Mtb-Selective 5-Aminomethyl Oxazolidinone Prodrugs: Robust Potency and Potential Liabilities.

Linezolid is a drug with proven human antitubercular activity whose use is limited to highly drug-resistant patients because of its toxicity. This toxicity is related to its mechanism of action─linezolid inhibits protein synthesis in both bacteria and eukaryotic mitochondria. A highly selective and potent series of oxazolidinones, bearing a 5-aminomethyl moiety (in place of the typical 5-acetamidomethyl moiety of linezolid), was identified. Linezolid-resistant mutants were cross-resistant to these molecules but not vice versa. Resistance to the 5-aminomethyl molecules mapped to an N-acetyl transferase (Rv0133) and these mutants remained fully linezolid susceptible. Purified Rv0133 was shown to catalyze the transformation of the 5-aminomethyl oxazolidinones to their corresponding N-acetylated metabolites, and this transformation was also observed in live cells of Mycobacterium tuberculosis. Mammalian mitochondria, which lack an appropriate N-acetyltransferase to activate these prodrugs, were not susceptible to inhibition with the 5-aminomethyl analogues. Several compounds that were more potent than linezolid were taken into C3HeB/FeJ mice and were shown to be highly efficacious, and one of these (9) was additionally taken into marmosets and found to be highly active. Penetration of these 5-aminomethyl oxazolidinone prodrugs into caseum was excellent. Unfortunately, these compounds were rapidly converted into the corresponding 5-alcohols by mammalian metabolism which retained antimycobacterial activity but resulted in substantial mitotoxicity.

Identification of an inhibitory pocket in falcilysin provides a new avenue for malaria drug development.

Identification of new druggable protein targets remains the key challenge in the current antimalarial development efforts. Here we used mass-spectrometry-based cellular thermal shift assay (MS-CETSA) to identify potential targets of several antimalarials and drug candidates. We found that falcilysin (FLN) is a common binding partner for several drug candidates such as MK-4815, MMV000848, and MMV665806 but also interacts with quinoline drugs such as chloroquine and mefloquine. Enzymatic assays showed that these compounds can inhibit FLN proteolytic activity. Their interaction with FLN was explored systematically by isothermal titration calorimetry and X-ray crystallography, revealing a shared hydrophobic pocket in the catalytic chamber of the enzyme. Characterization of transgenic cell lines with lowered FLN expression demonstrated statistically significant increases in susceptibility toward MK-4815, MMV000848, and several quinolines. Importantly, the hydrophobic pocket of FLN appears amenable to inhibition and the structures reported here can guide the development of novel drugs against malaria.

COVID-19 Vaccines and Heavy Menstrual Bleeding: The Impact of Media Attention on Reporting to EudraVigilance.

BACKGROUND AND OBJECTIVE: During the COVID-19 vaccination campaign, over 34,000 reports of heavy menstrual bleeding following the administration of COVID-19 vaccines originating in the Economic European Area were submitted to EudraVigilance, the European Union database of suspected adverse drug reactions. More than 90% of these reports were sent by consumers while the remaining by healthcare professionals. Public concerns regarding menstruation disorders in COVID-19 vaccinees were also covered by the media. We investigated the impact of media attention on the reporting trends of heavy menstrual bleeding to EudraVigilance. METHODS: We used media outlets published in the Economic European Area on menstrual disorders and COVID-19 vaccines from the beginning of the vaccination campaign in the Economic European Area (1 January, 2021) until December 2022 (i.e., after the regulatory request to add the adverse event to the product information) and spontaneous reports from EudraVigilance. RESULTS: We found that the publication of safety updates from regulatory authorities and subsequent coverage in media outlets preceded increased reporting to EudraVigilance. Furthermore, the heavy menstrual bleeding reported in the cases occurred several weeks or months earlier and were not submitted to the respective date. The analysis suggests that the spikes in reporting of heavy menstrual bleeding were to some extent influenced by media coverage in some countries. CONCLUSIONS: Consumer reporting to the European Union spontaneous data collection system, EudraVigilance, was of high value for regulatory safety reviews, albeit the reporting behaviours were not free of the influence of the media. These sources of information can be investigated to understand the context of safety concerns of public health interest.

Orally available nucleoside analog UMM-766 provides protection in a murine model of orthopox disease.

Although smallpox has been eradicated, other orthopoxviruses continue to be a public health concern as exemplified by the ongoing Mpox (formerly monkeypox) global outbreak. While medical countermeasures (MCMs) previously approved by the Food and Drug Administration for the treatment of smallpox have been adopted for Mpox, previously described vulnerabilities coupled with the questionable benefit of at least one of the therapeutics during the 2022 Mpox outbreak reinforce the need for identifying and developing other MCMs against orthopoxviruses. Here, we screened a panel of Merck proprietary small molecules and identified a novel nucleoside inhibitor with potent broad-spectrum antiviral activity against multiple orthopoxviruses. Efficacy testing of a 7-day dosing regimen of the orally administered nucleoside in a murine model of severe orthopoxvirus infection yielded a dose-dependent increase in survival. Treated animals had greatly reduced lesions in the lung and nasal cavity, particularly in the 10 µg/mL dosing group. Viral levels were also markedly lower in the UMM-766-treated animals. This work demonstrates that this nucleoside analog has anti-orthopoxvirus efficacy and can protect against severe disease in a murine orthopox model.IMPORTANCEThe recent monkeypox virus pandemic demonstrates that members of the orthopoxvirus, which also includes variola virus, which causes smallpox, remain a public health issue. While currently FDA-approved treatment options exist, risks that resistant strains of orthopoxviruses may arise are a great concern. Thus, continued exploration of anti-poxvirus treatments is warranted. Here, we developed a template for a high-throughput screening assay to identify anti-poxvirus small-molecule drugs. By screening available drug libraries, we identified a compound that inhibited orthopoxvirus replication in cell culture. We then showed that this drug can protect animals against severe disease. Our findings here support the use of existing drug libraries to identify orthopoxvirus-targeting drugs that may serve as human-safe products to thwart future outbreaks.

Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.

Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus.

The Mycobacterium abscessus drug development pipeline is poorly populated, with particularly few validated target-lead couples to initiate de novo drug discovery. Trimethoprim, an inhibitor of dihydrofolate reductase (DHFR) used for the treatment of a range of bacterial infections, is not active against M. abscessus. Thus, evidence that M. abscessus DHFR is vulnerable to pharmacological intervention with a small molecule inhibitor is lacking. Here, we show that the pyrrolo-quinazoline PQD-1, previously identified as a DHFR inhibitor active against Mycobacterium tuberculosis, exerts whole cell activity against M. abscessus. Enzyme inhibition studies showed that PQD-1, in contrast to trimethoprim, is a potent inhibitor of M. abscessus DHFR and over-expression of DHFR causes resistance to PQD-1, providing biochemical and genetic evidence that DHFR is a vulnerable target and mediates PQD-1's growth inhibitory activity in M. abscessus. As observed in M. tuberculosis, PQD-1 resistant mutations mapped to the folate pathway enzyme thymidylate synthase (TYMS) ThyA. Like trimethoprim in other bacteria, PQD-1 synergizes with the dihydropteroate synthase (DHPS) inhibitor sulfamethoxazole (SMX), offering an opportunity to exploit the successful dual inhibition of the folate pathway and develop similarly potent combinations against M. abscessus. PQD-1 is active against subspecies of M. abscessus and a panel of clinical isolates, providing epidemiological validation of the target-lead couple. Leveraging a series of PQD-1 analogs, we have demonstrated a dynamic structure-activity relationship (SAR). Collectively, the results identify M. abscessus DHFR as an attractive target and PQD-1 as a chemical starting point for the discovery of novel drugs and drug combinations that target the folate pathway in M. abscessus.

The Invention of WM382, a Highly Potent PMIX/X Dual Inhibitor toward the Treatment of Malaria.

Drug resistance to first-line antimalarials-including artemisinin-is increasing, resulting in a critical need for the discovery of new agents with novel mechanisms of action. In collaboration with the Walter and Eliza Hall Institute and with funding from the Wellcome Trust, a phenotypic screen of Merck's aspartyl protease inhibitor library identified a series of plasmepsin X (PMX) hits that were more potent than chloroquine. Inspired by a PMX homology model, efforts to optimize the potency resulted in the discovery of leads that, in addition to potently inhibiting PMX, also inhibit another essential aspartic protease, plasmepsin IX (PMIX). Further potency and pharmacokinetic profile optimization efforts culminated in the discovery of WM382, a very potent dual PMIX/X inhibitor with robust in vivo efficacy at multiple stages of the malaria parasite life cycle and an excellent resistance profile.

Activity of Tricyclic Pyrrolopyrimidine Gyrase B Inhibitor against Mycobacterium abscessus.

Tricyclic pyrrolopyrimidines (TPPs) are a new class of antibacterials inhibiting the ATPase of DNA gyrase. TPP8, a representative of this class, is active against Mycobacterium abscessus in vitro. Spontaneous TPP8 resistance mutations mapped to the ATPase domain of M. abscessus DNA gyrase, and the compound inhibited DNA supercoiling activity of recombinant M. abscessus enzyme. Further profiling of TPP8 in macrophage and mouse infection studies demonstrated proof-of-concept activity against M. abscessus ex vivo and in vivo.

Prescribing Patterns of Codeine and Alternative Medicines in Children in Europe.

INTRODUCTION: Concerns over serious respiratory depression in children led to two European Union (EU) referral procedures (in 2013 and 2015) to review the benefit-risk balance of codeine in this population when used for pain relief, cough or cold. Consequently, codeine should no longer be used in children aged < 12 years and restrictions were introduced for treatment in children ≥ 12 years. OBJECTIVE: This multinational collaborative study aimed to assess the effectiveness of these risk minimisation measures by evaluating changes in prescribing of codeine and alternative treatments. METHOD: Children under 12 and 12-18 years old were followed between 2010 and 2017 to analyse quarterly trends in prescribing of codeine and alternative treatments in electronic health records from France, Germany, Norway, Spain and the United Kingdom using interrupted time series analysis. RESULTS: Overall prescribing of codeine in children decreased in all five countries, reaching near zero prevalence in children under 12 years of age. This was accompanied by an increase in use of other opioid analgesics in France (from 0.15 to 0.56 prevalence per 100 person-years immediately after the first referral), Norway (from 0.0006 to 0.0013 at the end of the study), the United Kingdom (from 0.018 to 0.05 at the end of the study), and an increase in non-opioid analgesics in Norway (from 0.045 to 0.075 at the end of the study) after the referral on pain relief indication. The referral on cough/cold indication led to a decrease in use of opioid and non-opioid antitussives in children aged < 12 years in France (from 10 to 7 and 20 to 16, respectively) and had no impact in other countries. Overall prescribing trends for codeine and alternatives were similar across both age groups within each country. CONCLUSION: The decrease in use of codeine shows that healthcare professionals followed the adopted measures and switched prescribing practices for pain management in children aged < 18 years towards opioid or non-opioid analgesics depending on national clinical and reimbursement settings. Whist the magnitude of the first referral on pain differed between countries, the second referral on cough/cold had only a minimal impact on the use of codeine and antitussives.

Structure activity relationship of N-1 substituted 1,5-naphthyrid-2-one analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-9).

Novel bacterial topoisomerase inhibitors (NBTIs) are the newest members of gyrase inhibitor broad-spectrum antibacterial agents, represented by the most advanced member, gepotidacin, a 4-amino-piperidine linked NBTI, which is undergoing phase III clinical trials for treatment of urinary tract infections (UTI). We have extensively reported studies on oxabicyclooctane linked NBTIs, including AM-8722. The present study summarizes structure activity relationship (SAR) of AM-8722 leading to identification of 7-fluoro-1-cyanomethyl-1,5-naphthyridin-2-one based NBTI (16, AM-8888) with improved potency and spectrum (MIC values of 0.016-4 µg/mL), with Pseudomonas aeruginosa being the least sensitive strain (MIC 4 µg/mL).

Basis for drug selectivity of plasmepsin IX and X inhibition in Plasmodium falciparum and vivax.

Plasmepsins IX (PMIX) and X (PMX) are essential aspartyl proteases for Plasmodium spp. egress, invasion, and development. WM4 and WM382 inhibit PMIX and PMX in Plasmodium falciparum and P. vivax. WM4 inhibits PMX, while WM382 is a dual inhibitor of PMIX and PMX. To understand their function, we identified protein substrates. Enzyme kinetic and structural analyses identified interactions responsible for drug specificity. PMIX and PMX have similar substrate specificity; however, there are distinct differences for peptide and protein substrates. Differences in WM4 and WM382 binding for PMIX and PMX map to variations in the S' region and engagement of the active site S3 pocket. Structures of PMX reveal interactions and mechanistic detail of drug binding important for development of clinical candidates against these targets.

Identification of ß-Lactams Active against Mycobacterium tuberculosis by a Consortium of Pharmaceutical Companies and Academic Institutions.

Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19. Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of ß-lactams to kill Mycobacterium tuberculosis (Mtb). In the TB Drug Accelerator (TBDA), a consortium organized by the Bill & Melinda Gates Foundation, individual pharmaceutical companies collaborate with academic screening laboratories. We developed a higher order consortium within the TBDA in which four pharmaceutical companies (GlaxoSmithKline, Sanofi, MSD, and Lilly) collectively collaborated with screeners at Weill Cornell Medicine, the Infectious Disease Research Institute (IDRI), and the National Institute of Allergy and Infectious Diseases (NIAID), pharmacologists at Rutgers University, and medicinal chemists at the University of North Carolina to screen ∼8900 ß-lactams, predominantly cephalosporins, and characterize active compounds. In a striking contrast to historical expectation, 18% of ß-lactams screened were active against Mtb, many without a ß-lactamase inhibitor. One potent cephaloporin was active in Mtb-infected mice. The steps outlined here can serve as a blueprint for multiparty, intra- and intersector collaboration in the development of anti-infective agents.

DNA-Dependent Binding of Nargenicin to DnaE1 Inhibits Replication in Mycobacterium tuberculosis.

Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.

Antisynthetase Syndrome with Severe Interstitial Lung Disease in Pregnancy.

Antisynthetase syndrome is a rare multisystem autoimmune disorder which clinically manifests with myositis, arthritis, interstitial lung disease, Raynaud phenomenon, and skin hyperkeratosis. Lung involvement represents the most severe form of disease and has rarely been reported in pregnancy. We present the case of a 22-year-old woman with antisynthetase syndrome and severe restrictive pulmonary disease who experienced a successful pregnancy and delivery. We discuss anesthetic considerations and highlight the importance of a multidisciplinary team approach in caring for parturients with multifactorial medical conditions.

Multimodal Analgesia Decreases Postoperative Opioid Consumption in Living Liver Donation.

OBJECTIVE: To evaluate the effects of multimodal analgesia on postoperative opioid consumption and perioperative pain management in patients undergoing living liver donation. METHODS: A retrospective study was conducted of 129 patients who underwent living liver donation between 2006 and 2015. Patients were separated into 2 cohorts, pre-multimodal analgesia and multimodal analgesia, to allow intergroup analysis. All patients received an intrathecal opioid injection and underwent donor hepatectomy. Primary outcome data compared opioid consumption in oral morphine equivalents for postoperative days (PODs) 0 to 4 between the cohorts. Secondary outcomes compared yearly averaged cumulative opioid consumption on PODs 0 to 4 in oral morphine equivalents; yearly averaged numeric rating scale pain scores; hospital length of stay; and percentage of patients receiving intravenous ketorolac, ketamine, or transversus abdominis plane blocks. RESULTS: For PODs 0 to 4, a 50% reduction in overall opioids administered postoperatively (359 mg vs 179 mg; P<.01) was observed in the multimodal analgesia cohort, whereas no significant difference was found in year-to-year average postoperative pain scores (4.5 vs 3.6). The proportion of patients receiving ketorolac increased to more than 90% by 2013. More than 40% of all patients in the multimodal analgesia group received a perioperative regimen of acetaminophen, gabapentin, ketamine, and transverse abdominal plane blocks (0% in pre-multimodal analgesia). Mean hospital length of stay was reduced from 7.7 to 6.6 days (P<.01). CONCLUSION: Implementation of multimodal analgesia to manage perioperative pain in living liver donation resulted in a 50% reduction of postoperative opioid consumption. Clinically satisfactory average pain scores were maintained for PODs 0 to 4.

Ultrasound-Guided Local Anesthetic Infiltration Between the Popliteal Artery and the Capsule of the Posterior Knee (IPACK) Block for Primary Total Knee Arthroplasty: A Systematic Review of Randomized Controlled Trials.

Posterior knee pain after total knee arthroplasty (TKA) is common despite multimodal analgesia and regional anesthesia use. This review included randomized controlled trials (RCTs) comparing analgesic outcomes after inclusion of local anesthetic infiltration between the popliteal artery and capsule of the knee (iPACK) block versus pathways without iPACK. Electronic databases (MEDLINE, Cochrane Library, Web of Science, Scopus) were searched from inception to 10/11/2020. Eligible studies evaluated iPACK use on primary outcomes: opioid consumption and pain scores with movement. Secondary outcomes included rest pain, patient satisfaction, length of stay (LOS), gait distance, knee range of motion (ROM), and complications. Bias and quality were appraised using the Cochrane Risk of Bias tool and Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) guidelines. Eight RCTs (777 patients) were included. iPACK block use demonstrated similar opioid consumption in the PACU (4/7 RCTs) and 24 hours after TKA (5/7 RCTs) compared to without iPACK (moderate-quality GRADE evidence). Additionally, iPACK block use demonstrated lower movement pain scores in PACU (3/5 RCTs) but similar or higher pain scores after 24 hours (5/7 RCTs; low-quality GRADE evidence). Studies consistently reported no difference in gait distance (4/4 RCTs) or complications (7/7 RCTs) between treatment arms (high-quality GRADE evidence), although differing effect estimates were observed with resting pain, satisfaction, LOS, and knee ROM. This review provides a foundation of knowledge on iPACK efficacy. While evidence does not currently support widespread inclusion of iPACK within enhanced recovery pathways for TKA, limitations suggest further study is warranted.

Collagen analogs with phosphorylcholine are inflammation-suppressing scaffolds for corneal regeneration from alkali burns in mini-pigs.

The long-term survival of biomaterial implants is often hampered by surgery-induced inflammation that can lead to graft failure. Considering that most corneas receiving grafts are either pathological or inflamed before implantation, the risk of rejection is heightened. Here, we show that bioengineered, fully synthetic, and robust corneal implants can be manufactured from a collagen analog (collagen-like peptide-polyethylene glycol hybrid, CLP-PEG) and inflammation-suppressing polymeric 2-methacryloyloxyethyl phosphorylcholine (MPC) when stabilized with the triazine-based crosslinker 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The resulting CLP-PEG-MPC implants led to reduced corneal swelling, haze, and neovascularization in comparison to CLP-PEG only implants when grafted into a mini-pig cornea alkali burn model of inflammation over 12 months. Implants incorporating MPC allowed for faster nerve regeneration and recovery of corneal sensation. CLP-PEG-MPC implants appear to be at a more advanced stage of regeneration than the CLP-PEG only implants, as evidenced by the presence of higher amounts of cornea-specific type V collagen, and a corresponding decrease in the presence of extracellular vesicles and exosomes in the corneal stroma, in keeping with the amounts present in healthy, unoperated corneas.

Generation of SARS-CoV-2 reporter replicon for high-throughput antiviral screening and testing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.

SARS-CoV-2 tropism, entry, replication, and propagation: Considerations for drug discovery and development.

Since the initial report of the novel Coronavirus Disease 2019 (COVID-19) emanating from Wuhan, China, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally. While the effects of SARS-CoV-2 infection are not completely understood, there appears to be a wide spectrum of disease ranging from mild symptoms to severe respiratory distress, hospitalization, and mortality. There are no Food and Drug Administration (FDA)-approved treatments for COVID-19 aside from remdesivir; early efforts to identify efficacious therapeutics for COVID-19 have mainly focused on drug repurposing screens to identify compounds with antiviral activity against SARS-CoV-2 in cellular infection systems. These screens have yielded intriguing hits, but the use of nonhuman immortalized cell lines derived from non-pulmonary or gastrointestinal origins poses any number of questions in predicting the physiological and pathological relevance of these potential interventions. While our knowledge of this novel virus continues to evolve, our current understanding of the key molecular and cellular interactions involved in SARS-CoV-2 infection is discussed in order to provide a framework for developing the most appropriate in vitro toolbox to support current and future drug discovery efforts.