Metabolic processes required for bacterial pathogens to adapt in the host are potential targets for the development of much-needed new antimicrobial agents. The bacterial central metabolic enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is required for the synthesis of essential isoprenoids, thiamin diphosphate (ThDP), and pyridoxal phosphate (PLP) and is believed to function in bacterial adaptations requiring these metabolites. DXPS inhibition impairs a PLP-dependent adaptation of Uropathogenic Escherichia coli (UPEC) to d-serine, a bacteriostatic host metabolite in urine that inhibits pantothenate production in Coenzyme A (CoA) biosynthesis. CoA is required for a functioning tricarboxylic acid (TCA cycle, which is critical for UPEC survival in the urinary tract. Accordingly, inhibition of DXPS also sensitizes UPEC to N-pentylpantothenamide (N5-Pan, 1), an inhibitor of CoA synthesis. However, 1 is enzymatically hydrolyzed by pantetheinases, presenting challenges for its use in in vivo studies. We sought to identify a pantothenamide inhibitor candidate for studies to explore the in vivo efficacy of inhibitor combinations targeting DXPS and CoA syntheses. Here, we describe studies that highlight a truncated analog of 1, pantetheinase-resistant N5-α-Pan (6), as a promising candidate for in vivo studies. Analog 6 exhibited comparable antimicrobial activity with 1 against UPEC grown in nutrient-limited culture conditions, including urine, and displayed enhanced activity in the presence of a DXPS inhibitor. In contrast to 1, analog 6 was stable in the presence of mouse plasma and liver enzymes, making it suitable for studies to investigate an inhibitor combination targeting DXPS and CoA synthesis in UPEC by the in vivo ascending UTI assay.IMPORTANCENew approaches are needed to control the emergence of drug resistance in bacterial pathogens that cause life-threatening infections. Targeting DXPS-dependent synthesis of vitamins is a promising approach to prevent pathogen metabolic adaptation. Metabolic processes requiring DXPS-dependent synthesis of pyridoxal phosphate (PLP) or thiamin diphosphate (ThDP) can become particularly vulnerable in a specific pathogen and/or host environment, under conditions of DXPS inhibition. We previously observed that UPEC grown in urine is particularly sensitive to an inhibitor combination targeting DXPS and CoA synthesis; however, the CoA inhibitor is readily hydrolyzed by a host pantetheinase. This study is significant, as it identifies a pantothenamide inhibitor of CoA synthesis, N5-α-Pan (6), that is stable to mouse plasma and liver enzymes and whose activity is enhanced in the presence of a DXPS inhibitor. Thus, 6 is suitable for studies to explore how a pathogen can become sensitized in vivo under conditions of DXPS inhibition.

Synaptic plasticity in the central nervous system enables the encoding, storing, and integrating new information. AMPA (alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionic acid)-type glutamate receptors (AMPARs) are ligand-gated ion channels that mediate most fast excitatory synaptic transmission in the brain, and plasticity of AMPARs signaling underlies the long-lasting changes in synaptic efficacy and strength important for learning and memory. Recent work has indicated that the enigmatic N-terminal domain (NTD) of AMPARs may be a critical regulator of synaptic targeting and plasticity of AMPARs. However, few synaptic proteins have been identified that regulate AMPAR plasticity through interactions with AMPAR NTDs. Moreover, the scope of AMPAR NTD interactors that are important for synaptic plasticity remains unknown. Here, we present the dynamic, extracellular interactome for AMPARs during synaptic plasticity. Using surface-restricted proximity labeling and BioSITe-based proteomics, we identified 70 proteins that were differentially labeled by APEX2-tagged AMPARs after induction of chemical long-term potentiation of synapses in cultured neurons. Included in this list, were four members of the IgLON family of GPI-anchored proteins (Ntm, OBCAM/Opcml, Negr1, Lsamp). We show OBCAM and NTM directly interact with the extracellular domains of AMPARs. Moreover, overexpression of NTM significantly attenuates the mobility of surface AMPARs in dendritic spines. These data represent a significant step at uncovering the unexplored extracellular regulation of AMPARs, with broad implications for synapse function and synaptic plasticity.

The atomic-level detail provided by nuclear magnetic resonance (NMR) allows for the monitoring of biochemical reactions as substrates and intermediates are converted by enzymes. NMR signals are sensitive to chemical environments and this feature allows for scrutiny of the integrity of reactions. Routinely, time-consuming diversions are necessary with traditional approaches to monitor biochemical transformations as many require additional sample manipulations or their readouts are limited. Here, we highlight the advantages of NMR to both monitor and rescue a complex chemoenzymatic reaction that modifies an isotopically labeled nonribosomal peptide synthetase (NRPS) carrier protein with an unlabeled phosphopantetheine moiety through an enzymatic cascade. We present protocols to monitor this reaction that implement various 1D NMR experiments tailored to the detection of specific steps in the enzymatic cascade and can be easily applied even to reactions using unlabeled domains. Additionally, to highlight the signals of the unlabeled modification once attached to the labeled biomolecule, we present a modified diffusion and isotope-filtered pulse sequence. Importantly, when chemoenzymatic reactions are slow or are stalled, the technique provides an atomic-level readout of reaction byproducts and limiting or damaged reagents, and thus allows for quick interventions to rescue them. The advantages of using NMR highlighted in this study more generally serve as a dogma to monitor and restore complex chemoenzymatic reactions.

Supplemental Methods and Figure Legends

Supplementary Table Legends 1-5 from Inhibition of the Acetyltransferases p300 and CBP Reveals a Targetable Function for p300 in the Survival and Invasion Pathways of Prostate Cancer Cell Lines

Supplementary Figure 2 from Inhibition of the Acetyltransferases p300 and CBP Reveals a Targetable Function for p300 in the Survival and Invasion Pathways of Prostate Cancer Cell Lines

Long-acting injectable (LAI) formulations promise to deliver patient benefits by overcoming issues associated with non-adherence. A preclinical assessment of semi-solid prodrug nanoparticle (SSPN) LAI formulations of emtricitabine (FTC) is reported here. Pharmacokinetics over 28 days were assessed in Wistar rats, New Zealand white rabbits, and Balb/C mice following intramuscular injection. Two lead formulations were assessed for the prevention of an HIV infection in NSG-cmah(-/-) humanised mice to ensure antiviral activities were as anticipated according to the pharmacokinetics. Cmax was reached by 12, 48, and 24 h in rats, rabbits, and mice, respectively. Plasma concentrations were below the limit of detection (2 ng/mL) by 21 days in rats and rabbits, and 28 days in mice. Mice treated with SSPN formulations demonstrated undetectable viral loads (700 copies/mL detection limit), and HIV RNA remained undetectable 28 days post-infection in plasma, spleen, lung, and liver. The in vivo data presented here demonstrate that the combined prodrug/SSPN approach can provide a dramatically extended pharmacokinetic half-life across multiple preclinical species. Species differences in renal clearance of FTC mean that longer exposures are likely to be achievable in humans than in preclinical models.

Hepatocellular carcinoma (HCC) is a major cause of cancer mortality worldwide and available therapies, including immunotherapies, are ineffective for many patients. HCC is characterized by intratumoral hypoxia, and increased expression of hypoxia-inducible factor 1α (HIF-1α) in diagnostic biopsies is associated with patient mortality. Here we report the development of 32-134D, a low-molecular-weight compound that effectively inhibits gene expression mediated by HIF-1 and HIF-2 in HCC cells, and blocks human and mouse HCC tumor growth. In immunocompetent mice bearing Hepa1-6 HCC tumors, addition of 32-134D to anti-PD1 therapy increased the rate of tumor eradication from 25% to 67%. Treated mice showed no changes in appearance, behavior, body weight, hemoglobin, or hematocrit. Compound 32-134D altered the expression of a large battery of genes encoding proteins that mediate angiogenesis, glycolytic metabolism, and responses to innate and adaptive immunity. This altered gene expression led to significant changes in the tumor immune microenvironment, including a decreased percentage of tumor-associated macrophages and myeloid-derived suppressor cells, which mediate immune evasion, and an increased percentage of CD8+ T cells and natural killer cells, which mediate antitumor immunity. Taken together, these preclinical findings suggest that combining 32-134D with immune checkpoint blockade may represent a breakthrough therapy for HCC.

Immunization efforts for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been successful in reducing disease severity. Yet new variants continue to emerge, as well as vaccine hesitancy from some, requiring ongoing efforts to identify antiviral agents for SARS-CoV-2 treatment. Here, we evaluated the in vitro activities of piperidine-4-carboxamide compound (NCGC2955) against human α- coronavirus NL63, β-coronaviruses OC43, and the alpha and delta variants of SARS-CoV-2 in several cell lines. NCGC2955 showed antiviral activity in NL63-infected Vero and MK2 cells: EC 50 2.5± 0.15 µM and 1.5 ± 0.2 µM, respectively. The cellular toxicity in both cell types was > 300 µM. The EC 50 of NCGC2955 in OC43-infected human foreskin fibroblasts was 1.5 ± 0.01 µM, and a dose-response in reducing OC43 antigen was observed in Western blot analysis. NCGC2955 inhibited SARS-CoV-2 in both Vero E6 and Calu-3 cells. A comparison of the activity of NCGC2955 and a structurally related analog (153) in SARS-CoV-2-infected Calu-3 cells revealed similar EC 50 (0.2 ± 0.02 µM and 0.11 ± 0.04 µM, respectively). Both compounds inhibited the delta variant in Calu-3 cells. This class of agents may be promising broad-spectrum antivirals that can be further developed for clinical use.

Treatment options for human cytomegalovirus (CMV) remain limited and are associated with significant adverse effects and the selection of resistant CMV strains in transplant recipients and congenitally infected infants. Although most approved drugs target and inhibit the CMV DNA polymerase, additional agents with distinct mechanisms of action are needed for the treatment and prevention of CMV. In a large high throughput screen using our CMV-luciferase reporter Towne, we identified several unique inhibitors of CMV replication. Here, we synthesize and test in vitro 13 analogs of the original NCGC2955 hit (1). Analogs with no activity against the CMV-luciferase at 10 µM and 30 µM (2–6, 10–14) were removed from further analysis. Three analogs (7–9) inhibited CMV replication in infected human foreskin fibroblasts. The EC50 of (1) was 1.7 ± 0.6 µM and 1.99 ± 0.15 µM, based on luciferase and plaque assay, respectively. Compounds 7, 8, and 9 showed similar activities: the EC50 values of 7 were 0.21 ± 0.06 µM (luciferase) and 0.55 ± 0.06 (plaque), of 8: 0.28 ± 0.06 µM and 0.42 ± 0.07, and of 9: 0.30 ± 0.05 µM (luciferase) and 0.35 ± 0.07 (plaque). The CC50 for 7, 8, and 9 in non-infected human foreskin fibroblasts was > 500µM, yielding a selectivity index of >1500. Compounds 1, 7, and 8 were also tested in CMV-infected primary human hepatocytes and showed a dose–response against CMV by luciferase activity and viral protein expression. None of the active compounds inhibited herpes simplex virus 1 or 2. Compounds 7 and 8 inhibited mouse CMV replication in vitro. Both inhibited CMV at late stages of replication; 7 reduced virus yield at all late time points, although not to the same degree as letermovir. Finally, the activity of analog 8 was additive with newly identified CMV inhibitors (MLS8969, NFU1827, MSL8554, and MSL8091) and with ganciclovir. Further structural activity development should provide promising anti-CMV agents for use in clinical studies.

The non-invasive nature of NMR offers a means to monitor biochemical reactions in situ at the atomic level. We harness this advantage to monitor a complex chemoenzymatic reaction that sequentially modifies reagents and loads the product on a nonribosomal peptide synthetase carrier protein. We present a protocol including a pulse sequence that permits to assess both the integrity of reagents and the completion of each step in the reaction, thus alleviating otherwise time-consuming and costly approaches to debug and repeat inefficient reactions. This study highlights the importance of NMR as a tool to establish reliable and reproducible experimental conditions in biochemical studies.

The critical consequences of human cytomegalovirus (HCMV) infection in the transplant population and in congenitally infected infants, the limited treatment options for HCMV, and the rise of resistant mutants toward existing therapies has fueled the search for new anti-HCMV agents. A pp28-luciferase recombinant HCMV was used as a reporter system for high-throughput screening of HCMV inhibitors. Approximately 400 000 compounds from existing libraries were screened. Subsequent validation assays using resynthesized compounds, several virus strains, and detailed virology assays resulted in the identification of five structurally unique and selective HCMV inhibitors, active at sub to low micromolar concentrations. Further characterization revealed that each compound inhibited a specific stage of HCMV replication. One compound was also active against herpes simplex virus (HSV1 and HSV2), and another compound was active against Epstein-Barr virus (EBV). Drug combination studies revealed that all five compounds were additive with ganciclovir or letermovir. Future studies will focus on optimization of these new anti-HCMV compounds along with mechanistic studies.

The use of biotin or biotin-containing reagents is an essential component of many protein purification and labeling technologies. Owing to its small size and high affinity to the avidin family of proteins, biotin is a versatile molecular handle that permits both enrichment and purity that is not easily achieved by other reagents. Traditionally, the use of biotinylation to enrich for proteins has not required the detection of the site of biotinylation. However, newer technologies for discovery of protein-protein interactions, such as APEX and BioID, as well as some of the click chemistry-based labeling approaches have underscored the importance of determining the exact residue that is modified by biotin. Anti-biotin antibody-based enrichment of biotinylated peptides (e.g., BioSITe) coupled to LC-MS/MS permit large-scale detection and localization of sites of biotinylation. As with any chemical modification of peptides, understanding the fragmentation patterns that result from biotin modification is essential to improving its detection by LC-MS/MS. Tandem mass spectra of biotinylated peptides has not yet been studied systematically. Here, we describe the various signature fragment ions generated with collision-induced dissociation of biotinylated peptides. We focused on biotin adducts attached to peptides generated by BioID and APEX experiments, including biotin, isotopically heavy biotin, and biotin-XX-phenol, a nonpermeable variant of biotin-phenol. We also highlight how the detection of biotinylated peptides in high-throughput studies poses certain computational challenges for accurate quantitation which need to be addressed. Our findings about signature fragment ions of biotinylated peptides should be helpful in the confirmation of biotinylation sites.

Abstract Histone modifications, largely regulated by histone acetyltransferases (HAT) and histone deacetylases, have been recognized as major regulatory mechanisms governing human diseases, including cancer. Despite significant effort and recent advances, the mechanism by which the HAT and transcriptional coactivator p300 mediates tumorigenesis remains unclear. Here, we use a genetic and chemical approach to identify the microphthalmia-associated transcription factor (MITF) as a critical downstream target of p300 driving human melanoma growth. Direct transcriptional control of MITF by p300-dependent histone acetylation within proximal gene regulatory regions was coupled to cellular proliferation, suggesting a significant growth regulatory axis. Further analysis revealed forkhead box M1 (FOXM1) as a key effector of the p300–MITF axis driving cell growth that is selectively activated in human melanomas. Targeted chemical inhibition of p300 acetyltransferase activity using a potent and selective catalytic p300/CBP inhibitor demonstrated significant growth inhibitory effects in melanoma cells expressing high levels of MITF. Collectively, these data confirm the critical role of the p300–MITF–FOXM1 axis in melanoma and support p300 as a promising novel epigenetic therapeutic target in human melanoma. Significance: These results show that MITF is a major downstream target of p300 in human melanoma whose expression is predictive of melanoma response to small-molecule inhibition of p300 HAT activity.

OBJECTIVES:Efficacy is determined not only by size, but also by shape, of drug exposure. Here the critical importance of the temporal pattern of drug concentrations (pharmacokinetic profile) is examined for antitrypanosomals in vitro.METHODS:An in vitro hollow-fibre cartridge system was used to study contrasting drug profiles with four clinically used agents and two experimental candidates against the deadly parasite Trypanosoma brucei. Artificial kinetics were employed intentionally to favour either high peak concentration or sustained duration of drug.RESULTS:Changing the shape of drug exposure significantly impacted drug efficacy. Suramin, melarsoprol and pentamidine were concentration-driven and therefore more efficacious when applied as short-lived high peaks. In contrast, difluoromethylornithine (DFMO) was time-driven, and therefore maximally effective as a constant infusion. Kinetic preference was robust over a wide range of drug exposures. Promising clinical candidates SCYX-7158 (acoziborole) and fexinidazole (parent and sulfone) were concentration-driven, suggesting optimal clinical regimens would involve relatively high but intermittent dosing.CONCLUSIONS:Antitrypanosomals have an intrinsic pharmacokinetic driver for optimal efficacy, with important implications for clinical management and future candidate development.

The increasing global prevalence of human immunodeficiency virus (HIV) is estimated at 36.7 million people currently infected. Lifelong antiretroviral (ARV) drug combination dosing allows management as a chronic condition by suppressing circulating viral load to allow for a near-normal life; however, the daily burden of oral administration may lead to non-adherence and drug resistance development. Long-acting (LA) depot injections of nanomilled poorly water-soluble ARVs have shown highly promising clinical results with drug exposure largely maintained over months after a single injection. ARV oral combinations rely on water-soluble backbone drugs which are not compatible with nanomilling. Here, we evaluate a unique prodrug/nanoparticle formation strategy to facilitate semi-solid prodrug nanoparticles (SSPNs) of the highly water-soluble nucleoside reverse transcriptase inhibitor (NRTI) emtricitabine (FTC), and injectable aqueous nanodispersions; in vitro to in vivo extrapolation (IVIVE) modelling predicts sustained prodrug release, with activation in relevant biological environments, representing a first step towards complete injectable LA regimens containing NRTIs.

Non-ribosomal peptides are a class of microbial secondary metabolites synthesized by non-ribosomal peptide synthetases (NRPSs) often with pharmaceutical and medicinal properties. There is an emerging demand to engineer NRPSs to develop novel pharmaceuticals but gaps in understanding molecular mechanisms hamper progress. During synthesis, substrates are attached to 20 Å phosphopantetheinyl moieties of carrier protein (CP) domains via thioester bonds. CPs then visit partner domains in a series of sequential transient interactions. Substrates were seen to dock with CP protein cores in a manner that may affect domain interactions. Further, such docking events are yet to be observed for the canonical NRPS substrates, amino acids. However, substrates often rapidly fall off CPs due to hydrolysis of the thioester bond, thus challenging structural studies. Here, we employ a chemo-enzymatic route to load a cysteine onto a carrier protein through an inert amide bond. This approach overcomes disulfide bond formation, transthioesterification and disulfide-thiol exchange inherent to the thiol functional group of cysteines. Using NMR spectroscopy, we identified weak but well-defined chemical shift perturbations indicating a transient interaction between the substrate and the protein core. To describe this interaction at the molecular level, we developed a time-shared filtered NOESY NMR experiment to measure distances between substrates and CP cores. Our results will help understand whether and how different substrates interact with CPs as they are presented to partner domains, thus guiding NRPS engineering to produce novel metabolites.

p300 and CBP are highly related histone acetyltransferase (HAT) enzymes that regulate gene expression, and their dysregulation has been linked to cancer and other diseases. p300/CBP is composed of a number of domains including a HAT domain, which is inhibited by the small molecule A-485, and an acetyl-lysine binding bromodomain, which was recently found to be selectively antagonized by the small molecule I-CBP112. Here we show that the combination of I-CBP112 and A-485 can synergize to inhibit prostate cancer cell proliferation. We find that the combination confers a dramatic reduction in p300 chromatin occupancy compared to the individual effects of blocking either domain alone. Accompanying this loss of p300 on chromatin, combination treatment leads to the reduction of specific mRNAs including androgen-dependent and pro-oncogenic prostate genes such as KLK3 (PSA) and c-Myc. Consistent with p300 directly affecting gene expression, mRNAs that are significantly reduced by combination treatment also exhibit a strong reduction in p300 chromatin occupancy at their gene promoters. The relatively few mRNAs that are up-regulated upon combination treatment show no correlation with p300 occupancy. These studies provide support for the pharmacologic advantage of concurrent targeting of two domains within one key epigenetic modification enzyme.

Similarities in fungal and animal cells make antifungal discovery efforts more difficult than those for other classes of antimicrobial drugs. Currently, there are only three major classes of antifungal drugs used for the treatment of systemic fungal diseases: polyenes, azoles, and echinocandins. Even in situations where the offending fungal organism is susceptible to the available drugs, treatment courses can be lengthy and unsatisfactory, since eradication of infection is often very difficult, especially in individuals with impaired immunity. Consequently, there is a need for new and more effective antifungal drugs. We have identified compounds with significant antifungal activity in the Malaria Box (Medicines for Malaria Ventures, Geneva, Switzerland) that have higher efficacy than some of the currently used antifungal drugs. Our best candidate, MMV665943 (IUPAC name 4-[6-[[2-(4-aminophenyl)-3H-benzimidazol-5-yl]methyl]-1H-benzimidazol-2-yl]aniline), here referred to as DM262, showed 16- to 32-fold-higher activity than fluconazole against Cryptococcus neoformans. There was also significant antifungal activity in other fungal species with known antifungal resistance, such as Lomentospora prolificans and Cryptococcus gattii. Antifungal activity was also observed against a common fungus, Candida albicans. These results are important because they offer a potentially new class of antifungal drugs and the repurposing of currently available therapeutics. IMPORTANCE Much like the recent increase in drug-resistant bacteria, there is a rise in antifungal-resistant strains of pathogenic fungi. There is a need for novel and more potent antifungal therapeutics. Consequently, we investigated a mixed library of drug-like and probe-like compounds with activity in Plasmodium spp. for activity against two common fungal pathogens, Cryptococcus neoformans and Candida albicans, along with two less common pathogenic species, Lomentospora prolificans and Cryptococcus gattii. We uncover a previously uncharacterized drug with higher broad-spectrum antifungal activity than some current treatments. Our findings may eventually lead to a compound added to the arsenal of antifungal therapeutics.

Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is unclear. Here, we identify a novel activity of the histone acetyltransferase p300/CREB-binding protein (CBP) in regulating promoter-proximal paused Pol II. We find that Drosophila CBP inhibition results in "dribbling'' of Pol II from the pause site to positions further downstream but impedes transcription through the + 1 nucleosome genome-wide. Promoters strongly occupied by CBP and GAGA factor have high levels of paused Pol II, a unique chromatin signature, and are highly expressed regardless of cell type. Interestingly, CBP activity is rate limiting for Pol II recruitment to these highly paused promoters through an interaction with TFIIB but for transit into elongation by histone acetylation at other genes. Thus, CBP directly stimulates both Pol II recruitment and the ability to traverse the first nucleosome, thereby promoting transcription of most genes.