Using risk-adjusted cumulative sum to evaluate surgeon, divisional, and institutional outcomes-a feasibility study.

BACKGROUND: Few objective, real-time measurements of surgeon performance exist. The risk-adjusted cumulative sum is a novel method that can track surgeon-level outcomes on a continuous basis. The objective of this study was to demonstrate the feasibility of using risk-adjusted cumulative sum to monitor outcomes after colorectal operations and identify clinically relevant performance variations. METHODS: The National Surgical Quality Improvement Program was queried to obtain patient-level data for 1,603 colorectal operations at a high-volume center from 2011 to 2020. For each case, expected risks of morbidity, mortality, reoperation, readmission, and prolonged length of stay were estimated using the National Surgical Quality Improvement Program risk calculator. Risk-adjusted cumulative sum curves were generated to signal observed-to-expected odds ratios of 1.5 (poor performance) and 0.5 (exceptional performance). Control limits were set based on a false positive rate of 5% (α = 0.05). RESULTS: The cohort included data on 7 surgeons (those with more than 20 cases in the study period). Institutional observed versus expected outcomes were the following: morbidity 12.5% (vs 15.0%), mortality 2.5% (vs 2.0%), prolonged length of stay 19.7% (vs 19.1%), reoperation 11.1% (vs 11.3%), and 30-day readmission 6.1% (vs 4.8%). Risk-adjusted cumulative sum accurately demonstrated within- and between-surgeon performance variations across these metrics and proved effective when considering division-level data. CONCLUSION: Risk-adjusted cumulative sum adjusts for patient-level risk factors to provide real-time data on surgeon-specific outcomes. This approach enables prompt identification of performance outliers and can contribute to quality assurance, root-cause analysis, and incentivization not only at the surgeon level but at divisional and institutional levels as well.

Physiotherapy-led, community-based airway clearance services for people with chronic lung conditions: a retrospective descriptive evaluation of an existing model of care.

OBJECTIVES: Airway clearance interventions are recommended for people with chronic lung conditions and mucus hypersecretion, but there are few published models of care or descriptions of airway clearance service provision. This evaluation describes a dedicated, physiotherapy-led, community-based airway clearance service in a metropolitan local health network. DESIGN: Retrospective evaluation using existing airway clearance service administrative database. PARTICIPANTS: All first referrals to the airway clearance service in a 5-year period (1/1/2017 to 31/12/2021). MAIN OUTCOME MEASURES: Available service data grouped into four domains: participant demographics, referral demographics, service provision and outcomes. RESULTS: Of the 1335 first referrals eligible for inclusion, 1157 (87%) people attended. Bronchiectasis was the commonest condition (n = 649/1135, 49%). A total of 2996 occasions of service (face to face clinic n = 2108, 70%, phone n = 736, 25%, telehealth n = 99, 3%, home visit n = 53, 2%) were delivered. Airway clearance devices frequently prescribed were the Aerobika (525/1157, 45%), bubble-positive expiratory pressure (263/1157, 23%) and the Acapella (127/1157, 11%). On average, initial appointment with the airway clearance service occurred within 36 days of referral and people attended the service three times. Individuals voluntarily completed both pre/post service questionnaires around a third of the time. At least half of responders reported an improvement in respiratory symptom outcome measures consistent with the minimum clinically important difference. CONCLUSIONS: This evaluation describes an airway clearance service as it exists, providing an example from which airway clearance services can be planned, implemented and improved.

Early Implementation of Robotic Training in Surgical and Surgical Subspecialty Residency.

BACKGROUND: Robotic surgery has emerged as an operative tool for many elective and urgent surgical procedures. The purpose of this study was to evaluate early surgical trainees' experiences and opinions of robotic surgery. METHODS: An introductory robotic training course consisting of online da Vinci Xi/X training and in-person, hands on training was implemented for residents and medical students across surgical subspecialties at a single institution. A voluntary survey evaluating perceptions of and interest in robotic surgery and prior robotic surgery experience, as well as a basics of robotics quiz, was distributed to participants prior to the start of the in-person session. Descriptive statistics were used to evaluate the cohort. RESULTS: 85 trainees participated in the course between 2020 and 2023, including 58 first- and second-year surgical residents (general surgery, urology, OB/GYN, and thoracic surgery) and 27 fourth-year medical students. 9.4% of participants reported any formal robotic surgery training prior to the session, with only 19% of participants reporting robotic operative experience. 52% of the participants knew of and/or had completed the da Vinci online course modules prior to the scheduled training session. Participants unanimously (100%) agreed that robotic surgery should be implemented into surgical training. CONCLUSIONS: There is rising enthusiasm for robotic surgery, yet early exposure and training remain infrequent and inconsistent amongst medical students and new surgical residents. A standardized introduction of multi-disciplinary robotic surgery training should be incorporated into medical school and/or early residency education to ensure surgical residents receive appropriate exposure and training to achieve competency.

The Role of Artificial Intelligence in Surgery: What do General Surgery Residents Think?

BACKGROUND: Artificial intelligence (AI) holds significant potential in medical education and patient care, but its rapid emergence presents ethical and practical challenges. This study explored the perspectives of surgical residents on AI's role in medicine. METHODS: We performed a cross-sectional study surveying general surgery residents at a university-affiliated teaching hospital about their views on AI in medicine and surgical training. The survey covered demographics, residents' understanding of AI, its integration into medical practice, and use of AI tools like ChatGPT. The survey design was inspired by a recent national survey and underwent pretesting before deployment. RESULTS: Of the 31 participants surveyed, 24% identified diagnostics as AI's top application, 12% favored its use in identifying anatomical structures in surgeries, and 20% endorsed AI integration into EMRs for predictive models. Attitudes toward AI varied based on its intended application: 77.41% expressed concern about AI making life decisions and 70.97% felt excited about its application for repetitive tasks. A significant 67.74% believed AI could enhance the understanding of medical knowledge. Perception of AI integration varied with AI familiarity (P = .01), with more knowledgeable respondents expressing more positivity. Moreover, familiarity influenced the perceived academic use of ChatGPT (P = .039) and attitudes toward AI in operating rooms (P = .032). Conclusion: This study provides insights into surgery residents' perceptions of AI in medical practice and training. These findings can inform future research, shape policy decisions, and guide AI development, promoting a harmonious collaboration between AI and surgeons to improve both training and patient care.

Analysis of 10 years of open, laparoscopic, and robotic rectal surgeries in the community setting.

Background: Colorectal cancer is the fourth most common cancer in the US. Many of these patients will require operations. Although there is significant data in the literature that supports minimally invasive colorectal operations in the academic setting, few studies have examined their performance in community hospitals. Methods: Data was collected from a high-volume, university-affiliated, community center. Our Cancer Registry Database was queried to include any patients that had rectal surgery at our institution from 2010 to 2020. One hundred-twenty-two patients were identified and reviewed retrospectively. Main outcome measures include estimated blood loss (EBL), blood transfusion, time to first bowel movement, oncologic resection, length of stay (LOS), survival, and cost analysis. Results: Both robotic and laparoscopic operations resulted in lower average EBL, less blood transfusions, and less time to first bowel movement (p = 0.003, 0.006, 0.003, respectively). There was no significant difference in ability to achieve R0 resection, adequate lymph node retrieval, and adequate total mesorectal excision (TME, p = 0.856, 0.489, 0.500, respectively). LOS was significantly shorter for minimally invasive operations, 4.35 vs 8.48 days, and average survival was longest for laparoscopic operations at 7.19 years as compared to 5.55 years for open operations (p < 0.001, 0.026, respectively). Cost was lowest for robotic operations (0.003). Conclusions: Minimally invasive rectal operations, especially robotic, lead to better short- and long-term outcomes, equivalent oncologic resection, and are more cost-effective as compared to open operations even in the community setting, supporting continued performance and growth of robotic colorectal operations in the community setting. Key message: Although there is significant data in the literature that supports minimally invasive colorectal operations in the academic setting, few studies have examined their performance in community hospitals as this study does. This study found that minimally invasive rectal operations, especially robotic, lead to better short- and long-term outcomes, equivalent oncologic resection, and are more cost-effective as compared to open operations even in the community setting, supporting continued performance and growth of robotic colorectal operations in the community setting.

Cecal-Colon Intussusception due to Appendiceal Mucinous Adenocarcinoma.

Intussusception is a rare presentation in adults and describes when one portion of the intestine telescopes into another portion. Intussusception is associated with malignancies serving as the lead point in adults. Appendiceal mucinous neoplasms are uncommon tumors often incidentally discovered during appendectomy procedures to manage acute appendicitis. Here we present a case report of an instance of mucinous adenocarcinoma of the appendix that manifested as a large bowel obstruction with intussusception limited to the colon, underscoring the possibility of concurrent intussusception and mucinous neoplasms. The case highlights the importance of meticulous diagnostic evaluation and management, particularly without well-defined treatment protocols. Appropriate diagnostic workup and management, including surgical intervention, are critical for patient outcomes and overall prognosis. The study recommends that patients diagnosed with confirmed or suspected appendiceal neoplasms undergo upfront oncologic resection where aggressive malignancy is a concern. Colonoscopy should be performed postoperatively for all patients to identify synchronous lesions.

Sulforaphane is a reversible covalent inhibitor of 3-chymotrypsin-like protease of SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a major public health threat worldwide and emphasizes an urgent need for effective therapeutics. Recently, Ordonez et al. identified sulforaphane (SFN) as a novel coronavirus inhibitor both in vitro and in mice, but the mechanism of action remains elusive. In this study, we independently discovered SFN for its inhibitory effect against SARS-CoV-2 using a target-based screening approach, identifying the viral 3-chymotrypsin-like protease (3CLpro ) as a target of SFN. Mechanistically, SFN inhibits 3CLpro in a reversible, mixed-type manner. Moreover, enzymatic kinetics studies reveal that SFN is a slow-binding inhibitor, following a two-step interaction. Initially, an encounter complex forms by specific binding of SFN to the active pocket of 3CLpro ; subsequently, the isothiocyanate group of SFN as "warhead" reacts covalently to the catalytic cysteine in a slower velocity, stabilizing the SFN-3CLpro complex. Our study has identified a new lead of the covalent 3CLpro inhibitors which has potential to be developed as a therapeutic agent to treat SARS-CoV-2 infection.

Australian airway clearance services for adults with chronic lung conditions: A national survey.

BACKGROUND: Physiotherapy-led airway clearance interventions are indicated for some people with chronic lung conditions. This study describes Australian clinical models for the provision of adult airway clearance services. METHODS: This cross-sectional national study recruited public and private health care providers (excluding cystic fibrosis-specific services) identified by a review of websites. Providers were invited to complete an electronic 61-item survey with questions about airway clearance service context, referral demographics, service provision and program metrics. Data were reported descriptively with differences between metropolitan and non-metropolitan services explored with chi-square tests. RESULTS: Between October-December 2019, the survey was disseminated to 131 providers with 91 responses received (69% response rate; 87 (96%) public (34 metropolitan; 53 non-metropolitan) and 4 (4%) private). Intent (chronic condition self-management) and types of intervention provided (education, breathing techniques, exercise prescription) were common across all services. Geographic location was associated with differences in airway clearance service models (greater use of regular clinics, telephone/telehealth consultations and dedicated cardiorespiratory physiotherapists in metropolitan locations versus clients incurring service and device provision costs in non-metropolitan regions). CONCLUSIONS: While similarities in airway clearance interventions exist, differences in service models may disadvantage people living with chronic lung conditions, especially in non-metropolitan regions of Australia.

An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) neutralize infection and are efficacious for the treatment of COVID-19. However, SARS-CoV-2 variants, notably sublineages of B.1.1.529/omicron, have emerged that escape antibodies in clinical use. As an alternative, soluble decoy receptors based on the host entry receptor ACE2 broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22 .v2.4-IgG1 was previously shown to be effective against SARS-CoV-2 variants when administered intravenously. Here, inhalation of aerosolized sACE22 .v2.4-IgG1 increased survival and ameliorated lung injury in K18-hACE2 mice inoculated with P.1/gamma virus. Loss of catalytic activity reduced the decoy's therapeutic efficacy, which was further confirmed by intravenous administration, supporting dual mechanisms of action: direct blocking of S and turnover of ACE2 substrates associated with lung injury and inflammation. Furthermore, sACE22 .v2.4-IgG1 tightly binds and neutralizes BA.1, BA.2, and BA.4/BA.5 omicron and protects K18-hACE2 mice inoculated with a high dose of BA.1 omicron virus. Overall, the therapeutic potential of sACE22 .v2.4-IgG1 is demonstrated by the inhalation route and broad neutralization potency persists against highly divergent SARS-CoV-2 variants.

Analysis of the Utility of CO2 and Pulse-Dye Lasers Together and Separately in the Treatment of Hypertrophic Burn Scars.

INTRODUCTION: Hypertrophic burn scars (HTBSs) remain a significant source of morbidity. Contemporary treatment has evolved to use CO2 lasers and/or pulse-dye lasers (PDLs) to reduce scar thickness (ST) and erythema. This study seeks to compare treatment efficacy with CO2 or PDL individually and in combination. METHODS: Patients undergoing laser treatments for HTBSs were enrolled. Three 3 × 3 cm squares of HTBSs were randomized to receive treatment with CO2 laser, PDL or CO2 + PDL. Patients underwent 3 treatments, 4 to 6 weeks apart and were followed up over 3 to 6 months. Scar assessments occurred at each visit before treatment and consisted of photographs, ultrasound, colorimetry, and the Patient and Observer Scar Assessment Score. RESULTS: Twenty-five patients were enrolled. Twenty completed 2 treatments (80%) and 11 completed all 3 treatments (44%). Median initial ST was 0.3 cm. Median time since injury was 8 months. Hypertrophic burn scars treated with CO2 or PDL showed a significant decrease in Patient and Observer Scar Assessment Scale score from visit 1 to 3 (P = 0.01 and 0.01, respectively). When separated by ST, thick scars (≥0.3 cm) showed a significant decrease in thickness between visit 1 and 2 using all laser modalities (CO2 + PDL, P = 0.01; CO2, P = 0.02; PDL, P = 0.03). Thin scars (<0.3 cm) showed a reduction in thickness by visit 3 after CO2 + PDL or PDL alone (P = 0.01 and 0.04, respectively). Separating scars by age, younger scars (<9 months) showed a significant reduction in thickness between visit 1 and 2 for CO2 treatment (P = 0.04), and between visit 2 and 3 for CO2 + PDL treatment (P = 0.04). Hypertrophic burn scars treated with PDL did not demonstrate a significant reduction in thickness until visit 3 (P = 0.002). Older scars (≥9 months) showed a significant reduction in thickness between visit 1 and 2 only after CO2 + PDL (P = 0.01). CONCLUSIONS: Hypertrophic burn scars of varying ages, etiologies, and thicknesses were examined in this study with greater degree of early reduction seen in thicker scars using all laser modalities of CO2, PDL or in combination. However, there was no clinically meaningful benefit found with combination as compared with individual treatment. These data support the use of laser to improve HTBS but does not support one modality or combination of modalities over another.

Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants.

Vaccine hesitancy and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) escaping vaccine-induced immune responses highlight the urgency for new COVID-19 therapeutics. Engineered angiotensin-converting enzyme 2 (ACE2) proteins with augmented binding affinities for SARS-CoV-2 spike (S) protein may prove to be especially efficacious against multiple variants. Using molecular dynamics simulations and functional assays, we show that three amino acid substitutions in an engineered soluble ACE2 protein markedly augmented the affinity for the S protein of the SARS-CoV-2 WA-1/2020 isolate and multiple VOCs: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). In humanized K18-hACE2 mice infected with the SARS-CoV-2 WA-1/2020 or P.1 variant, prophylactic and therapeutic injections of soluble ACE22.v2.4-IgG1 prevented lung vascular injury and edema formation, essential features of CoV-2-induced SARS, and above all improved survival. These studies demonstrate broad efficacy in vivo of an engineered ACE2 decoy against SARS-CoV-2 variants in mice and point to its therapeutic potential.

Design of SARS-CoV-2 PLpro Inhibitors for COVID-19 Antiviral Therapy Leveraging Binding Cooperativity.

Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.

Pilot trial of remote monitoring to prevent malnutrition after hepatopancreatobiliary surgery.

BACKGROUND: Patients undergoing hepatopancreatobiliary (HPB) surgery, such patients with pancreatic, periampullary, and liver cancer, are at high risk for malnutrition. Malnutrition increases surgical complications and reduces overall survival. Despite its severity, there are limited interventions addressing malnutrition after HPB surgery. The aim of this pilot trial was to examine feasibility, acceptability, usability, and preliminary efficacy of a remote nutrition monitoring intervention after HPB surgery. METHODS: Participants received tailored nutritional counseling before and after surgery at 2 and 4 weeks after hospital discharge. Participants also recorded nutritional intake daily for 30 days, and these data were reviewed remotely by registered dietitians before nutritional counseling visits. Descriptive statistics were used to describe study outcomes. RESULTS: All 26 patients approached to participate consented to the trial before HPB surgery. Seven were excluded after consent for failing to meet eligibility criteria (e.g., did not receive surgery). Nineteen participants (52.6% female, median age = 65 years) remained eligible for remote monitoring post-surgery. Nineteen used the mobile app food diary, 79% of participants recorded food intake for greater than 80% of study days, 95% met with the dietitian for all visits, and 89% were highly satisfied with the intervention. Among participants with complete data, the average percent caloric goal obtained was 82.4% (IQR: 21.7). CONCLUSIONS: This intervention was feasible and acceptable to patients undergoing HPB surgery. Preliminary efficacy data showed most participants were able to meet calorie intake goals. Future studies should examine intervention efficacy in a larger, randomized controlled trial. TRIAL REGISTRATION: Clinicaltrials.gov. Registered 16 September 2019, https://clinicaltrials.gov/ct2/show/NCT04091165 .

Direct detection of human adenovirus or SARS-CoV-2 with ability to inform infectivity using DNA aptamer-nanopore sensors.

Viral infections are a major global health issue, but no current method allows rapid, direct, and ultrasensitive quantification of intact viruses with the ability to inform infectivity, causing misdiagnoses and spread of the viruses. Here, we report a method for direct detection and differentiation of infectious from noninfectious human adenovirus and SARS-CoV-2, as well as from other virus types, without any sample pretreatment. DNA aptamers are selected from a DNA library to bind intact infectious, but not noninfectious, virus and then incorporated into a solid-state nanopore, which allows strong confinement of the virus to enhance sensitivity down to 1 pfu/ml for human adenovirus and 1 × 104 copies/ml for SARS-CoV-2. Applications of the aptamer-nanopore sensors in different types of water samples, saliva, and serum are demonstrated for both enveloped and nonenveloped viruses, making the sensor generally applicable for detecting these and other emerging viruses of environmental and public health concern.

A recombinant Cedar virus based high-throughput screening assay for henipavirus antiviral discovery.

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic, bat-borne paramyxoviruses in the genus Henipavirus that cause severe and often fatal acute respiratory and/or neurologic diseases in humans and livestock. There are currently no approved antiviral therapeutics or vaccines for use in humans to treat or prevent NiV or HeV infection. To facilitate development of henipavirus antivirals, a high-throughput screening (HTS) platform was developed based on a well-characterized recombinant version of the nonpathogenic Henipavirus, Cedar virus (rCedV). Using reverse genetics, a rCedV encoding firefly luciferase (rCedV-Luc) was rescued and its utility evaluated for high-throughput antiviral compound screening. The luciferase reporter gene signal kinetics of rCedV-Luc in different human cell lines was characterized and validated as an authentic real-time measure of viral growth. The rCedV-Luc platform was optimized as an HTS assay that demonstrated high sensitivity with robust Z' scores, excellent signal-to-background ratios and coefficients of variation. Eight candidate compounds that inhibited rCedV replication were identified for additional validation and demonstrated that 4 compounds inhibited authentic NiV-Bangladesh replication. Further evaluation of 2 of the 4 validated compounds in a 9-point dose response titration demonstrated potent antiviral activity against NiV-Bangladesh and HeV, with minimal cytotoxicity. This rCedV reporter can serve as a surrogate yet authentic BSL-2 henipavirus platform that will dramatically accelerate drug candidate identification in the development of anti-henipavirus therapies.

Discovery of chebulagic acid and punicalagin as novel allosteric inhibitors of SARS-CoV-2 3CLpro.

The emerging SARS-CoV-2 infection is the cause of the global COVID-19 pandemic. To date, there are limited therapeutic options available to fight this disease. Here we examined the inhibitory abilities of two broad-spectrum antiviral natural products chebulagic acid (CHLA) and punicalagin (PUG) against SARS-CoV-2 viral replication. Both CHLA and PUG reduced virus-induced plaque formation in Vero-E6 monolayer at noncytotoxic concentrations, by targeting the enzymatic activity of viral 3-chymotrypsin-like cysteine protease (3CLpro) as allosteric regulators. Our study demonstrates the potential use of CHLA and PUG as novel COVID-19 therapies.

Utilization of a Novel Negative Pressure Platform Wound Dressing on Surgical Incisions: A Case Series.

Closed incision negative pressure therapy (ciNPT) has been shown to improve wound healing for patients at high risk for wound complications. Current devices consist of opaque interface dressings that do not allow ongoing visual evaluation of the surgical incision and utilize a negative pressure of -80 mm Hg to -125 mm Hg. The Negative Pressure Platform Wound Dressing (NP-PWD) was developed to address these aspects. This case series is the first evaluation of the NP-PWD in a clinical setting. METHODS: Patients aged 18-85 undergoing an operation with an anticipated incision and primary closure were screened. Demographics, comorbidities, and operation performed were recorded. Following closure, the incision was measured and photographed before NP-PWD placement. The NP-PWD was removed at the first postoperative check (POC) between postoperative days (PODs) 3-5. Subjects were followed until PODs 9-14. POCs consisted of incision assessment, measurement, photography, and adverse event monitoring. RESULTS: A total of 8 patients with 10 incisions were included in the study. Five patients were men. Median age was 56 years (IQR 53-74 years). All incisions were intact and without inflammation or infection at all POCs. Three adverse events, including small blisters and interruption of therapy, were noted. CONCLUSIONS: This case series reports that patients tolerated the NP-PWD on closed surgical incisions well and that all incisions were intact without evidence of inflammation or infection after 2 weeks of follow-up. Future controlled, clinical studies should further examine the safety and efficacy of the use of the NP-PWD.

Evidence for distinct mechanisms of small molecule inhibitors of filovirus entry.

Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinctive mechanisms of action and lay the groundwork for future development of anti-filoviral agents. The three mechanisms identified here include: (1) direct binding to the internal fusion loop region of Ebola virus glycoprotein (GP); (2) the HR2 domain is likely the main binding site for Marburg virus GP inhibitors and a secondary binding site for some EBOV GP inhibitors; (3) lysosome trapping of GP inhibitors increases drug exposure in the lysosome and further improves the viral inhibition. Importantly, small molecules targeting different domains on GP are synergistic in inhibiting EBOV entry suggesting these two mechanisms of action are distinct. Our findings provide important mechanistic insights into filovirus entry and rational drug design for future antiviral development.

Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures.

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 lower case Greek µM). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a 'BL2 groove' that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

Ginkgolic acid and anacardic acid are specific covalent inhibitors of SARS-CoV-2 cysteine proteases.

BACKGROUND: In the urgent campaign to develop therapeutics against SARS-CoV-2, natural products have been an important source of new lead compounds. RESULTS: We herein identified two natural products, ginkgolic acid and anacardic acid, as inhibitors using a high-throughput screen targeting the SARS-CoV-2 papain-like protease (PLpro). Moreover, our study demonstrated that the two hit compounds are dual inhibitors targeting the SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) in addition to PLpro. A mechanism of action study using enzyme kinetics further characterized the two compounds as irreversible inhibitors against both 3CLpro and PLpro. Significantly, both identified compounds inhibit SARS-CoV-2 replication in vitro at nontoxic concentrations. CONCLUSIONS: Our finding provides two novel natural products as promising SARS-CoV-2 antivirals.