New Insights into the Mechanism of Action of L-681,217, a Medicinally Promising Polyketide Inhibitor of Bacterial Protein Translation.

An attractive strategy for combating antibacterial resistance involves the development of new antibiotics whose mechanisms differ from those of existing ones in the clinic. Elfamycin antibiotics, whose prototypes include kirromycin and aurodox, are illustrative examples based on their ability to target EF-Tu, an essential component for protein translation in bacteria. Our efforts to revisit this antibiotic class were enabled by two developments.

Structural basis for intermodular communication in assembly-line polyketide biosynthesis.

Assembly-line polyketide synthases (PKSs) are modular multi-enzyme systems with considerable potential for genetic reprogramming. Understanding how they selectively transport biosynthetic intermediates along a defined sequence of active sites could be harnessed to rationally alter PKS product structures. To investigate functional interactions between PKS catalytic and substrate acyl carrier protein (ACP) domains, we employed a bifunctional reagent to crosslink transient domain-domain interfaces of a prototypical assembly line, the 6-deoxyerythronolide B synthase, and resolved their structures by single-particle cryogenic electron microscopy (cryo-EM).

Structural and mechanistic analysis of Ca-dependent regulation of transglutaminase 2 activity using a Ca-bound intermediate state.

Mammalian transglutaminases, a family of Ca-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates.

Past, Present, and Future of Noninvasive Tests to Assess Gluten Exposure, Celiac Disease Activity, and End-Organ Damage.

Although many biomarkers have been proposed, and several are in widespread clinical use, there is no single readout or combination of readouts that correlates tightly with gluten exposure, disease activity, or end-organ damage in treated patients with celiac disease. Challenges to developing and evaluating better biomarkers include significant interindividual variability-related to immune amplification of gluten exposure and how effects of immune activation are manifest. Furthermore, the current "gold standard" for assessment of end-organ damage, small intestinal biopsy, is itself highly imperfect, such that a marker that is a better reflection of the "ground truth" may indeed appear to perform poorly.

Structure and Mechanisms of Assembly-Line Polyketide Synthases.

Three decades of studies on the multifunctional 6-deoxyerythronolide B synthase have laid a foundation for understanding the chemistry and evolution of polyketide antibiotic biosynthesis by a large family of versatile enzymatic assembly lines. Recent progress in applying chemical and structural biology tools to this prototypical assembly-line polyketide synthase (PKS) and related systems has highlighted several features of their catalytic cycles and associated protein dynamics. There is compelling evidence that multiple mechanisms have evolved in this enzyme family to channel growing polyketide chains along uniquely defined sequences of 10-100 active sites, each of which is used only once in the overall catalytic cycle of an assembly-line PKS.

Celiac disease: mechanisms and emerging therapeutics.

Celiac disease (CeD) is a widespread, gluten-induced, autoimmune disorder that lacks any medicinal therapy. Towards the goal of developing non-dietary treatments for CeD, research has focused on elucidating its molecular and cellular etiology. A model of pathogenesis has emerged centered on interactions between three molecular families: specific class II MHC proteins on antigen-presenting cells (APCs), deamidated gluten-derived peptides, and T cell receptors (TCRs) on inflammatory CD4 T cells.

Targeted Lysosomal Degradation of Secreted and Cell Surface Proteins through the LRP-1 Pathway.

Protein dysregulation has been characterized as the cause of pathogenesis in many different diseases. For proteins lacking easily druggable pockets or catalytically active sites, targeted protein degradation is an attractive therapeutic approach. While several methods for targeted protein degradation have been developed, there remains a demand for lower molecular weight molecules that promote efficient degradation of their targets.

Genomic mining and diversity of assembly line polyketide synthases.

Assembly line polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for synthesizing many medicinally relevant natural products with remarkable structural variety and biological activity. The decrease in cost of genomic sequencing paired with development of computational tools like antiSMASH presents an opportunity to survey the vast diversity of assembly line PKS. Mining the genomic data in the National Center for Biotechnology Information database, our updated catalogue (https://orphanpkscatalog2022.

Discovery and Characterization of Antibody Probes of Module 2 of the 6-Deoxyerythronolide B Synthase.

Fragment antigen-binding domains of antibodies (Fs) are powerful probes of structure-function relationships of assembly line polyketide synthases (PKSs). We report the discovery and characterization of Fs interrogating the structure and function of the ketosynthase-acyltransferase (KS-AT) core of Module 2 of the 6-deoxyerythronolide B synthase (DEBS). Two Fs (AC2 and BB1) were identified to potently inhibit the catalytic activity of Module 2.

Evaluation of Acebilustat, a Selective Inhibitor of Leukotriene B4 Biosynthesis, for Treatment of Outpatients With Mild-Moderate Coronavirus Disease 2019: A Randomized, Double-Blind, Placebo-Controlled Phase 2 Trial.

Background: The vast majority of coronavirus disease 2019 (COVID-19) disease occurs in outpatients where treatment is limited to antivirals for high-risk subgroups. Acebilustat, a leukotriene B4 inhibitor, has potential to reduce inflammation and symptom duration.

Methods: In a single-center trial spanning Delta and Omicron variants, outpatients were randomized to 100 mg/d of oral acebilustat or placebo for 28 days.

Challenges in Harnessing Shared Within-Host Severe Acute Respiratory Syndrome Coronavirus 2 Variation for Transmission Inference.

Background: The limited variation observed among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consensus sequences makes it difficult to reconstruct transmission linkages in outbreak settings. Previous studies have recovered variation within individual SARS-CoV-2 infections but have not yet measured the informativeness of within-host variation for transmission inference.

Methods: We performed tiled amplicon sequencing on 307 SARS-CoV-2 samples, including 130 samples from 32 individuals in 14 households and 47 longitudinally sampled individuals, from 4 prospective studies with household membership data, a proxy for transmission linkage.

LRP-1 links post-translational modifications to efficient presentation of celiac disease-specific T cell antigens.

Celiac disease (CeD) is an autoimmune disorder in which gluten-derived antigens trigger inflammation. Antigenic peptides must undergo site-specific deamidation to be presentable to CD4 T cells in an HLA-DQ2 or -DQ8 restricted manner. While the biochemical basis for this post-translational modification is understood, its localization in the patient's intestine remains unknown.

Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells.

In eukaryotes, carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for β-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for posttranslational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood.

Early immune markers of clinical, virological, and immunological outcomes in patients with COVID-19: a multi-omics study.

Background: The great majority of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections are mild and uncomplicated, but some individuals with initially mild COVID-19 progressively develop more severe symptoms. Furthermore, there is substantial heterogeneity in SARS-CoV-2-specific memory immune responses following infection. There remains a critical need to identify host immune biomarkers predictive of clinical and immunological outcomes in SARS-CoV-2-infected patients.

Structure-Based Prototyping of Allosteric Inhibitors of Human Uridine/Cytidine Kinase 2 (UCK2).

Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2.

A Mouse Model of Celiac Disease.

The design and use of mouse models that reproduce key features of human diseases are critical to advance our understanding of the pathogenesis of autoimmune diseases and to test new therapeutic strategies. Celiac disease is a unique organ-specific autoimmune-like disorder occurring in genetically susceptible individuals carrying HLA-DQ2 or HLA-DQ8 molecules who consume gluten. The key histological characteristic of the disease in humans is the destruction of the lining of the small intestine, a feature that has been difficult to reproduce in immunocompetent animal models.

In vivo visualization and molecular targeting of the cardiac conduction system.

Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against the CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects on CCS function.

Latiglutenase Protects the Mucosa and Attenuates Symptom Severity in Patients With Celiac Disease Exposed to a Gluten Challenge.

Background & Aims: Gluten ingestion in patients with celiac disease can lead to gastrointestinal symptoms and small intestinal mucosal injury.

Methods: This gluten challenge phase 2 trial was double blind and placebo controlled, and it assessed the efficacy and safety of a 1200-mg dose of IMGX003 in patients with celiac disease exposed to 2 g of gluten per day for 6 weeks. The change in the ratio of villus height to crypt depth was the primary endpoint.

Engineering site-selective incorporation of fluorine into polyketides.

Although natural products and synthetic small molecules both serve important medicinal functions, their structures and chemical properties are relatively distinct. To expand the molecular diversity available for drug discovery, one strategy is to blend the effective attributes of synthetic and natural molecules. A key feature found in synthetic compounds that is rare in nature is the use of fluorine to tune drug behavior.

Favipiravir for Treatment of Outpatients With Asymptomatic or Uncomplicated Coronavirus Disease 2019: A Double-Blind, Randomized, Placebo-Controlled, Phase 2 Trial.

Background: Favipiravir, an oral, RNA-dependent RNA polymerase inhibitor, has in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite limited data, favipiravir is administered to patients with coronavirus disease 2019 (COVID-19) in several countries.

Methods: We conducted a phase 2, double-blind, randomized controlled outpatient trial of favipiravir in asymptomatic or mildly symptomatic adults with a positive SARS-CoV-2 reverse-transcription polymerase chain reaction assay (RT-PCR) within 72 hours of enrollment.

KIRCD8 T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19.

In this work, we find that CD8 T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49CD8 regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8 T cells efficiently eliminated pathogenic gliadin-specific CD4 T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIRCD8 T cells, but not CD4 regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis.

An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease.

Celiac disease (CeD) is an autoimmune disorder induced by consuming gluten proteins from wheat, barley, and rye. Glutens resist gastrointestinal proteolysis, resulting in peptides that elicit inflammation in patients with CeD. Despite well-established connections between glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner.