SARS-CoV-2 Main Protease Drug Design, Assay Development, and Drug Resistance Studies.

SARS-CoV-2 is the etiological pathogen of the COVID-19 pandemic, which led to more than 6.5 million deaths since the beginning of the outbreak in December 2019. The unprecedented disruption of social life and public health caused by COVID-19 calls for fast-track development of diagnostic kits, vaccines, and antiviral drugs. Small molecule antivirals are essential complements of vaccines and can be used for the treatment of SARS-CoV-2 infections. Currently, there are three FDA-approved antiviral drugs, remdesivir, molnupiravir, and paxlovid. Given the moderate clinical efficacy of remdesivir and molnupiravir, the drug-drug interaction of paxlovid, and the emergence of SARS-CoV-2 variants with potential drug-resistant mutations, there is a pressing need for additional antivirals to combat current and future coronavirus outbreaks.In this Account, we describe our efforts in developing covalent and noncovalent main protease (Mpro) inhibitors and the identification of nirmatrelvir-resistant mutants. We initially discovered GC376, calpain inhibitors II and XII, and boceprevir as dual inhibitors of Mpro and host cathepsin L from a screening of a protease inhibitor library. Given the controversy of targeting cathepsin L, we subsequently shifted the focus to designing Mpro-specific inhibitors. Specifically, guided by the X-ray crystal structures of these initial hits, we designed noncovalent Mpro inhibitors such as Jun8-76-3R that are highly selective toward Mpro over host cathepsin L. Using the same scaffold, we also designed covalent Mpro inhibitors with novel cysteine reactive warheads containing di- and trihaloacetamides, which similarly had high target specificity. In parallel to our drug discovery efforts, we developed the cell-based FlipGFP Mpro assay to characterize the cellular target engagement of our rationally designed Mpro inhibitors. The FlipGFP assay was also applied to validate the structurally disparate Mpro inhibitors reported in the literature. Lastly, we introduce recent progress in identifying naturally occurring Mpro mutants that are resistant to nirmatrelvir from genome mining of the nsp5 sequences deposited in the GISAID database. Collectively, the covalent and noncovalent Mpro inhibitors and the nirmatrelvir-resistant hot spot residues from our studies provide insightful guidance for future work aimed at developing orally bioavailable Mpro inhibitors that do not have overlapping resistance profile with nirmatrelvir.

Inhibitors of Keap1-Nrf2 protein-protein interaction reduce estrogen responsive gene expression and oxidative stress in estrogen receptor-positive breast cancer.

Estrogen contributes to the development of breast cancer through estrogen receptor (ER) signaling and by generating genotoxic metabolites that cause oxidative DNA damage. To protect against oxidative stress, cells activate nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream cytoprotective genes that initiate antioxidant responses and detoxify xenobiotics. Nrf2 activation occurs by inhibiting the protein-protein interaction (PPI) between Nrf2 and its inhibitor Keap1, which otherwise targets Nrf2 for ubiquitination and destruction. In this study, we examined a series of novel direct inhibitors of Keap1-Nrf2 PPI in their role in promoting the availability of Nrf2 for antioxidant activity and attenuating estrogen-mediated responses in breast cancer. ER-positive human breast cancer cells MCF-7 were treated with 17ß-estradiol (E2) in the presence or absence of selected Keap1-Nrf2 PPI inhibitors. Keap1-Nrf2 PPI inhibitors suppressed the mRNA and protein levels of estrogen responsive genes induced by E2 exposure, such as PGR. Keap1-Nrf2 PPI inhibitors caused significant activation of Nrf2 target genes. E2 decreased the mRNA and protein level of the Nrf2 target gene NQO1, and the Keap1-Nrf2 PPI inhibitors reversed this effect. The reversal of E2 action by these compounds was not due to binding to ER as ER antagonists. Further, a selected compound attenuated oxidative stress induced by E2, determined by the level of a biomarker 8-oxo-deoxyguanosine. These findings suggest that the Keap1-Nrf2 PPI inhibitors have potent antioxidant activity by activating Nrf2 pathways and inhibit E2-induced gene and protein expression. These compounds may serve as potential chemopreventive agents in estrogen-stimulated breast cancer.

Grade V renal trauma management: results from the multi-institutional genito-urinary trauma study.

PURPOSE: To investigate management trends for American Association for the Surgery of Trauma (AAST) grade V renal trauma with focus on non-operative management. METHODS: We used prospectively collected data as part of the Multi-institutional Genito-Urinary Trauma Study (MiGUTS). We included patients with grade V renal trauma according to the AAST Injury Scoring Scale 2018 update. All cases submitted by participating centers with radiology images available were independently reviewed to confirm renal trauma grade. Management was classified as expectant, conservative (minimally invasive, endoscopic or percutaneous procedures), or operative (renal-related surgery). RESULTS: Eighty patients were included, 25 of whom had complete imaging and had independent confirmation of AAST grade V renal trauma. Median age was 35 years (Interquartile range (IQR) 25-50) and 23 (92%) had blunt trauma. Ten patients (40%) were managed operatively with nephrectomy. Conservative management was used in nine patients (36%) of which six received angioembolization and three had a stent or drainage tube placed. Expectant management was followed in six (24%) patients. Transfusion requirements were progressively higher with groups requiring more aggressive treatment, and injury characteristics differed significantly across management groups in terms of hematoma size and laceration size. Vascular contrast extravasation was more likely in operatively managed patients though a statistically significant association was not found. CONCLUSION: Successful use of nonoperative management for grade V injuries is used for a substantial subset of patients. Lower transfusion requirement and less severe injury radiologic phenotype appear to be important characteristics delineating this group.

The history, mechanism, and perspectives of nirmatrelvir (PF-07321332): an orally bioavailable main protease inhibitor used in combination with ritonavir to reduce COVID-19-related hospitalizations.

The rapid development of effective vaccines to combat the SARS-CoV-2 virus has been an effective counter measure to decrease hospitalization and the mortality rate in many countries. However, with the risk of mutated strains decreasing the efficacy of the vaccine, there has been an increasing demand for antivirals to treat COVID-19. While antivirals, such as remdesivir, have had some success treating COVID-19 patients in hospital settings, there is a need for orally bioavailable, cost-effective antivirals that can be administered in outpatient settings to minimize COVID-19-related hospitalizations and death. Nirmatrelvir (PF-07321332) is an orally bioavailable Mpro (also called 3CLpro) inhibitor developed by Pfizer. It is administered in combination with ritonavir, a potent CYP3A4 inhibitor that decreases the metabolism of nirmatrelvir. This review seeks to outline the history of the rational design, the target selectivity, synthesis, drug resistance, and future perspectives of nirmatrelvir. Graphical abstract.

The American Association for the Surgery of Trauma Renal Grading System-Should Segmental Kidney Infarction be Classified as a Grade IV Injury?

PURPOSE: In 2018 the American Association for the Surgery of Trauma revised renal injury grading. One change was inclusion of segmental kidney infarction under grade IV injuries. We aimed to assess how segmental kidney infarction will change the scope of grade IV injuries and compare bleeding control interventions in those with and without isolated segmental kidney infarction. METHODS: We used high grade renal trauma data from 7 level 1 trauma centers from 2013 to 2018 as part of the Multi-institutional Genito-Urinary Trauma Study. Initial computerized tomography scans were reviewed to regrade the injuries. Injuries were categorized as isolated segmental kidney infarction if segmental parenchymal infarction was the only reason for inclusion under grade IV injury. All other grade IV injuries (including combined injury patterns) were categorized as without isolated segmental kidney infarction. Bleeding interventions were compared between those with and without isolated segmental kidney infarction. RESULTS: From 550 patients with high grade renal trauma and available computerized tomography, 250 (45%) were grade IV according to the 2018 American Association for the Surgery of Trauma grading system. Of these, 121 (48%) had isolated segmental kidney infarction. The majority of patients with isolated segmental kidney infarction (88%) would have been assigned a lower grade using the original 1989 grading system. Rate of bleeding control interventions was lower in isolated segmental kidney infarction compared to other grade IV injuries (7% vs 21%, p=0.002). Downgrading all patients with isolated segmental kidney infarction to grade III did not change the grading system's associations with bleeding interventions. CONCLUSIONS: Approximately half of the 2018 American Association for the Surgery of Trauma grade IV injuries have isolated segmental kidney infarction. Including isolated segmental kidney infarction in grade IV injuries increases the heterogeneity of these injuries without increasing the grading system's ability to predict bleeding interventions. In future iterations of the American Association for the Surgery of Trauma renal trauma grading isolated segmental kidney infarction could be reclassified as grade III injury.

Characterization of an l-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations.

l-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. Here we report that Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699) uses l-ascorbate as sole carbon source via a novel catabolic pathway. RNaseq identified eight candidate catabolic genes, sequence similarity networks, and genome neighborhood networks guided predictions for function of the encoded proteins, and the predictions were confirmed by in vitro assays and in vivo growth phenotypes of gene deletion mutants. l-Ascorbate, a lactone, is oxidized and ring-opened by enzymes in the cytochrome b561 and gluconolactonase families, respectively, to form 2,3-diketo-l-gulonate. A protein predicted to have a WD40-like fold catalyzes an unprecedented benzilic acid rearrangement involving migration of a carboxylate group to form 2-carboxy-l-lyxonolactone; the lactone is hydrolyzed by a member of the amidohydrolase superfamily to yield 2-carboxy-l-lyxonate. A member of the PdxA family of oxidative decarboxylases catalyzes a novel decarboxylation that uses NAD+ catalytically. The product, l-lyxonate, is catabolized to α-ketoglutarate by a previously characterized pathway. The pathway is found in hundreds of bacteria, including the pathogens Pseudomonas aeruginosa and Acinetobacter baumannii.

Quick Guide: Programming a Gaming Keyboard for PACS to Optimize Radiology Workflow.

The surging demand for diagnostic imaging has highlighted inefficiencies with traditional input devices. Radiologists, using conventional mice and keyboards, grapple with cumbersome shortcuts leading to fatigue, errors, and possible injuries. Gaming keyboards, designed for gamers' precision and adaptability, feature customizable keys that simplify complex tasks into single-touch actions, offering radiologists a more efficient workflow with less physical and mental strain. Incorporating these keyboards could revolutionize radiologists' engagement with PACS. The customizable feature significantly trims time spent searching, ushering in swifter, ergonomic interactions. This manuscript delineates a guide for adapting a Logitech gaming keyboard to radiology needs, from profile creations and shortcut mapping to intricate macro setups. Although the guide uses a Logitech gaming keyboard for demonstration, it is designed to be intuitive, helping users adapt to their unique needs across different modalities, subspecialties, and various radiology viewer software. Furthermore, its fundamental concepts are transferrable to other mouse brands or models with similar customization software. As radiology pivots toward utmost efficiency, gaming keyboards emerge as invaluable assets, promising significant workflow enhancements.

Proposed revision of the American Association for Surgery of Trauma Renal Organ Injury Scale: Secondary analysis of the Multi-institutional Genitourinary Trauma Study.

BACKGROUND: This study updates the American Association for the Surgery of Trauma (AAST) Organ Injury Scale (OIS) for renal trauma using evidence-based criteria for bleeding control intervention. METHODS: This was a secondary analysis of a multicenter retrospective study including patients with high-grade renal trauma from seven level 1 trauma centers from 2013 to 2018. All eligible patients were assigned new renal trauma grades based on revised criteria. The primary outcome used to measure injury severity was intervention for renal bleeding. Secondary outcomes included intervention for urinary extravasation, units of packed red blood cells transfused within 24 hours, and mortality. To test the revised grading system, we performed mixed-effect logistic regression adjusted for multiple baseline demographic and trauma covariates. We determined the area under the curve (AUC) to assess accuracy of predicting bleeding interventions from the revised grading system and compared this to 2018 AAST OIS. RESULTS: Based on the 2018 OIS grading system, we included 549 patients with AAST grades III to V injuries and computed tomography scans (III, 52% [n = 284]; IV, 45% [n = 249]; and V, 3% [n = 16]). Among these patients, 89% experienced blunt injury (n = 491), and 12% (n = 64) underwent intervention for bleeding. After applying the revised grading criteria, 60% (n = 329) of patients were downgraded, and 4% (n = 23) were upgraded; 2.8% (n = 7) downgraded from grade V to IV, and 69.5% (n = 173) downgraded from grade IV to III. The revised renal trauma grading system demonstrated improved predictive ability for bleeding interventions (2018 AUC, 0.805; revised AUC, 0.883; p = 0.001) and number of units of packed red blood cells transfused. When we removed urinary injury from the revised system, there was no difference in its predictive ability for renal hemorrhage intervention. CONCLUSION: A revised renal trauma grading system better delineates the need for hemostatic interventions than the current AAST OIS renal trauma grading system. LEVEL OF EVIDENCE: Diagnostic Test/Criteria; Level III.

Unwarranted variability in antibiotic prophylaxis for cesarean section delivery: a national survey of anesthesiologists.

BACKGROUND: Current guidelines from the American College of Obstetricians and Gynecologists recommend antibiotic prophylaxis for cesarean delivery immediately before incision. The purpose of this study was to measure and describe correlates of adherence to these guidelines in a sample of United States anesthesiologists. METHODS: We invited a random sample of the membership of the American Society of Anesthesiologists (n = 10,000) to complete an online survey. RESULTS: Of 1052 respondents (10.5%) with complete information for analysis, 63.5% (95% confidence interval 60.6%, 66.3%, n = 668) reported preincision prophylaxis as the standard of care for scheduled cesarean delivery. Twenty-eight percent (n = 299) agreed that the anesthesiologist should take primary responsibility for prophylaxis timing. In a multivariable model, significant variability in preincision prophylaxis was noted for hospital type (community versus teaching, 62% vs 70%, P = 0.004), region (West versus Southeast, 70% vs 59%, P = 0.01; West versus Southwest, 70% vs 58%, P = 0.02), and respondents' belief in appropriate preincision timing (those endorsing routine preincision administration [80%], routine after cord clamp administration [17%], at the discretion of the obstetrician [47%], and the belief that more information was needed [43%]) (P < 0.001 all comparisons). Respondents' belief about appropriate preincision timing was the strongest discriminator in the model (change in area under the receiver operating characteristic curve = 0.13 vs ≤0.02 for all others). CONCLUSION: Adherence with current prophylactic antibiotic administration guidelines for cesarean delivery is not uniform. Education initiatives, regulatory maneuvers, and process improvement should be targeted at sites where anesthesiologists do not comply with current guidelines.

An Overview of Antivirals against Monkeypox Virus and Other Orthopoxviruses.

The current monkeypox outbreaks during the COVID-19 pandemic have reignited interest in orthopoxvirus antivirals. Monkeypox belongs to the Orthopoxvirus genus of the Poxviridae family, which also includes the variola virus, vaccinia virus, and cowpox virus. Two orally bioavailable drugs, tecovirimat and brincidofovir, have been approved for treating smallpox infections. Given their human safety profiles and in vivo antiviral efficacy in animal models, both drugs have also been recommended to treat monkeypox infection. To facilitate the development of additional orthopoxvirus antivirals, we summarize the antiviral activity, mechanism of action, and mechanism of resistance of orthopoxvirus antivirals. This perspective covers both direct-acting and host-targeting antivirals with an emphasis on drug candidates showing in vivo antiviral efficacy in animal models. We hope to speed the orthopoxvirus antiviral drug discovery by providing medicinal chemists with insights into prioritizing proper drug targets and hits for further development.

Exploring diverse reactive warheads for the design of SARS-CoV-2 main protease inhibitors.

SARS-CoV-2 main protease (Mpro) is a validated antiviral drug target of nirmatrelvir, the active ingredient in Pfizer's oral drug Paxlovid. Drug-drug interactions limit the use of Paxlovid. In addition, drug-resistant Mpro mutants against nirmatrelvir have been identified from cell culture viral passage and naturally occurring variants. As such, there is a need for a second generation of Mpro inhibitors. In this study, we explored several reactive warheads in the design of Mpro inhibitors. We identified Jun11119R (vinyl sulfonamide warhead), Jun10221R (propiolamide warhead), Jun1112R (4-chlorobut-2-ynamide warhead), Jun10541R (nitrile warhead), and Jun10963R (dually activated nitrile warhead) as potent Mpro inhibitors. Jun10541R and Jun10963R also had potent antiviral activity against SARS-CoV-2 in Calu-3 cells with EC50 values of 2.92 and 6.47 µM, respectively. X-ray crystal structures of Mpro with Jun10541R and Jun10221 revealed covalent modification of Cys145. These Mpro inhibitors with diverse reactive warheads collectively represent promising candidates for further development.

Shattered Kidney After Renal Trauma: Should It Be Classified As an American Association for the Surgery of Trauma Grade V Injury?

OBJECTIVE: To study the prevalence and management of shattered kidney and to evaluate if the new description of "loss of identifiable renal anatomy" in the 2018 American Association for the Surgery of Trauma (AAST) organ injury scale (OIS) would improve the ability to predict bleeding control interventions. METHODS: We used high-grade renal trauma data from 21 Level-1 trauma centers from 2013 to 2018. Initial CT scans were reviewed to identify shattered kidneys, defined as a kidney having ≥3 parenchymal fragments displaced by blood or fluid on cross-sectional imaging. We further categorized patients with shattered kidney in two models based on loss of identifiable renal parenchymal anatomy and presence or absence of vascular contrast extravasation (VCE). Bleeding interventions were compared between the groups. RESULTS: From 861 high-grade renal trauma patients, 41 (4.8%) had shattered kidney injury. 25 (61%) underwent a bleeding control intervention including 18 (43.9%) nephrectomies and 11 (26.8%) angioembolizations. 18 (41%) had shattered kidney with "loss of identifiable parenchymal renal anatomy" per 2018 AAST OIS (model-1). 28 (68.3%) had concurrent VCE (model-2). Model-2 had a statistically significant improvement in area under the curve over model-1 in predicting bleeding interventions (0.75 vs 0.72; P = .01). CONCLUSION: Shattered kidney is associated with high rates of active bleeding, urinary extravasation, and interventions including nephrectomy. The definition of shattered kidney is vague and subjective and our definition might be simpler and more reproducible. Loss of identifiable renal anatomy per the 2018 AAST OIS did not provide better distinction for bleeding control interventions over presence of VCE.

External validation of a nomogram predicting risk of bleeding control interventions after high-grade renal trauma: The Multi-institutional Genito-Urinary Trauma Study.

BACKGROUND: Renal trauma grading has a limited ability to distinguish patients who will need intervention after high-grade renal trauma (HGRT). A nomogram incorporating both clinical and radiologic factors has been previously developed to predict bleeding control interventions after HGRT. We aimed to externally validate this nomogram using multicenter data from level 1 trauma centers. METHODS: We gathered data from seven level 1 trauma centers. Patients with available initial computed tomography (CT) scans were included. Each CT scan was reviewed by two radiologists blinded to the intervention data. Nomogram variables included trauma mechanism, hypotension/shock, concomitant injuries, vascular contrast extravasation (VCE), pararenal hematoma extension, and hematoma rim distance (HRD). Mixed-effect logistic regression was used to assess the associations between the predictors and bleeding intervention. The prediction accuracy of the nomogram was assessed using the area under the receiver operating characteristic curve and its 95% confidence interval (CI). RESULTS: Overall, 569 HGRT patients were included for external validation. Injury mechanism was blunt in 89%. Using initial CT scans, 14% had VCE and median HRD was 1.7 (0.9-2.6) cm. Overall, 12% underwent bleeding control interventions including 34 angioembolizations and 24 nephrectomies. In the multivariable analysis, presence of VCE was associated with a threefold increase in the odds of bleeding interventions (odds ratio, 3.06; 95% CI, 1.44-6.50). Every centimeter increase in HRD was associated with 66% increase in odds of bleeding interventions. External validation of the model provided excellent discrimination in predicting bleeding interventions with an area under the curve of 0.88 (95% CI, 0.84-0.92). CONCLUSION: Our results reinforce the importance of radiologic findings such as VCE and hematoma characteristics in predicting bleeding control interventions after renal trauma. The prediction accuracy of the proposed nomogram remains high using external data. These variables can help to better risk stratify high-grade renal injuries. LEVEL OF EVIDENCE: Prognostic and epidemiological study, level III.

Clinical and Radiographic Factors Associated With Failed Renal Angioembolization: Results From the Multi-institutional Genitourinary Trauma Study (Mi-GUTS).

OBJECTIVE: To find clinical or radiographic factors that are associated with angioembolization failure after high-grade renal trauma. MATERIAL AND METHODS: Patients were selected from the Multi-institutional Genito-Urinary Trauma Study. Included were patients who initially received renal angioembolization after high-grade renal trauma (AAST grades III-V). This cohort was dichotomized into successful or failed angioembolization. Angioembolization was considered a failure if angioembolization was followed by repeat angiography and/or an exploratory laparotomy. RESULTS: A total of 67 patients underwent management initially with angioembolization, with failure in 18 (27%) patients. Those with failed angioembolization had a larger proportion ofgrade IV (72% vs 53%) and grade V (22% vs 12%) renal injuries. A total of 53 patients underwent renal angioembolization and had initial radiographic data for review, with failure in 13 cases. The failed renal angioembolization group had larger perirenal hematoma sizes on the initial trauma scan. CONCLUSION: Angioembolization after high-grade renal trauma failed in 27% of patients. Failed angioembolization was associated with higher injury grade and a larger perirenal hematoma. Likely these characteristics are associated with high-grade renal trauma that may be less amenable to successful treatment after a single renal angioembolization.

3T Magnetic Resonance Imaging of the Wrist Tendons.

Magnetic resonance imaging (MRI) is frequently used in the imaging evaluation of wrist pain. The complex anatomy of the wrist can be demonstrated by MRI. Three tesla (3 T) MRI offers increased signal-to-noise ratio relative to 1.5 T MRI allowing for higher soft tissue contrast and better spatial resolution. The resulting increase in conspicuity of fine anatomic detail may improve the detection and characterization of wrist pathology. In this article, we will review the anatomy, normal variants, and common pathologies of the wrist tendons as evaluated on 3 T MRI.

Hypercapnic cerebral edema presenting in a woman with asthma: a case report.

INTRODUCTION: Common causes of non-traumatic acute cerebral edema include malignant hypertension, hyponatremia, anoxia, and cerebral vascular accident. The computed tomographic images and data obtained during care of the patient described in this case report provide evidence that hypercarbia can cause increased intracranial pressure and coma without permanent brain injury. Partial pressure of carbon dioxide evaluation for coma is essential to provide faster diagnosis and therapeutic correction in certain common critical disease states. We present the case of a patient in a coma associated with cerebral edema during a typical asthma exacerbation with hypercapnic respiratory failure. CASE PRESENTATION: An obese 63-year-old African American woman with asthma presented to our hospital with facial swelling and shortness of breath. Immediately following intubation for hypercapnic respiratory failure, she was noted to have a dilated, unresponsive right pupil. An emergent computed tomographic head scan revealed that she had increased intracranial pressure. A neurosurgeon agreed with the computed tomography interpretation and recommended no surgical intervention. The patient's respiratory acidosis was corrected with ventilatory management over several hours in the intensive care unit. Nine and one-half hours later a follow-up head computed tomographic scan was read as normal without cerebral edema. At 12 hours, the patient's right pupil was 5 mm in diameter and reactive. By 24 hours, her pupils were symmetrically equal and reactive. Her symptoms had improved, and she was extubated. A brain magnetic resonance imaging scan revealed no abnormalities. CONCLUSION: Alteration of consciousness related to hypercapnia during respiratory failure is not generally thought to be related to cerebral edema. Respiratory acidosis resulting from hypercarbia is known to produce carbon dioxide narcosis and coma, but no current treatment algorithm suggests that rapid hypercapnia correction can be critical to neurologic outcome. To the best of our knowledge, our case is a unique example of the physiological changes that may occur in relation to arterial carbon dioxide concentration in the normal brain in the setting of typical hypercapnic respiratory failure. Correction of respiratory acidosis reversed the neurologic symptoms and physiology causing cerebral edema and coma in our patient. Rare similar cases have been sporadically reported in the medical literature, typically in children. Our case is also unusual in that rapid deterioration and clinical status were directly observed on simultaneous computed tomographic scans. Had this patient been found unresponsive, or had she had brief respiratory or cardiac arrest, the scan could have been interpreted as global anoxic injury leading to a different therapeutic course.