annotate Bioactives in Botanical Extracts: Case Study Analysis of C. asiatica Extract Associates Dicaffeoylquinic Acids with Protection against Amyloid-ß Toxicity.

Rapid screening of botanical extracts for the discovery of bioactive natural products was performed using a fractionation approach in conjunction with flow-injection high-resolution mass spectrometry for obtaining chemical fingerprints of each fraction, enabling the correlation of the relative abundance of molecular features (representing individual phytochemicals) with the read-outs of bioassays. We applied this strategy for discovering and identifying constituents of Centella asiatica (C. asiatica) that protect against Aß cytotoxicity in vitro. C. asiatica has been associated with improving mental health and cognitive function, with potential use in Alzheimer's disease. Human neuroblastoma MC65 cells were exposed to subfractions of an aqueous extract of C. asiatica to evaluate the protective benefit derived from these subfractions against amyloid ß-cytotoxicity. The % viability score of the cells exposed to each subfraction was used in conjunction with the intensity of the molecular features in two computational models, namely Elastic Net and selectivity ratio, to determine the relationship of the peak intensity of molecular features with % viability. Finally, the correlation of mass spectral features with MC65 protection and their abundance in different sub-fractions were visualized using GNPS molecular networking. Both computational methods unequivocally identified dicaffeoylquinic acids as providing strong protection against Aß-toxicity in MC65 cells, in agreement with the protective effects observed for these compounds in previous preclinical model studies.

The Neonatal and Paediatric Pharmacokinetics of Antimicrobials study (NAPPA): investigating amoxicillin, benzylpenicillin, flucloxacillin and piperacillin pharmacokinetics from birth to adolescence.

BACKGROUND: Pharmacokinetic (PK) data underlying paediatric penicillin dosing remain limited, especially in critical care. OBJECTIVES: The primary objective of the Neonatal and Paediatric Pharmacokinetics of Antimicrobials study (NAPPA) was to characterize PK profiles of commonly used penicillins using data obtained during routine care, to further understanding of PK variability and inform future evidence-based dosing. METHODS: NAPPA was a multicentre study of amoxicillin, co-amoxiclav, benzylpenicillin, flucloxacillin and piperacillin/tazobactam. Patients were recruited with informed consent. Antibiotic dosing followed standard of care. PK samples were obtained opportunistically or at optimal times, frozen and analysed using UPLC with tandem MS. Pharmacometric analysis was undertaken using NONMEM software (v7.3). Model-based simulations (n = 10 000) tested PTA with British National Formulary for Children (BNFC) and WHO dosing. The study had ethical approval. RESULTS: For the combined IV PK model, 963 PK samples from 370 participants were analysed simultaneously incorporating amoxicillin, benzylpenicillin, flucloxacillin and piperacillin data. BNFC high-dose regimen simulations gave these PTA results (median fT>MIC at breakpoints of specified pathogens): amoxicillin 100% (Streptococcus pneumoniae); benzylpenicillin 100% (Group B Streptococcus); flucloxacillin 48% (MSSA); and piperacillin 100% (Pseudomonas aeruginosa). Oral population PK models for flucloxacillin and amoxicillin enabled estimation of first-order absorption rate constants (1.16 h-1 and 1.3 h-1) and bioavailability terms (62.7% and 58.7%, respectively). CONCLUSIONS: NAPPA represents, to our knowledge, the largest prospective combined paediatric penicillin PK study undertaken to date, and the first paediatric flucloxacillin oral PK model. The PTA results provide evidence supportive of BNFC high-dose IV regimens for amoxicillin, benzylpenicillin and piperacillin.

Uncovering the brain-wide pattern of synaptic input to vasopressin-expressing neurons in the paraventricular nucleus of the hypothalamus.

Arginine vasopressin (AVP) is a neuropeptide critical for the mammalian stress response and social behavior. AVP produced in the hypothalamus regulates water osmolality and vasoconstriction in the body, and in the brain, it regulates social behavior, aggression, and anxiety. However, the circuit mechanisms that link AVP to social behavior, homeostatic function, and disease are not well understood. This study investigates the circuit configurations of AVP-expressing neurons in the rodent hypothalamus and characterizes synaptic input from the entire brain. We targeted the paraventricular nucleus (PVN) using retrograde viral tracing techniques to identify direct afferent synaptic connections made onto AVP-expressing neurons. AVP neurons in the PVN display region-specific anatomical configurations that reflect their unique contributions to homeostatic function, motor behaviors, feeding, and affiliative behavior. The afferent connections identified were similar in both sexes and subsequent molecular investigation of these inputs shows that those local hypothalamic inputs are overwhelmingly nonpeptidergic cells indicating a potential interneuron nexus between hormone cell activation and broader cortical connection. This proposed work reveals new insights into the organization of social behavior circuits in the brain, and how neuropeptides act centrally to modulate social behaviors.

The Association between Caffeine Intake and the Colonic Mucosa-Associated Gut Microbiota in Humans-A Preliminary Investigation.

We examined the association between caffeine and coffee intake and the community composition and structure of colonic microbiota. A total of 34 polyp-free adults donated 97 colonic biopsies. Microbial DNA was sequenced for the 16S rRNA gene V4 region. The amplicon sequence variant was assigned using DADA2 and SILVA. Food consumption was ascertained using a food frequency questionnaire. We compared the relative abundance of taxonomies by low (<82.9 mg) vs. high (≥82.9 mg) caffeine intake and by never or <2 cups vs. 2 cups vs. ≥3 cups coffee intake. False discovery rate-adjusted p values (q values) <0.05 indicated statistical significance. Multivariable negative binomial regression models were used to estimate the incidence rate ratio and its 95% confidence interval of having a non-zero count of certain bacteria by intake level. Higher caffeine and coffee intake was related to higher alpha diversity (Shannon index p < 0.001), higher relative abundance of Faecalibacterium and Alistipes, and lower relative abundance of Erysipelatoclostridium (q values < 0.05). After adjustment of vitamin B2 in multivariate analysis, the significant inverse association between Erysipelatoclostridium count and caffeine intake remained statistically significant. Our preliminary study could not evaluate other prebiotics in coffee.

LC-QToF-Based Metabolomics Identifies Aberrant Tissue Metabolites Associated with a Higher-Fat Diet and Their 'Reversion to Healthy' with Dietary Probiotic Supplementation.

Over 33% of Americans are labeled as obese, leading the World Health Organization to designate obesity as a major public health problem. One consequence of obesity is the development of metabolic syndrome, a condition which has been correlated to an increased risk for developing cardiovascular disease and Type 2 diabetes. Prolonged ingestion of a higher-fat diet, one cause of obesity, results in alterations to the gut microbiome. These alterations are implicated to have a profound role in the evolution and progression of obesity-linked diseases. Probiotics are associated with positive health effects such as limiting pathogen colonization, aiding in digestion, and vitamin synthesis. Using Ossabaw pigs as a model for obesity, and in conjunction with our previous research, we performed an in-depth, nontargeted, metabolomic analysis on select organs to elucidate the effects of dietary supplementation with the probiotic Lacticaseibacillus paracasei. We focused our analysis on the effects of probiotic supplementation on a higher-fat (obesogenic) diet and a nutritionally balanced diet. Notably, our findings reveal that the brain cortex is highly sensitive to dietary influencers, and with probiotic supplementation, several aberrant metabolites associated with a higher-fat diet revert to healthy levels, thus demonstrating the potential for a probiotic intervention for obesity-linked disease.

The Controversy of Chronotherapy: Emerging Evidence regarding Bedtime Dosing of Antihypertensive Medications in Non-Arteritic Anterior Ischemic Optic Neuropathy.

"Blindness upon awakening" occurs in a significant proportion of patients with non-arteritic anterior ischemic optic neuropathy (NAION). This observation has led to a notion that nocturnal hypotension is a significant contributor and, perhaps, the final insult in a multifactorial process leading to the development of NAION, as has been proposed in other ischemic events like strokes, myocardial infarction, and ischemic rest pain. An extension of this concept has led to the recommendation that patients who have experienced NAION avoid taking blood pressure medications at bedtime. However, mounting evidence in the cardiology literature suggests that nocturnal hypertension is associated with increased risk of cardiovascular morbidity. In two prospective blood pressure monitoring studies in 1994 and 1999, Hayreh observed an extreme dipping pattern in nocturnal systolic blood pressure in NAION patients compared to reported normal values. Yet, two subsequent ambulatory blood pressure studies found either normal or non-dipping patterns in NAION patients. The majority of clinical trials published since 1976 that have studied nocturnal administration of antihypertensives have reported enhanced blood pressure control and reduced cardiovascular risk. Most notably, the large, prospective 2020 Hygia Chronotherapy Trial reported a statistically-significant beneficial effect of nocturnal antihypertensive dosing on cardiovascular outcomes and mortality. The controversy regarding nocturnal hypotension and NAION is of increasing relevance as there is new evidence to suggest a beneficial effect of nocturnal antihypertensive dosing in cardiovascular risk. This new information should prompt a re-evaluation of the relevant risk-to-benefit of reducing the risk of NAION on one hand, and the potential increase of cardiovascular risk on the other. Definitive resolution of this question would require a prospective, randomized control study with input from both cardiology and ophthalmology.

Complementary and Integrative Medicine in Head and Neck Cancer.

Complementary/integrative medicine (CIM) is an evolving area of collaboration between oncology, patient and their beliefs, and practitioners of complementary medicine. Evidence-informed decision-making is necessary to advise patients on which treatments may be incorporated into standard of care treatments for cancer. Patients use CIM for a variety of reasons and often have unrealistic expectations of cure or disease modifications; on the other hand, there is increasing evidence that symptoms, side effects, and dysfunction related to cancer and its treatment can be ameliorated by CIM approaches to improve patient satisfaction and quality of life. Open communication between patients and providers is paramount.

Predictors of nonresponse to dupilumab in patients with atopic dermatitis: A machine learning analysis.

BACKGROUND: Many patients with atopic dermatitis (AD) have a suboptimal response to systemic therapy. OBJECTIVE: This study assessed predictors of nonresponse to dupilumab in patients with AD. METHODS: Data (April 2017 through June 2019) for patients aged 12 years and above with AD (International Classification of Diseases-9/10-Clinical Modification: 691.8/L20.x) who initiated dupilumab on or after April 1, 2017 (index date) were collected from an electronic health record and insurance claims database. Nonresponse indicators (dupilumab discontinuation, addition of another systemic therapy or phototherapy, addition of a high-potency topical corticosteroid, AD-related hospital visit, AD-related emergency department visit, incident skin infection) were predicted from available demographic and clinical variables using machine learning. RESULTS: Among 419 patients (mean age: 45 years), 145 (35%) experienced at least 1 indicator of nonresponse in the 6-month postindex period. In patients with at least 1 indicator, the most common was dupilumab discontinuation (47% [68/145]). Of note, this analysis could not capture nonmedical reasons for dupilumab discontinuation (eg, cost, access). The most common predictors of nonresponse were a claim for ibuprofen (in 69% of patients with a nonresponse indicator) and a Quan-Charlson Comorbidity Index value of 3 to 4 (59%). CONCLUSION: Systemic dupilumab therapy for AD can be associated with a relatively high prevalence of nonresponse indicators. Factors associated with these indicators-that is, predictors of nonresponse-may be used to optimize disease management.

Bimekizumab Safety in Patients With Moderate to Severe Plaque Psoriasis: Pooled Results From Phase 2 and Phase 3 Randomized Clinical Trials.

Importance: Psoriasis is a chronic disease requiring long-term management; understanding the long-term safety profiles of psoriasis treatments, such as bimekizumab, is important. Objective: To evaluate the 2-year safety profile of bimekizumab in patients with moderate to severe plaque psoriasis. Design, Setting, and Participants: Safety data were pooled from a cohort of patients from 4 phase 2 randomized clinical trials (BE ABLE 1, BE ABLE 2, PS0016, and PS0018) and 4 phase 3 randomized clinical trials (BE VIVID, BE READY, BE SURE, and BE BRIGHT) to include 2 years of study treatment. Data were obtained on adults with moderate to severe plaque psoriasis (Psoriasis Area and Severity Index level ≥12, ≥10% body surface area affected by psoriasis, and an Investigator's Global Assessment score ≥3 on a 5-point scale) who were eligible for systemic psoriasis therapy and/or phototherapy. Interventions: Included patients received 1 or more doses of bimekizumab during the phase 2 or phase 3 trials. Main Outcomes and Measures: Treatment-emergent adverse events (TEAEs), serious TEAEs, and TEAEs leading to treatment discontinuation are reported using exposure-adjusted incidence rates (EAIRs) per 100 person-years. Results: A total of 1789 patients (1252 [70.0%] men; mean [SD] age, 45.2 [13.5] years) were treated with 1 or more doses of bimekizumab across the phase 2/3 trials and were included in these analyses; total bimekizumab exposure was 3109.7 person-years. TEAEs occurred at an EAIR of 202.4 per 100 person-years and did not increase with longer duration of bimekizumab exposure. The 3 most frequently reported TEAEs were nasopharyngitis (19.1 per 100 person-years; 95% CI, 17.4-20.9 per 100 person-years), oral candidiasis (12.6 per 100 person-years; 95% CI, 11.3-14.0 per 100 person-years), and upper respiratory tract infection (8.9 per 100 person-years; 95% CI, 7.8-10.1 per 100 person-years). Most oral candidiasis events were mild or moderate; 3 events led to discontinuation. The EAIRs of inflammatory bowel disease (0.1 per 100 person-years; 95% CI, 0.0-0.3 per 100 person-years), adjudicated suicidal ideation and behavior (0.0 per 100 person-years; 95% CI, 0.0-0.2 per 100 person-years), and adjudicated major adverse cardiac events (0.5 per 100 person-years; 95% CI, 0.3-0.8 per 100 person-years) were low. Conclusions and Relevance: In these pooled analyses of data from a cohort of patients from 8 randomized clinical trials, bimekizumab was well tolerated aside from an increased incidence of mild to moderate oral candidiasis. No safety signals were observed compared with previous reports, and there was no increased risk of AEs with longer duration of bimekizumab exposure.

Shoe Allergens: A Retrospective Analysis of Cross-sectional Data From the North American Contact Dermatitis Group, 2005-2018.

BACKGROUND: Shoe contact allergy can be difficult to diagnose and manage. OBJECTIVE: The aim of the study was to characterize demographics, clinical characteristics, patch test results, and occupational data for the North American Contact Dermatitis Group patients with shoe contact allergy. METHODS: This is a retrospective study of 33,661 patients, patch tested from 2005 to 2018, with a shoe source, foot as 1 of 3 sites of dermatitis, and final primary diagnosis of allergic contact dermatitis. RESULTS: Three hundred fifty-two patients met the inclusion criteria. They were more likely to be male (odds ratio = 3.36, confidence interval = 2.71-4.17) and less likely to be older than 40 years (odds ratio = 0.49, confidence interval = 0.40-0.61) compared with others with positive patch test reactions. The most common relevant North American Contact Dermatitis Group screening allergens were potassium dichromate (29.8%), p-tert-butylphenol formaldehyde resin (20.1%), thiuram mix (13.3%), mixed dialkyl thioureas (12.6%), and carba mix (12%). A total of 29.8% (105/352) had positive patch test reactions to supplemental allergens, and 12.2% (43/352) only had reactions to supplemental allergens. CONCLUSIONS: Shoe contact allergy was more common in younger and male patients. Potassium dichromate and p-tert-butylphenol formaldehyde resin were the top shoe allergens. Testing supplemental allergens, personal care products, and shoe components should be part of a comprehensive evaluation of suspected shoe contact allergy.

Efficacy of ixekizumab in patients with moderate-to-severe plaque psoriasis and prediabetes or type 2 diabetes.

Objective: Patients with psoriasis have an increased prevalence of type 2 diabetes when compared to the general population. Research suggests that type 2 diabetes (T2D) as well as obesity may have an impact on patients' response to treatment. This post-hoc analysis reports the efficacy of ixekizumab in treating moderate-to-severe psoriasis in patients with prediabetes or T2D. Method and materials: UNCOVER-1, UNCOVER-2, and UNCOVER-3 were three Phase 3, multicenter, randomized, double-blind, placebo-controlled trials that evaluated the efficacy and safety of ixekizumab in adult patients with moderate-to-severe psoriasis. Patients were aged ≥18 years with chronic moderate-to-severe psoriasis (defined as ≥10% body surface area affected, static Physician Global Assessment ≥3, and Psoriasis Area and Severity Index [PASI] ≥12 at screening and baseline) who were candidates for phototherapy or systemic therapy. UNCOVER-1, UNCOVER-2, and UNCOVER-3 participants received ixekizumab as per label (that is, an initial dose of two subcutaneous injections [160 mg in total] at Week 0, followed by 80 mg every 2 weeks through Week 12 and 80 mg every 4 weeks thereafter through Week 60). Results: The proportions of patients with prediabetes, T2D and normoglycemia that achieved PASI75, PASI90, and PASI100 at Week 60 were similar. Results suggest that patients with T2D were slower to achieve PASI100 than patients with prediabetes or those with normoglycemia. Ixekizumab had no effect on any metabolic markers in patients receiving the treatment. Conclusions: Despite the higher rate of obesity and extreme obesity in patients with prediabetes and T2D, ixekizumab was an efficacious treatment in treating patients with psoriasis.

Does biologic therapy impact the development of PsA among patients with psoriasis?

OBJECTIVE: To examine the association of biologic therapy use for psoriasis with incident psoriatic arthritis (PsA) diagnosis. METHODS: A retrospective cohort study was conducted in the OptumInsights Electronic Health Record Database between 2006 and 2017 among patients with psoriasis between the ages of 16 and 90 initiating a therapy for psoriasis (oral, biologic or phototherapy). The incidence of PsA was calculated within each therapy group. Multivariable Cox models were used to calculate the HR for biologic versus oral or phototherapy using biologics as a time-varying exposure and next in a propensity score-matched cohort. RESULTS: Among 1 93 709 patients with psoriasis without PsA, 14 569 biologic and 20 321 cumulative oral therapy and phototherapy initiations were identified. Mean age was lower among biologic initiators compared with oral/phototherapy initiators (45.9 vs 49.8). The incidence of PsA regardless of therapy exposure was 9.75 per 1000 person-years compared with 77.26 among biologic users, 61.99 among oral therapy users, 26.11 among phototherapy users and 5.85 among those without a prescription for one of the target therapies. Using a multivariable adjustment approach with time-varying exposure, adjusted HR (95% CI) for biologic users was 4.48 (4.23 to 4.75) compared with oral or phototherapy users. After propensity score matching, the HR (95% CI) was 2.14 (2.00 to 2.28). CONCLUSIONS: In this retrospective cohort study, biologic use was associated with the development of PsA among patients with psoriasis. This may be related to confounding by indication and protopathic bias. Prospective studies are needed to address this important question.

Dietary Docosahexaenoic Acid as a Potential Treatment for Semi-acute and Chronic Particle-Induced Pulmonary Inflammation in Balb/c Mice.

Acute and chronic inflammation are vital contributing factors to pulmonary diseases which can be triggered by exposure to occupational and man-made particles; however, there are no established treatments. One potential treatment shown to have anti-inflammatory capabilities is the dietary supplement docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid found in fish oil. DHA's anti-inflammatory mechanisms are unclear for particle-induced inflammation; therefore, this study evaluated DHA as a prophylactic treatment for semi-acute and chronic particle-induced inflammation in vivo. Balb/c mice were fed a control or 1% DHA diet and exposed to dispersion media, an inflammatory multi-walled carbon nanotube (MWCNT), or crystalline silica (SiO2) either once (semi-acute) or once a week for 4 weeks (chronic). The hypothesis was that DHA will decrease pulmonary inflammatory markers in response to particle-induced inflammation. Results indicated that DHA had a trending anti-inflammatory effect in mice exposed to MWCNT. There was a general decrease in inflammatory signals within the lung lavage fluid and upregulation of M2c macrophage gene expression in the spleen tissue. In contrast, mice exposed to SiO2 while on the DHA diet significantly increased most inflammatory markers. However, DHA stabilized the phagolysosomal membrane upon prolonged treatment. This indicated that DHA treatment may depend upon certain inflammatory particle exposures as well as the length of the exposure.

Morning light therapy in adults with Tourette's disorder.

BACKGROUND: Sleep disturbance is common among individuals with Tourette's Disorder (TD). Given that sleep is influenced by the circadian system, this study examined circadian rhythms and sleep in adults with TD, and explored the possible benefit of short-wavelength wearable morning light therapy. METHODS: Participants were 34 adults with TD (n = 14) and age- and sex-matched healthy controls (HC; n = 20). Participants were screened using clinician-rated diagnostic and tic severity interviews, and procedures lasted 3 consecutive weeks. Participants completed a baseline week of actigraphy. Adults with TD completed 2 weeks of Re-Timer™ morning light therapy and continued actigraphy monitoring. Dim light melatonin-onset (DLMO) phase assessment, tic severity interview, and measures of chronotype, sleep disturbance, daytime sleepiness, disability, depression, anxiety, and stress were completed at baseline and post-intervention. RESULTS: Adults with TD reported significantly greater eveningness and sleep disturbance relative to controls. Per wrist actigraphy, adults with TD exhibited significantly longer sleep-onset latency, lower sleep efficiency, and greater sleep fragmentation than HC. Following morning light therapy, there was a significant advance in DLMO phase, but not self-report or actigraphy sleep variables. There were small, statistically significant decreases in tic severity and impairment. There were also significant reductions in daytime sleepiness, and self-reported anxiety, but not depression, stress, or disability. Participants reported minimal side effects and rated light therapy as acceptable and comfortable. CONCLUSIONS: Findings showed some benefits following brief light therapy in TD; further exploration of the impact of spectral tuning the photic environment as part of treatment for TD subjects is warranted.

Fruit and Vegetable Supplemented Diet Modulates the Pig Transcriptome and Microbiome after a Two-Week Feeding Intervention.

A study was conducted to determine the effects of a diet supplemented with fruits and vegetables (FV) on the host whole blood cell (WBC) transcriptome and the composition and function of the intestinal microbiome. Nine six-week-old pigs were fed a pig grower diet alone or supplemented with lyophilized FV equivalent to half the daily recommended amount prescribed for humans by the Dietary Guideline for Americans (DGA) for two weeks. Host transcriptome changes in the WBC were evaluated by RNA sequencing. Isolated DNA from the fecal microbiome was used for 16S rDNA taxonomic analysis and prediction of metabolomic function. Feeding an FV-supplemented diet to pigs induced differential expression of several genes associated with an increase in B-cell development and differentiation and the regulation of cellular movement, inflammatory response, and cell-to-cell signaling. Linear discriminant analysis effect size (LEfSe) in fecal microbiome samples showed differential increases in genera from Lachnospiraceae and Ruminococcaceae families within the order Clostridiales and Erysipelotrichaceae family with a predicted reduction in rgpE-glucosyltransferase protein associated with lipopolysaccharide biosynthesis in pigs fed the FV-supplemented diet. These results suggest that feeding an FV-supplemented diet for two weeks modulated markers of cellular inflammatory and immune function in the WBC transcriptome and the composition of the intestinal microbiome by increasing the abundance of bacterial taxa that have been associated with improved intestinal health.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14/nsp10 exoribonuclease.

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease.

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.