Molecular mechanisms of SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir is a specific antiviral drug that targets the main protease (Mpro) of SARS-CoV-2 and has been approved to treat COVID-191,2. As an RNA virus characterized by high mutation rates, whether SARS-CoV-2 will develop resistance to nirmatrelvir is a question of concern. Our previous studies have shown that several mutational pathways confer resistance to nirmatrelvir, but some result in a loss of viral replicative fitness, which is then compensated for by additional alterations3. The molecular mechanisms for this observed resistance are unknown. Here we combined biochemical and structural methods to demonstrate that alterations at the substrate-binding pocket of Mpro can allow SARS-CoV-2 to develop resistance to nirmatrelvir in two distinct ways. Comprehensive studies of the structures of 14 Mpro mutants in complex with drugs or substrate revealed that alterations at the S1 and S4 subsites substantially decreased the level of inhibitor binding, whereas alterations at the S2 and S4' subsites unexpectedly increased protease activity. Both mechanisms contributed to nirmatrelvir resistance, with the latter compensating for the loss in enzymatic activity of the former, which in turn accounted for the restoration of viral replicative fitness, as observed previously3. Such a profile was also observed for ensitrelvir, another clinically relevant Mpro inhibitor. These results shed light on the mechanisms by which SARS-CoV-2 evolves to develop resistance to the current generation of protease inhibitors and provide the basis for the design of next-generation Mpro inhibitors.

Characterization and antiviral susceptibility of SARS-CoV-2 Omicron BA.2.

The recent emergence of SARS-CoV-2 Omicron (B.1.1.529 lineage) variants possessing numerous mutations has raised concerns of decreased effectiveness of current vaccines, therapeutic monoclonal antibodies and antiviral drugs for COVID-19 against these variants1,2. The original Omicron lineage, BA.1, prevailed in many countries, but more recently, BA.2 has become dominant in at least 68 countries3. Here we evaluated the replicative ability and pathogenicity of authentic infectious BA.2 isolates in immunocompetent and human ACE2-expressing mice and hamsters. In contrast to recent data with chimeric, recombinant SARS-CoV-2 strains expressing the spike proteins of BA.1 and BA.2 on an ancestral WK-521 backbone4, we observed similar infectivity and pathogenicity in mice and hamsters for BA.2 and BA.1, and less pathogenicity compared with early SARS-CoV-2 strains. We also observed a marked and significant reduction in the neutralizing activity of plasma from individuals who had recovered from COVID-19 and vaccine recipients against BA.2 compared to ancestral and Delta variant strains. In addition, we found that some therapeutic monoclonal antibodies (REGN10987 plus REGN10933, COV2-2196 plus COV2-2130, and S309) and antiviral drugs (molnupiravir, nirmatrelvir and S-217622) can restrict viral infection in the respiratory organs of BA.2-infected hamsters. These findings suggest that the replication and pathogenicity of BA.2 is similar to that of BA.1 in rodents and that several therapeutic monoclonal antibodies and antiviral compounds are effective against Omicron BA.2 variants.

Oral Nirmatrelvir-Ritonavir as Postexposure Prophylaxis for Covid-19.

BACKGROUND: Clinical trials of treatments for coronavirus disease 2019 (Covid-19) have not shown a significant benefit of postexposure prophylaxis. METHODS: We conducted a phase 2-3 double-blind trial to assess the efficacy and safety of nirmatrelvir-ritonavir in asymptomatic, rapid antigen test-negative adults who had been exposed to a household contact with Covid-19 within 96 hours before randomization. The participants were randomly assigned in a 1:1:1 ratio to receive nirmatrelvir-ritonavir (300 mg of nirmatrelvir and 100 mg of ritonavir) every 12 hours for 5 days or for 10 days or matching placebo for 5 or 10 days. The primary end point was the development of symptomatic SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection, confirmed on reverse-transcriptase-polymerase-chain-reaction (RT-PCR) or rapid antigen testing, through 14 days in participants who had a negative RT-PCR test at baseline. RESULTS: A total of 2736 participants were randomly assigned to a trial group - 921 to the 5-day nirmatrelvir-ritonavir group, 917 to the 10-day nirmatrelvir-ritonavir group, and 898 to the placebo group. Symptomatic, confirmed SARS-CoV-2 infection developed by day 14 in 2.6% of the participants in the 5-day nirmatrelvir-ritonavir group, 2.4% of those in the 10-day nirmatrelvir-ritonavir group, and 3.9% of those in the placebo group. In each nirmatrelvir-ritonavir group, the percentage of participants in whom symptomatic, confirmed SARS-CoV-2 infection developed did not differ significantly from that in the placebo group, with risk reductions relative to placebo of 29.8% (95% confidence interval [CI], -16.7 to 57.8; P = 0.17) in the 5-day nirmatrelvir-ritonavir group and 35.5% (95% CI, -11.5 to 62.7; P = 0.12) in the 10-day nirmatrelvir-ritonavir group. The incidence of adverse events was similar across the trial groups, with dysgeusia being the most frequently reported adverse event (in 5.9% and 6.8% of the participants in the 5-day and 10-day nirmatrelvir-ritonavir groups, respectively, and in 0.7% of those in the placebo group). CONCLUSIONS: In this placebo-controlled trial, postexposure prophylaxis with nirmatrelvir-ritonavir for 5 or 10 days did not significantly reduce the risk of symptomatic SARS-CoV-2 infection. (Funded by Pfizer; ClinicalTrials.gov number, NCT05047601.).

The CLIP1-LTK fusion is an oncogenic driver in non-small-cell lung cancer.

Lung cancer is one of the most aggressive tumour types. Targeted therapies stratified by oncogenic drivers have substantially improved therapeutic outcomes in patients with non-small-cell lung cancer (NSCLC)1. However, such oncogenic drivers are not found in 25-40% of cases of lung adenocarcinoma, the most common histological subtype of NSCLC2. Here we identify a novel fusion transcript of CLIP1 and LTK using whole-transcriptome sequencing in a multi-institutional genome screening platform (LC-SCRUM-Asia, UMIN000036871). The CLIP1-LTK fusion was present in 0.4% of NSCLCs and was mutually exclusive with other known oncogenic drivers. We show that kinase activity of the CLIP1-LTK fusion protein is constitutively activated and has transformation potential. Treatment of Ba/F3 cells expressing CLIP1-LTK with lorlatinib, an ALK inhibitor, inhibited CLIP1-LTK kinase activity, suppressed proliferation and induced apoptosis. One patient with NSCLC harbouring the CLIP1-LTK fusion showed a good clinical response to lorlatinib treatment. To our knowledge, this is the first description of LTK alterations with oncogenic activity in cancers. These results identify the CLIP1-LTK fusion as a target in NSCLC that could be treated with lorlatinib.

Proof-of-concept studies with a computationally designed Mpro inhibitor as a synergistic combination regimen alternative to Paxlovid.

As the SARS-CoV-2 virus continues to spread and mutate, it remains important to focus not only on preventing spread through vaccination but also on treating infection with direct-acting antivirals (DAA). The approval of Paxlovid, a SARS-CoV-2 main protease (Mpro) DAA, has been significant for treatment of patients. A limitation of this DAA, however, is that the antiviral component, nirmatrelvir, is rapidly metabolized and requires inclusion of a CYP450 3A4 metabolic inhibitor, ritonavir, to boost levels of the active drug. Serious drug-drug interactions can occur with Paxlovid for patients who are also taking other medications metabolized by CYP4503A4, particularly transplant or otherwise immunocompromised patients who are most at risk for SARS-CoV-2 infection and the development of severe symptoms. Developing an alternative antiviral with improved pharmacological properties is critical for treatment of these patients. By using a computational and structure-guided approach, we were able to optimize a 100 to 250 µM screening hit to a potent nanomolar inhibitor and lead compound, Mpro61. In this study, we further evaluate Mpro61 as a lead compound, starting with examination of its mode of binding to SARS-CoV-2 Mpro. In vitro pharmacological profiling established a lack of off-target effects, particularly CYP450 3A4 inhibition, as well as potential for synergy with the currently approved alternate antiviral, molnupiravir. Development and subsequent testing of a capsule formulation for oral dosing of Mpro61 in B6-K18-hACE2 mice demonstrated favorable pharmacological properties, efficacy, and synergy with molnupiravir, and complete recovery from subsequent challenge by SARS-CoV-2, establishing Mpro61 as a promising potential preclinical candidate.

Computational prediction of interactions between Paxlovid and prescription drugs.

Pfizer's Paxlovid has recently been approved for the emergency use authorization (EUA) from the US Food and Drug Administration (FDA) for the treatment of mild-to-moderate COVID-19. Drug interactions can be a serious medical problem for COVID-19 patients with underlying medical conditions, such as hypertension and diabetes, who have likely been taking other drugs. Here, we use deep learning to predict potential drug-drug interactions between Paxlovid components (nirmatrelvir and ritonavir) and 2,248 prescription drugs for treating various diseases.

Oral Nirmatrelvir for High-Risk, Nonhospitalized Adults with Covid-19.

BACKGROUND: Nirmatrelvir is an orally administered severe acute respiratory syndrome coronavirus 2 main protease (Mpro) inhibitor with potent pan-human-coronavirus activity in vitro. METHODS: We conducted a phase 2-3 double-blind, randomized, controlled trial in which symptomatic, unvaccinated, nonhospitalized adults at high risk for progression to severe coronavirus disease 2019 (Covid-19) were assigned in a 1:1 ratio to receive either 300 mg of nirmatrelvir plus 100 mg of ritonavir (a pharmacokinetic enhancer) or placebo every 12 hours for 5 days. Covid-19-related hospitalization or death from any cause through day 28, viral load, and safety were evaluated. RESULTS: A total of 2246 patients underwent randomization; 1120 patients received nirmatrelvir plus ritonavir (nirmatrelvir group) and 1126 received placebo (placebo group). In the planned interim analysis of patients treated within 3 days after symptom onset (modified intention-to treat population, comprising 774 of the 1361 patients in the full analysis population), the incidence of Covid-19-related hospitalization or death by day 28 was lower in the nirmatrelvir group than in the placebo group by 6.32 percentage points (95% confidence interval [CI], -9.04 to -3.59; P<0.001; relative risk reduction, 89.1%); the incidence was 0.77% (3 of 389 patients) in the nirmatrelvir group, with 0 deaths, as compared with 7.01% (27 of 385 patients) in the placebo group, with 7 deaths. Efficacy was maintained in the final analysis involving the 1379 patients in the modified intention-to-treat population, with a difference of -5.81 percentage points (95% CI, -7.78 to -3.84; P<0.001; relative risk reduction, 88.9%). All 13 deaths occurred in the placebo group. The viral load was lower with nirmatrelvir plus ritonavir than with placebo at day 5 of treatment, with an adjusted mean difference of -0.868 log10 copies per milliliter when treatment was initiated within 3 days after the onset of symptoms. The incidence of adverse events that emerged during the treatment period was similar in the two groups (any adverse event, 22.6% with nirmatrelvir plus ritonavir vs. 23.9% with placebo; serious adverse events, 1.6% vs. 6.6%; and adverse events leading to discontinuation of the drugs or placebo, 2.1% vs. 4.2%). Dysgeusia (5.6% vs. 0.3%) and diarrhea (3.1% vs. 1.6%) occurred more frequently with nirmatrelvir plus ritonavir than with placebo. CONCLUSIONS: Treatment of symptomatic Covid-19 with nirmatrelvir plus ritonavir resulted in a risk of progression to severe Covid-19 that was 89% lower than the risk with placebo, without evident safety concerns. (Supported by Pfizer; ClinicalTrials.gov number, NCT04960202.).

Nirmatrelvir Use and Severe Covid-19 Outcomes during the Omicron Surge.

BACKGROUND: The oral protease inhibitor nirmatrelvir has shown substantial efficacy in high-risk, unvaccinated patients infected with the B.1.617.2 (delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data regarding the effectiveness of nirmatrelvir in preventing severe coronavirus disease 2019 (Covid-19) outcomes from the B.1.1.529 (omicron) variant are limited. METHODS: We obtained data for all members of Clalit Health Services who were 40 years of age or older at the start of the study period and were assessed as being eligible to receive nirmatrelvir therapy during the omicron surge. A Cox proportional-hazards regression model with time-dependent covariates was used to estimate the association of nirmatrelvir treatment with hospitalization and death due to Covid-19, with adjustment for sociodemographic factors, coexisting conditions, and previous SARS-CoV-2 immunity status. RESULTS: A total of 109,254 patients met the eligibility criteria, of whom 3902 (4%) received nirmatrelvir during the study period. Among patients 65 years of age or older, the rate of hospitalization due to Covid-19 was 14.7 cases per 100,000 person-days among treated patients as compared with 58.9 cases per 100,000 person-days among untreated patients (adjusted hazard ratio, 0.27; 95% confidence interval [CI], 0.15 to 0.49). The adjusted hazard ratio for death due to Covid-19 was 0.21 (95% CI, 0.05 to 0.82). Among patients 40 to 64 years of age, the rate of hospitalization due to Covid-19 was 15.2 cases per 100,000 person-days among treated patients and 15.8 cases per 100,000 person-days among untreated patients (adjusted hazard ratio, 0.74; 95% CI, 0.35 to 1.58). The adjusted hazard ratio for death due to Covid-19 was 1.32 (95% CI, 0.16 to 10.75). CONCLUSIONS: Among patients 65 years of age or older, the rates of hospitalization and death due to Covid-19 were significantly lower among those who received nirmatrelvir than among those who did not. No evidence of benefit was found in younger adults.

An RNA replicon system to investigate promising inhibitors of feline coronavirus.

Feline infectious peritonitis (FIP) is a fatal feline disease, caused by a feline coronavirus (FCoV), namely feline infectious peritonitis virus (FIPV). We produced a baby hamster kidney 21 (BHK) cell line expressing a serotype I FCoV replicon RNA with a green fluorescent protein (GFP) reporter gene (BHK-F-Rep) and used it as an in vitro screening system to test different antiviral compounds. Two inhibitors of the FCoV main protease (Mpro), namely GC376 and Nirmatrelvir, as well as the nucleoside analog Remdesivir proved to be effective in inhibiting the replicon system. Different combinations of these compounds also proved to be potent inhibitors, having an additive effect when combined. Remdesivir, GC376, and Nirmatrelvir all have a 50% cytotoxic concentration (CC50) more than 200 times higher than their half-maximal inhibitory concentrations (IC50), making them important candidates for future in vivo studies as well as clinically implemented drug candidates. In addition, results were acquired with a virus infection system, where Felis catus whole fetus 4 (Fcwf-4) cells were infected with a previously described recombinant GFP-expressing FIPV (based on the laboratory-adapted serotype I FIPV strain Black) and treated with the most promising compounds. Results acquired with the replicon system were comparable to the results acquired with the virus infection system, demonstrating that we successfully implemented the FCoV replicon system for antiviral screening. We expect that this system will greatly facilitate future screens for anti-FIPV compounds and provide a non-infectious system to study and evaluate drug-resistant mutations that may emerge in the FIPV genome.IMPORTANCEFIPV is of great significance in the cat population around the world, causing 0.3%-1.4% of feline deaths in veterinary practices (2). As there are neither effective preventive measures nor approved treatment options available, there is an urgent need to identify antiviral drugs against FIPV. Our FCoV replicon system provides a valuable tool for drug discovery in vitro. Due to the lack of cell culture systems for serotype I FCoVs (the serotype most prevalent in the feline population) (2), a different system is needed to study these viruses. A viral replicon system is a valuable tool for studying FCoVs. Overall, our results demonstrate the utility of the serotype I feline coronavirus replicon system for antiviral screening as well as to study this virus in general. We propose several compounds representing promising candidates for future clinical trials and ultimately with the potential to save cats suffering from FIP.

In outpatients with COVID-19 during Omicron variant circulation, molnupiravir and nirmatrelvir-ritonavir improved outcomes.

SOURCE CITATION: Sommer I, Ledinger D, Thaler K, et al. Outpatient treatment of confirmed COVID-19: a living, rapid evidence review for the American College of Physicians (version 2). Ann Intern Med. 2023;176:1377-1385. 37722115.

Nirmatrelvir/Ritonavir Use and Hospitalizations or Death in a Previously Uninfected Nonhospitalized High-Risk Population With COVID-19: A Matched Cohort Study.

OBJECTIVE: To determine the association of nirmatrelvir/ritonavir (NMV/r) with hospitalization or death within 30 days as compared with untreated controls previously uninfected and nonhospitalized. METHODS: We used a matched cohort design using inverse probability of treatment weight (IPTW). Individuals prescribed NMV/r within 3 days of COVID-19 diagnosis were compared with IPTW-based untreated controls. Variables for IPTW included age, race, sex, body mass index, geographic location, vaccination status, and multiple comorbidities. Additional analyses were conducted on NMV/r-treated and propensity score-matched untreated controls. RESULTS: Among 7615 individuals prescribed NMV/r and 62 077 controls identified between 1 January 2022 and 25 February 2023, the risk of hospitalization/death was lower among NMV/r-treated persons vs untreated controls (243 vs 3468 events; absolute risk difference [ARD], -2.36 [95% CI, -2.57 to -2.14]). The difference was significant for those >60 and ≤60 years old (ARD, -3.86 [95% CI, -4.19 to -3.54] vs -0.27 [95% CI, -0.51 to -0.03]) and for persons asymptomatic and symptomatic (ARD, -7.09 [95% CI, -7.62 to -6.55] vs -1.46 [95% CI, -1.66 to -1.25]). Significant benefit was observed among individuals unvaccinated and vaccinated, with or without a booster dose. CONCLUSIONS: NMV/r is associated with a significant reduction in 30-day hospitalization or death among individuals previously uninfected and nonhospitalized.

Structural basis of nirmatrelvir and ensitrelvir activity against naturally occurring polymorphisms of the SARS-CoV-2 main protease.

SARS-CoV-2 is the causative agent of COVID-19. The main viral protease (Mpro) is an attractive target for antivirals. The clinically approved drug nirmatrelvir and the clinical candidate ensitrelvir have so far showed great potential for treatment of viral infection. However, the broad use of antivirals is often associated with resistance generation. Herein, we enzymatically characterized 14 naturally occurring Mpro polymorphisms that are close to the binding site of these antivirals. Nirmatrelvir retained its potency against most polymorphisms tested, while mutants G143S and Q189K were associated with diminished inhibition constants. For ensitrelvir, diminished inhibition constants were observed for polymorphisms M49I, G143S, and R188S, but not for Q189K, suggesting a distinct resistance profile between inhibitors. In addition, the crystal structures of selected polymorphisms revealed interactions that were critical for loss of potency. In conclusion, our data will assist the monitoring of potential resistant strains, support the design of combined therapy, as well as assist the development of the next generation of Mpro inhibitors.

Contribution of the catalytic dyad of SARS-CoV-2 main protease to binding covalent and noncovalent inhibitors.

The effect of mutations of the catalytic dyad residues of SARS-CoV-2 main protease (MProWT) on the thermodynamics of binding of covalent inhibitors comprising nitrile [nirmatrelvir (NMV), NBH2], aldehyde (GC373), and ketone (BBH1) warheads to MPro is examined together with room temperature X-ray crystallography. When lacking the nucleophilic C145, NMV binding is ∼400-fold weaker corresponding to 3.5 kcal/mol and 13.3 °C decrease in free energy (ΔG) and thermal stability (Tm), respectively, relative to MProWT. The H41A mutation results in a 20-fold increase in the dissociation constant (Kd), and 1.7 kcal/mol and 1.4 °C decreases in ΔG and Tm, respectively. Increasing the pH from 7.2 to 8.2 enhances NMV binding to MProH41A, whereas no significant change is observed in binding to MProWT. Structures of the four inhibitor complexes with MPro1-304/C145A show that the active site geometries of the complexes are nearly identical to that of MProWT with the nucleophilic sulfur of C145 positioned to react with the nitrile or the carbonyl carbon. These results support a two-step mechanism for the formation of the covalent complex involving an initial non-covalent binding followed by a nucleophilic attack by the thiolate anion of C145 on the warhead carbon. Noncovalent inhibitor ensitrelvir (ESV) exhibits a binding affinity to MProWT that is similar to NMV but differs in its thermodynamic signature from NMV. The binding of ESV to MProC145A also results in a significant, but smaller, increase in Kd and decrease in ΔG and Tm, relative to NMV.

Thermodynamic consequences of stapling side-chains on a peptide ligand using a lactam-bridge: A theoretical study on anti-angiogenic peptides targeting VEGF.

Peptides are promising therapeutic agents for various biological targets due to their high efficacy and low toxicity, and the design of peptide ligands with high binding affinity to the target of interest is of utmost importance in peptide-based drug design. Introducing a conformational constraint to a flexible peptide ligand using a side-chain lactam-bridge is a convenient and efficient method to improve its binding affinity to the target. However, in general, such a small structural modification to a flexible ligand made with the intent of lowering the configurational entropic penalty for binding may have unintended consequences in different components of the binding enthalpy and entropy, including the configurational entropy component, which are still not clearly understood. Toward probing this, we examine different components of the binding enthalpy and entropy as well as the underlying structure and dynamics, for a side-chain lactam-bridged peptide inhibitor and its flexible analog forming complexes with vascular endothelial growth factor (VEGF), using all-atom molecular dynamics simulations. It is found that introducing a side-chain lactam-bridge constraint into the flexible peptide analog led to a gain in configurational entropy change but losses in solvation entropy, solute internal energy, and solvation energy changes upon binding, pinpointing the opportunities and challenges in drug design. The present study features an interplay between configurational and solvation entropy changes, as well as the one between binding enthalpy and entropy, in ligand-target binding upon imposing a conformational constraint into a flexible ligand.

Characteristics and Outcomes of US Veterans With Immunocompromised Conditions at High Risk of SARS-CoV-2 Infection With or Without Receipt of Oral Antiviral Agents.

OBJECTIVES: Molnupiravir and nirmatrelvir-ritonavir were the first oral antiviral agents to demonstrate reduced hospitalization or death in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but patients with immunocompromised conditions were not well-represented. The objective of this study was to characterize and compare the clinical outcomes of US veterans with immunocompromised conditions prescribed oral antivirals with those who did not receive oral antivirals for mild-to-moderate SARS-CoV-2 active infection. METHODS: This was a retrospective, observational, nationwide propensity-matched analysis of US veterans with immunocompromised conditions who developed documented SARS-CoV-2 infection. The primary outcome was the composite of any hospitalization or death within 30 days of diagnosis. Secondary outcomes included 30-day comparative rates of (1) any hospitalization, (2) death, (3) intensive care requirement, and (4) subset analyses of outcomes by oral antiviral used and vaccination status. RESULTS: The composite primary outcome was significantly lower in patients receiving oral antiviral therapy compared with those who did not (23/390 [5.9%] vs 57/390 [14.6%]; odds ratio, 0.37; 95% confidence interval, .22-.61). This difference was driven largely by fewer deaths in the oral antiviral group (1/390 [0.3%] vs 19/390 [4.9%]; odds ratio, 0.05; 95% confidence interval, .007-.38). There was no significant difference in rate of intensive care requirement. The composite outcome was improved in vaccinated patients (completing the first series or first booster dose) who received oral antiviral agents compared with those who did not receive oral antiviral agents. Compared with those prescribed nirmatrelvir-ritonavir, patients given molnupiravir were older, had a higher incidence of cautions/contraindications, greater prevalence of tobacco use, and more cardiovascular complications. CONCLUSIONS: Use of molnupiravir or nirmatrelvir-ritonavir was associated with lower incidences of hospitalization or death within 30 days of diagnosis in US veterans with immunocompromised conditions, regardless of vaccination status. These findings support the use of either oral antiviral in this patient population.

Combined Protection of Vaccination and Nirmatrelvir-Ritonavir Against Hospitalization in Adults With COVID-19.

Among adults at risk for severe coronavirus disease 2019 (COVID-19), the lowest hospitalization rate was among those who received nirmatrelvir-ritonavir after 3 or more messenger RNA vaccine doses (adjusted hazard ratio, 0.22; 95% confidence interval, .19-.24). Eligible adults, including those previously vaccinated, should be considered for COVID-19 antiviral treatment.

Discovery of a Bacterial Hydrazine Transferase That Constructs the N-Aminolactam Pharmacophore in Albofungin Biosynthesis.

Structural motifs containing nitrogen-nitrogen (N-N) bonds are prevalent in a large number of clinical drugs and bioactive natural products. Hydrazine (N2H4) serves as a widely utilized building block for the preparation of these N-N-containing molecules in organic synthesis. Despite its common use in chemical processes, no enzyme has been identified to catalyze the incorporation of free hydrazine in natural product biosynthesis. Here, we report that a hydrazine transferase catalyzes the condensation of N2H4 and an aromatic polyketide pathway intermediate, leading to the formation of a rare N-aminolactam pharmacophore in the biosynthesis of broad-spectrum antibiotic albofungin. These results expand the current knowledge on the biosynthetic mechanism for natural products with N-N units and should facilitate future development of biocatalysts for the production of N-N-containing chemicals.

Conjugation to Native and Nonnative Triscatecholate Siderophores Enhances Delivery and Antibacterial Activity of a ß-Lactam to Gram-Negative Bacterial Pathogens.

Developing new antibiotics and delivery strategies is of critical importance for treating infections caused by Gram-negative bacterial pathogens. Hijacking bacterial iron uptake machinery, such as that of the siderophore enterobactin (Ent), represents one promising approach toward these goals. Here, we report a novel Ent-inspired siderophore-antibiotic conjugate (SAC) employing an alternative siderophore moiety as the delivery vector and demonstrate the potency of our SACs harboring the ß-lactam antibiotic ampicillin (Amp) against multiple pathogenic Gram-negative bacterial strains. We establish the ability of N,N',N''-(nitrilotris(ethane-2,1-diyl))tris(2,3-dihydroxybenzamide) (TRENCAM, hereafter TC), a synthetic mimic of Ent, to facilitate drug delivery across the outer membrane (OM) of Gram-negative pathogens. Conjugation of Amp to a new monofunctionalized TC scaffold affords TC-Amp, which displays markedly enhanced antibacterial activity against the gastrointestinal pathogen Salmonella enterica serovar Typhimurium (STm) compared with unmodified Amp. Bacterial uptake, antibiotic susceptibility, and microscopy studies with STm show that the TC moiety facilitates TC-Amp uptake by the OM receptors FepA and IroN and that the Amp warhead inhibits penicillin-binding proteins. Moreover, TC-Amp achieves targeted activity, selectively killing STm in the presence of a commensal lactobacillus. Remarkably, we uncover that TC-Amp and its Ent-based predecessor Ent-Amp achieve enhanced antibacterial activity against diverse Gram-negative ESKAPE pathogens that express Ent uptake machinery, including strains that possess intrinsic ß-lactam resistance. TC-Amp and Ent-Amp exhibit potency comparable to that of the FDA-approved SAC cefiderocol against Gram-negative pathogens. These results demonstrate the effective application of native and appropriately designed nonnative siderophores as vectors for drug delivery across the OM of multiple Gram-negative bacterial pathogens.

Public Health Impact of Paxlovid as Treatment for COVID-19, United States.

We evaluated the population-level benefits of expanding treatment with the antiviral drug Paxlovid (nirmatrelvir/ritonavir) in the United States for SARS-CoV-2 Omicron variant infections. Using a multiscale mathematical model, we found that treating 20% of symptomatic case-patients with Paxlovid over a period of 300 days beginning in January 2022 resulted in life and cost savings. In a low-transmission scenario (effective reproduction number of 1.2), this approach could avert 0.28 million (95% CI 0.03-0.59 million) hospitalizations and save US $56.95 billion (95% CI US $2.62-$122.63 billion). In a higher transmission scenario (effective reproduction number of 3), the benefits increase, potentially preventing 0.85 million (95% CI 0.36-1.38 million) hospitalizations and saving US $170.17 billion (95% CI US $60.49-$286.14 billion). Our findings suggest that timely and widespread use of Paxlovid could be an effective and economical approach to mitigate the effects of COVID-19.

Treatment sequencing after failure to alectinib in patients with anaplastic lymphoma kinase-positive non-small-cell lung cancer.

Alectinib is the first-line therapy for anaplastic lymphoma kinase-positive non-small-cell lung cancer. Although some guidelines have recommended using other anaplastic lymphoma kinase inhibitors after alectinib failure, evidence for such regimens in patients who fail to respond to alectinib is limited. This study involved using administrative claims data from acute care hospitals in Japan. We extracted the data of 634 patients diagnosed with lung cancer between September 1, 2014, and January 31, 2023, who received alectinib treatment before treatment with another anaplastic lymphoma kinase inhibitor. We assessed distributions of patients according to their treatment sequencing and prognosis among three periods defined based on the initial marketing dates of lorlatinib and brigatinib. The type of anaplastic lymphoma kinase inhibitors after alectinib failure changed over time. In the most recent period, lorlatinib (58%) and brigatinib (40%) became predominant. Two-year overall survival improved over time (47%-84%), accompanied by an increased 2-year proportion of patients who continuously used anaplastic lymphoma kinase inhibitors after alectinib failure (13%-44%). The times to treatment discontinuation of the regimen between patients treated with lorlatinib and brigatinib were similar, with a hazard ratio of 1.02 (95% confidence interval, 0.64-1.64) in the period after marketing brigatinib. This study provides insights into the evolving treatment landscape for patients with anaplastic lymphoma kinase-positive non-small-cell lung cancer who experience failed alectinib treatment and highlights the need for further studies and data accumulation to determine the optimal treatment strategy.