A synthetic antibiotic class overcoming bacterial multidrug resistance.

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern1. For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings2. Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the [Formula: see text] nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance.

Structural basis of Cfr-mediated antimicrobial resistance and mechanisms to evade it.

The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.

Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR.

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.

Expression of Bacillus subtilis ABCF antibiotic resistance factor VmlR is regulated by RNA polymerase pausing, transcription attenuation, translation attenuation and (p)ppGpp.

Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Thus, transcription in the presence of antibiotic induces vmlR expression. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression.

Invisible disabilities and college academic success: New evidence from a mediation analysis.

Students with "invisible" disabilities-including autism spectrum disorder (ASD), attention deficit disorder (ADD/ADHD), learning disorders, and mental health conditions-make up an increasingly large share of college students in the United States. Despite these gains in access, students with invisible disabilities remain disadvantaged relative to their neurotypical and non-disabled peers in many parts of the college experience, including academically. Researchers have hypothesized that inequalities in pre-college academic preparation, barriers to social integration, and lower levels of engagement on college campuses may be at least partially to blame. We test this hypothesis using newly available survey data on college students in the state of Indiana (n = 2728). Based on a series of decompositions, we show that students with invisible disabilities face a series of interrelated challenges, beginning with their academic preparation and extending into their social and academic experiences on college campuses. That these disadvantages feed into one another suggests the presence of a cumulative advantage/disadvantage process, in which early advantages and disadvantages compound as disabled and non-disabled students move through the educational system.

[3+2] Dipolar Cycloaddition of a Stabilized Azomethine Ylide and an Electron-Deficient Dipolarophile: Revision of Regioselectivity.

The regioselectivity of a [3+2] dipolar cycloaddition reaction of a stabilized azomethine ylide with an electron-deficient dipolarophile was found to be counter to a report published in this journal.

Proposed Resolution of a Mechanistic Puzzle of Long Duration: Self-Condensation of Cyclopentanone to Form an 11-Carbon Dienoic Acid.

The mechanism proposed for the transformation of cyclopentanone to the dienoic acid 1, as published in this journal, is revealed to be in error. We show that carbon 11 derives not from dimethyl sulfoxide as proposed but from the dichloromethane present in the "quenching" solution. The intermediacy of an α-chloromethyl ketone and its subsequent fragmentation in the presence of a hydroxide ion is supported by experiments described herein and by extensive literature precedent.

A method for tritiation of iboxamycin permits measurement of its ribosomal binding.

Hydrogen-tritium exchange is widely employed for radioisotopic labeling of molecules of biological interest but typically involves the metal-promoted exchange of sp2-hybridized carbon-hydrogen bonds, a strategy that is not directly applicable to the antibiotic iboxamycin, which possesses no such bonds. We show that ruthenium-induced 2'-epimerization of 2'-epi-iboxamycin in HTO (200 mCi) of low specific activity (10 Ci/g, 180 mCi/mmol) at 80 °C for 18 h affords after purification tritium-labeled iboxamycin (3.55 µCi) with a specific activity of 53 mCi/mmol. Iboxamycin displayed an apparent inhibition constant (Ki, app) of 41 ± 30 nM towards Escherichia coli ribosomes, binding approximately 70-fold more tightly than the antibiotic clindamycin (Ki, app = 2.7 ± 1.1 µM).

Evaluation of severe acute respiratory syndrome coronavirus 2 monoclonal antibodies in high-risk solid organ transplant recipients across three major coronavirus disease 2019 variant waves.

BACKGROUND: Coronavirus disease 2019 (COVID-19) continues to impact immunocompromised populations including solid organ transplant recipients (SOTRs). Monoclonal antibodies (mAbs) have shown effectiveness in reducing COVID-19-related hospitalizations and emergency department (ED) visits in SOTRs at different time frames in the COVID-19 pandemic; however, less data exist on the impact of mAbs for SOTRs across variant waves and with the advent of available COVID-19 vaccines. METHODS: This retrospective study included SOTR outpatients who tested positive for SARS-CoV-2 and received mAbs from December 2020 to February 2022 (n = 233); using in-house sequencing of clinical samples, we monitored the emergence of Alpha, Delta, and Omicron variants. The primary outcome was a composite of 29-day COVID-19-related hospitalizations and ED visits. Prespecified secondary outcomes included individual components of the primary endpoint; for patients requiring hospitalization post-mAb administration, we describe their inpatient treatment. RESULTS: A low percentage of SOTRs treated with mAb required hospitalization or an ED visit (14.6% overall); this did not differ across COVID-19 variants (p = .152). Hospitalization and ED visits did not significantly differ between abdominal and cardiothoracic SOTRs. For hospitalized patients, the majority received treatment with corticosteroids and few required intensive care unit (ICU) care. CONCLUSION: Among SOTR outpatients with mild or moderate COVID-19 symptoms, early administration of mAb minimizes the need for hospital care. For patients requiring hospitalization, corticosteroids were common but patients experienced low rates of oxygen supplementation and ICU care. Use of mAbs in SOTRs should be considered early in the disease when therapy is available.

"Patiently waiting": How do non-driving disabled adults get around in rural America?

Most mobility needs in rural America are primarily met via driving. Yet many disabled adults cannot drive. Lack of adequate public transportation in rural areas exacerbates transportation-related disparities. We interviewed 33 non-driving disabled adults throughout the U.S. to explore how they get around in their rural communities. Research questions primarily focused on the relationships between transportation, community participation, healthcare access, and social capital. We used thematic analysis to identify themes related to our research questions. In general, interviewees described a dearth of public transportation options. Even if public transit were available, other issues limited use such as inaccessibility, expenses, or schedules and routes. Many participants also described getting rides from others. While this was a critical component of many individuals' daily travel, it also introduced a layer of dependency and social pressure that otherwise would not exist with more independent means of transportation. Policy implications include improved public transit funding and strategies to promote universal auto access.

Discovery of Macrolide Antibiotics Effective against Multi-Drug Resistant Gram-Negative Pathogens.

Macrolides are among the most widely prescribed antibiotics, particularly for bacterial lung infections, due to their favorable safety, oral bioavailability, and spectrum of activity against Gram-positive pathogens such as Streptococcus pneumoniae, the most common cause of bacterial pneumonia. Their utility against Gram-negative bacteria is extremely limited and does not include the Enterobacteriaceae or other ESKAPE pathogens. With the increasing development of resistance to current therapies and the lack of safe, oral options to treat Gram-negative infections, extended-spectrum macrolides have the potential to provide valuable treatment options. While the bacterial ribosome, the target of macrolides, is highly conserved across Gram-positive and Gram-negative bacteria, traditional macrolides do not possess the proper physicochemical properties to cross the polar Gram-negative outer membrane and are highly susceptible to efflux. As with most natural product-derived compounds, macrolides are generally prepared through semisynthesis, which is limited in scope and lacks the ability to make the drastic physicochemical property changes necessary to overcome these hurdles.By using a fully synthetic platform technology to greatly expand structural diversity, novel macrolides were prepared with a focus on lowering the MW and increasing the polarity to achieve a physicochemical property profile more similar to that of traditional Gram-negative drug classes. In addition to the removal of lipophilic groups, a critical structural feature for obtaining Gram-negative activity in the macrolide class proved to be the introduction of small secondary or tertiary amines to yield polycationic species potentially capable of self-promoted uptake. Within the azithromycin-like 15-membered azalides, potent activity was seen when small alkyl amines were introduced at the 6'-position of desosamine. The biggest gains, however, were made by replacing the entire C10-C13 fragment of the macrolactone ring with commercially available or readily synthesized 1,2-aminoalcohols, leading to 13-membered azalides. The introduction of a tethered basic amine at the C10-position and systematic optimization of substitution and tether length and flexibility ultimately provided new macrolides that for the first time exhibit clinically relevant antibacterial activity against multi-drug resistant Gram-negative bacteria. A retrospective computational analysis of >1800 fully synthetic macrolides prepared during this effort identified key drivers and optimum ranges for improving permeability and avoiding efflux. In contrast to standard Gram-negative drugs which generally have MWs below 600 and clogD7.4 values below 0, we found that the ideal ranges for Gram-negative macrolides were MW between 600 and 720 and cLogD7.4 between -1 and 3. A total charge of between 2.5 and 3 was also required to provide optimal permeability and efflux avoidance. Thus, Gram-negative macrolides occupy a unique physicochemical property space that lies between traditional Gram-negative drug classes and Gram-positive macrolides.

A platform for the discovery of new macrolide antibiotics.

The chemical modification of structurally complex fermentation products, a process known as semisynthesis, has been an important tool in the discovery and manufacture of antibiotics for the treatment of various infectious diseases. However, many of the therapeutics obtained in this way are no longer effective, because bacterial resistance to these compounds has developed. Here we present a practical, fully synthetic route to macrolide antibiotics by the convergent assembly of simple chemical building blocks, enabling the synthesis of diverse structures not accessible by traditional semisynthetic approaches. More than 300 new macrolide antibiotic candidates, as well as the clinical candidate solithromycin, have been synthesized using our convergent approach. Evaluation of these compounds against a panel of pathogenic bacteria revealed that the majority of these structures had antibiotic activity, some efficacious against strains resistant to macrolides in current use. The chemistry we describe here provides a platform for the discovery of new macrolide antibiotics and may also serve as the basis for their manufacture.

A Practical, Component-Based Synthetic Route to Methylthiolincosamine Permitting Facile Northern-Half Diversification of Lincosamide Antibiotics.

The development of a flexible, component-based synthetic route to the amino sugar fragment of the lincosamide antibiotics is described. This route hinges on the application and extension of nitroaldol chemistry to forge strategic bonds within complex amino sugar targets and employs a glycal epoxide as a versatile glycosyl donor for the installation of anomeric groups. Through building-block exchange and late-stage functionalization, this route affords access to a host of rationally designed lincosamides otherwise inaccessible by semisynthesis and underpins a platform for the discovery of new lincosamide antibiotics.

Practical Gram-Scale Synthesis of Iboxamycin, a Potent Antibiotic Candidate.

A gram-scale synthesis of iboxamycin, an antibiotic candidate bearing a fused bicyclic amino acid residue, is presented. A pivotal transformation in the route involves an intramolecular hydrosilylation-oxidation sequence to set the ring-fusion stereocenters of the bicyclic scaffold. Other notable features of the synthesis include a high-yielding, highly diastereoselective alkylation of a pseudoephenamine amide, a convergent sp3-sp2 Negishi coupling, and a one-pot transacetalization-reduction reaction to form the target compound's oxepane ring. Implementation of this synthetic strategy has provided ample quantities of iboxamycin to allow for its in vivo profiling in murine models of infection.

Mediator kinase inhibition further activates super-enhancer-associated genes in AML.

Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.

Efficacy and Safety of a Nicotine Mouth Spray for Smoking Cessation: A Randomized, Multicenter, Controlled Study in a Naturalistic Setting.

BACKGROUND: Nicotine replacement therapy (NRT) has been demonstrated to be an effective pharmacological treatment for smoking cessation, and most types of NRT have been approved as over-the-counter (OTC) medications. In an effort to create a fast-acting, flexible, and discreet NRT, a nicotine mouth spray (NMS) has been developed. This study was designed to assess the efficacy and safety of NMS in a naturalistic setting in the United States. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled, parallel-group, 26-week study in 1198 smokers motivated to quit. The study was designed to resemble an OTC environment, and thus included limited intervention, limited motivational screening, and no behavioral support. The primary efficacy endpoint was carbon monoxide-verified, self-reported continuous abstinence from smoking from week 2 until week 6. The safety of NMS was assessed by measuring vital signs, visual mouth inspection, and collection of subject-reported adverse events (AEs). RESULTS: The percentage of subjects with carbon monoxide-verified continuous abstinence from week 2 to week 6 was statistically significantly greater in the NMS group compared with the placebo group (5.0% vs. 2.5%, p = .021). Statistically significant treatment effects for the NMS were maintained throughout the 26-week period. The study medications were generally well tolerated. The severity of AEs was similar for both treatment groups, and most AEs were of mild or moderate severity. CONCLUSIONS: These study results demonstrate that the NMS is an effective and safe smoking cessation option for smokers motivated to quit, even in a naturalistic setting and without behavioral support. IMPLICATIONS: This study demonstrated the safety, efficacy, and acceptability of an NMS in an OTC environment with no behavioral counseling or support. It provides an additional option for smokers motivated to quit. TRIAL REGISTRATION: ClinicalTrials.gov (number NCT02355665).

Life Starts at Home: Bathing, Exertion and Participation for People With Mobility Impairment.

OBJECTIVE: To investigate the relationship between perceived exertion while bathing/dressing/grooming and associations with social-recreational activities outside the home for individuals with mobility impairment (MI). DESIGN: A 2-study approach was used to examine data from the American Time Use Survey (ATUS) and primary data from the Health and Home Survey (HHS). The relationship between bathing/dressing/grooming and engagement in social-recreational activities was explored, as well as the role that exertion in the bathroom may play in participation in these activities. SETTING: General community setting. PARTICIPANTS: For the ATUS survey, participants (n=6002) included individuals who reported an MI. For the HHS, 2 mail-based recruitment methods were used to recruit a sample of individuals with MI (n=170) across 3 geographically diverse U.S. communities. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Participation in social and recreational activities. RESULTS: People with MI (relative to those without MI) were less likely to report spending any time bathing/dressing/grooming on a given day, but spent more time when they did. People with MI reported higher exertion while bathing/dressing/grooming than people without. People with MI were less likely to leave the house or engage in social-recreational activities on days where they did not engage in bathing activities. People who reported greater exertion in the bathroom engaged in fewer social-recreational activities. CONCLUSIONS: Exertion in the bathroom may present a barrier to participation, indicating a relationship between exertion in the bathroom and social-recreational participation. Research that examines the impact of home modifications on exertion and participation is needed.

Disability Items From the Current Population Survey (2008-2015) and Permanent Versus Temporary Disability Status.

OBJECTIVES: To examine longitudinal responses to the disability indicator questions that have been adopted as the standard across national surveys sponsored by the US Department of Health and Human Services. METHODS: Data from the Current Population Survey between 2008 and 2015 were linked to create a longitudinal sample of 721 178 individual respondents. RESULTS: Responses to the disability questions fluctuated significantly. Although 17% of all respondents reported a disability at some point, only 3% consistently reported the same set of disabilities. Demographic differences were found between people who always reported a consistent set of disabilities and those whose responses fluctuated. CONCLUSIONS: The disability questions capture 2 discrete groups: people who experience a permanent disability and those who experience a temporary disability. Demographic differences between these groups suggest that this is not simply due to measurement error.

Clinical Case of the Month: A 57-Year-Old Man with an Axillary Mass.

A 57-year-old man presented to the surgical oncology clinic with a mildly tender mass under his right arm. Four years prior, the patient had a melanoma removed from his right shoulder along with an ipsilateral right axillary sentinel lymph sampling. Computed tomography (CT) scan was negative for metastatic disease at that time. The patient did not undergo completion axillary node dissection and was lost to follow-up. The patient was originally from Australia, did not tan but reported multiple sunburns before age 18. He was of Irish ancestry. He denied weight gain, fever, fatigue, anorexia, or night sweats. The patient had a medical history of atrial fibrillation, hypertension, gout, melanoma, and benign prostatic hypertrophy. His surgical history included an appendectomy and a facial laceration repair. His brother died at 16 years old from leukemia and his mother died from colon cancer. He consumed 3 alcoholic beverages per day and denied tobacco or illicit drug use. On physical exam, the patient's temperature was 98.8° Fahrenheit, heart rate of 73 beats / minute, blood pressure of 121 / 59 mm Hg, respiratory rate of 18 / min. He appeared to be healthy and in no apparent distress. Cardiovascular, respiratory, breast, gastrointestinal, musculoskeletal, and neurological exam were unremarkable. His right axillary lymph node exam revealed a firm mass roughly 2.5 cm tall by 1.5 cm wide. This mass was biopsied and findings were consistent with metastatic melanoma. CT scan revealed small volume mediastinal adenopathy and a 4.5 cm right axillary mass. There was a 4.7 cm lesion within the anterior left lower lobe of the liver and periportal node conglomerate measuring 3.9 cm consistent with metastatic disease (Figure 1). He was negative for the BRAF V600E mutation. The patient was consented for treatment with combination immune checkpoint inhibition with ipilimumab and nivolumab. After two cycles the patient showed good response, but temporarily stopped treatment after complications related to a ST segment elevation myocardial infarction. He developed mild pneumonitis felt to be related to nivolumab, and recovered after a short course of glucocorticosteroids. Restaging CT scans were ordered after two cycles of therapy (Figure 2), which showed decrease in the size of the axillary and hepatic metastases. At six months, CT scans showed continued durable response (Figure 3).

Synthesis of D-Desosamine and Analogs by Rapid Assembly of 3-Amino Sugars.

D-Desosamine is synthesized in 4 steps from methyl vinyl ketone and sodium nitrite. The key step in this chromatography-free synthesis is the coupling of (R)-4-nitro-2-butanol and glyoxal (trimeric form) mediated by cesium carbonate, which affords in crystalline form 3-nitro-3,4,6-trideoxy-α-D-glucose, a nitro sugar stereochemically homologous to D-desosamine. This strategy has enabled the syntheses of an array of analogous 3-nitro sugars. In each case the 3-nitro sugars are obtained in pure form by crystallization.