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PURPOSE: Many countries recommend human papillomavirus (HPV) vaccination in female adolescents at an age when other vaccines are routinely administered. This open, randomized, multicenter study (108464/NCT00426361) evaluated coadministration of HPV-16/18 AS04-adjuvanted vaccine with diphtheria-tetanus-acellular pertussis-inactivated poliovirus vaccine (dTpa-IPV). METHODS: Healthy females aged 10-18 years were randomized to receive HPV vaccine at months 0, 1, and 6 (n = 248), HPV vaccine coadministered with dTpa-IPV at month 0 and HPV vaccine at months 1 and 6 (n = 255), or dTpa-IPV at month 0 followed by HPV vaccine at months 1, 2, and 7 (n = 248). Immunogenicity was evaluated at months 0, 1, and 7 or 8 (depending on group). Vaccine reactogenicity and safety were also assessed. RESULTS: Coadministered dTpa-IPV and HPV vaccine was noninferior to dTpa-IPV alone in terms of seroprotection against diphtheria (99.2% and 100%), tetanus (100% and 100%) and poliovirus types 1, 2, and 3 (> or = 99.6%), and geometric mean antibody concentrations (ELISA Units/mL) for pertussis toxoid (84 vs. 75), filamentous hemagglutinin (612 and 615) and pertactin (426 and 360) at month 1. Coadministered dTpa-IPV and HPV vaccine was noninferior to HPV vaccine alone in terms of seroconversion rates for HPV-16 (99.5% and 100%) and HPV-18 (99.5% and 100%) and geometric mean antibody titers (ELISA Units/mL) for HPV-16 (15,608 and 18,965) and HPV-18 (6,597 and 6,902) at month 7. Coadministration was generally well tolerated. The reactogenicity of dTpa-IPV and the first dose of HPV vaccine was similar. CONCLUSIONS: Results from this study support coadministration of the HPV-16/18 AS04-adjuvanted vaccine with dTpa-IPV vaccine in females aged 10-18 years. [ABSTRACT FROM AUTHOR]

PURPOSE: The human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine (Cervarix) has been shown to be well-tolerated and immunogenic in females aged 10 to 55 years, and up to 100% effective for the prevention of HPV-16/18 infection and associated precancerous cervical lesions in females aged 15 to 25 years. This study is the first to evaluate the immunogenicity and safety of the vaccine in males. METHODS: Healthy males aged 10 to 18 years were randomized (2:1 ratio) to receive HPV-16/18 AS04-adjuvanted vaccine (n = 181) or hepatitis B virus (HBV) control vaccine (n = 89) at 0, 1, and 6 months, and were followed for 7 months. RESULTS: All initially seronegative subjects in the HPV-16/18 group seroconverted for HPV-16 and 18 (ELISA) at month 2. At month 7, all subjects were seropositive, and the HPV-16 and -18 antibody levels were, respectively, four- and twofold higher than at month 2. The anti-HPV-16 and -18 antibody responses for males aged 10 to 18 years and 10 to 14 years, respectively, were higher than those reported for females aged 15 to 25 years and 10 to 14 years, respectively, in a previous study. The reactogenicity profiles of the HPV-16/18 AS04 and HBV vaccines were similar, except that pain and swelling at the injection site were more common in the HPV-16/18 group. However, vaccine-related symptoms did not affect compliance with the three-dose course, which was equally high (97%) in both groups. CONCLUSIONS: The HPV-16/18 AS04-adjuvanted vaccine is immunogenic and well tolerated in boys aged 10 to 18 years. However, further data on the potential public health benefits of vaccination of boys are required before any recommendations can be made. [ABSTRACT FROM AUTHOR]

Context: Viruslike particle human papillomavirus (HPV) vaccines were designed to prevent HPV infection and development of cervical precancers and cancer. Women with oncogenic HPV infections might consider vaccination as therapy.Objective: To determine whether vaccination against HPV types 16 and 18 increases the rate of viral clearance in women already infected with HPV.Design and Setting: Phase 3, masked, community-based randomized trial conducted in 2 provinces of Costa Rica.Participants: A total of 2189 women aged 18 to 25 years who were recruited between June 2004 and December 2005. Participants were positive for HPV DNA at enrollment, had at least 6 months of follow-up, and had follow-up HPV DNA results.Intervention: Participants were randomly assigned to receive 3 doses of a bivalent HPV-16/18 L1 protein viruslike particle AS04 candidate vaccine (n = 1088) or a control hepatitis A vaccine (n = 1101) over 6 months.Main Outcome Measures: Presence of HPV DNA was determined in cervical specimins by a molecular hybridization assay using chemiluminescence with HPV RNA probes and by polymerase chain reaction using SPF10 primers and a line probe assay detection system before vaccination and by polymerase chain reaction after vaccination. We compared rates of type-specific viral clearance using generalized estimating equations methods at the 6-month visit (after 2 doses) and 12-month visit (after 3 doses) in the 2 study groups.Results: There was no evidence of increased viral clearance at 6 or 12 months in the group who received HPV vaccine compared with the control group. Clearance rates for HPV-16/18 infections at 6 months were 33.4% (82/248) in the HPV vaccine group and 31.6% (95/298) in the control group (vaccine efficacy for viral clearance, 2.5%; 95% confidence interval, -9.8% to 13.5%). Human papillomavirus 16/18 clearance rates at 12 months were 48.8% (86/177) in the HPV vaccine group and 49.8% (110/220) in the control group (vaccine efficacy for viral clearance, -2.0%; 95% confidence interval, -24.3% to 16.3%). There was no evidence of a therapeutic effect for other oncogenic or nononcogenic HPV categories, among women receiving all vaccine doses, among women with single infections, or among women stratified by the following entry variables: HPV-16/18 serology, cytologic results, HPV DNA viral load, time since sexual debut, Chlamydia trachomatis or Neisseria gonorrhoeae infection, hormonal contraceptive use, or smoking.Conclusion: In women positive for HPV DNA, HPV-16/18 vaccination does not accelerate clearance of the virus and should not be used to treat prevalent infections.Trial Registration: clinicaltrials.gov Identifier: NCT00128661. [ABSTRACT FROM AUTHOR]

Studies of proteinaceous cysteine protease inhibitors originated with the discovery of cystatins in the 1960s. Since that time, a rich and fascinating world of proteins that control and regulate a multitude of important physiological processes, ranging from the basics of protein turnover to development and brain function, has been uncovered. Failures in such important and complex systems inevitably lead to pathologies. Many threatening diseases such as cancer or neurological disorders, to mention only some, are attributed to deregulation of proteaseinhibitor balance. Moreover, important aspects of infection pathology and host defense rely on proteolysis and protease inhibition. Recent advances in the field of protease inhibitors have drawn attention to the possible use of this collected knowledge to control related pathological processes. This review attempts to familiarize the reader with proteinaceous cysteine protease inhibitors by providing an overview of current knowledge. The work primarily highlights biological processes in which the inhibitors are involved and focuses on pathologies resulting from aberrant protease-inhibitor balance, pointing out emerging possibilities for their correction. [ABSTRACT FROM AUTHOR]

Describes a case of patient in Pennsylvania who experienced acute cholestatic hepatitis on initial exposure to zidovudine. Results of physical and laboratory examinations; Findings from liver function tests; Problems presented by zidovudine therapy.

Peroxisomal protein import has been identified as a valid target in trypanosomiases, an important health threat in Central and South America. The importomer is built of multiple peroxins (Pex) and structural characterization of these proteins facilitates rational inhibitor development. We report crystal structures of the Trypanosoma brucei and T. cruzi tetratricopeptide repeat domain (TPR) of the cytoplasmic peroxisomal targeting signal 1 (PTS1) receptor Pex5. The structure of the TPR domain of TbPex5 represents an apo-form of the receptor which, together with the previously determined structure of the complex of TbPex5 TPR and PTS1 demonstrate significant receptor dynamics associated with signal peptide recognition. The structure of the complex of TPR domain of TcPex5 with PTS1 provided in this study details the molecular interactions that guide signal peptide recognition at the atomic level in the pathogenic species currently perceived as the most relevant among Trypanosoma. Small - angle X - ray scattering (SAXS) data obtained in solution supports the crystallographic findings on the compaction of the TPR domains of TbPex5 and TcPex5 upon interaction with the cargo., Competing Interests: Declaration of competing interest None. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. It bears a significant global health burden with limited treatment options, thus calling for the development of new and effective drugs. Certain trypanosomal metabolic enzymes have been suggested to be druggable and valid for subsequent inhibition. In this study, the crystal structure of glycerol kinase from T. cruzi, a key enzyme in glycerol metabolism in this parasite, is presented. Structural analysis allowed a detailed description of the glycerol binding pocket, while comparative assessment pinpointed a potential regulatory site which may serve as a target for selective inhibition. These findings advance the understanding of glycerol metabolism in eukaryotes and provide a solid basis for the future treatment of Chagas disease.

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The PD-1/PD-L1 complex belongs to the group of inhibitory immune checkpoints and plays a critical role in immune regulation. The PD-1/PD-L1 axis is also responsible for immune evasion of cancer cells, and this complex is one of the main targets of immunotherapies used in oncology. Treatment using immune checkpoint inhibitors is mainly based on antibodies. This approach has great therapeutic potential; however, it also has major drawbacks and can induce immune-related adverse events. Thus, there is a strong need for alternative, non-antibody-based therapies using small molecules, peptides, or peptidomimetics. In the present study, we designed, synthesized, and evaluated a set of PD-1-targeting peptides based on the sequence and structure of PD-L1. The binding of these peptides to PD-1 was investigated using SPR and ELISA. We also assessed their ability to compete with PD-L1 for binding to PD-1 and their inhibitory properties against the PD-1/PD-L1 complex at the cellular level. The best results were obtained for the peptide PD-L1(111-127) (Y112C-I126C) , named (L11), which displaced PD-L1 from binding to PD-1 in the competitive assay and inhibited the formation of the PD-1/PD-L1 complex. The (L11) peptide also exhibited strong affinity for PD-1. NMR studies revealed that (L11) does not form a well-defined secondary structure; however, MD simulation indicated that (L11) binds to PD-1 at the same place as PD-L1. After further optimization of the structure, the peptide inhibitor obtained in this study could also be used as a potential therapeutic compound targeting the PD-1/PD-L1 axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Alphaviruses pose a significant threat to public health. Capsid protein encoded in the alphaviral genomes constitutes an interesting therapy target, as it also serves as a protease (CP). Remarkably, it undergoes autoproteolysis, leading to the generation of the C-terminal tryptophan that localizes to the active pocket, deactivating the enzyme. Lack of activity hampers the viral replication cycle, as the virus is not capable of producing the infectious progeny. We investigated the structure and function of the CP encoded in the genome of O'nyong'nyong virus (ONNV), which has instigated outbreaks in Africa. Our research provides a high-resolution crystal structure of the ONNV CP in its active state and evaluates the enzyme's activity. Furthermore, we demonstrated a dose-dependent reduction in ONNV CP proteolytic activity when exposed to indole, suggesting that tryptophan analogs may be a promising basis for developing small molecule inhibitors. It's noteworthy that the capsid protease plays an essential role in virus assembly, binding viral glycoproteins through its glycoprotein-binding hydrophobic pocket. We showed that non-aromatic cyclic compounds like dioxane disrupt this vital interaction. Our findings provide deeper insights into ONNV's biology, and we believe they will prove instrumental in guiding the development of antiviral strategies against arthritogenic alphaviruses., Competing Interests: Declaration of competing interest Krzysztof Pyrc, Adam Lesner, and Marcin Sienczyk reports financial support was provided by National Science Centre Poland., (Copyright © 2024. Published by Elsevier B.V.)

Here, we report the fragment-based drug discovery of potent and selective fragments that disrupt the Spire2-FMN2 but not the Spire1-FMN2 interaction. Hit fragments were identified in a differential scanning fluorimetry-based screen of an in-house library of 755 compounds and subsequently validated in multiple orthogonal biophysical assays, including fluorescence polarization, microscale thermophoresis, and 1 H- 15 N HSQC nuclear magnetic resonance. Extensive structure-activity relationships combined with molecular docking followed by chemical optimization led to the discovery of compound 13 , which exhibits micromolar potency and high ligand efficiency (LE = 0.38). Therefore, this fragment represents a validated starting point for the future development of selective chemical probes targeting the Spire2-FMN2 interaction.

The selective inhibition of kinases from the diabetic kinome is known to promote the regeneration of beta cells and provide an opportunity for the curative treatment of diabetes. The effect can be achieved by carefully tailoring the selectivity of inhibitor toward a particular kinase, especially DYRK1A, previously associated with Down syndrome and Alzheimer's disease. Recently DYRK1A inhibition has been shown to promote both insulin secretion and beta cells proliferation. Here, we show that commonly available flavones are effective inhibitors of DYRK1A. The observed biochemical activity of flavone compounds is confirmed by crystal structures solved at 2.06 Å and 2.32 Å resolution, deciphering the way inhibitors bind in the ATP-binding pocket of the kinase, which is driven by the arrangement of hydroxyl moieties. We also demonstrate antidiabetic properties of these biomolecules and prove that they could be further improved by therapy combined with TGF-β inhibitors. Our data will allow future structure-based optimization of the presented scaffolds toward potent, bioavailable and selective anti-diabetic drugs., (© 2023. Springer Nature Limited.)

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation, and tissue remodeling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used the systematic evolution of ligands by exponential enrichment to develop ssDNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Staphylococcus aureus is a common opportunistic pathogen of humans and livestock that causes a wide variety of infections. The success of S. aureus as a pathogen depends on the production of an array of virulence factors including cysteine proteases (staphopains)-major secreted proteases of certain strains of the bacterium. Here, we report the three-dimensional structure of staphopain C (ScpA2) of S. aureus , which shows the typical papain-like fold and uncovers a detailed molecular description of the active site. Because the protein is involved in the pathogenesis of a chicken disease, our work provides the foundation for inhibitor design and potential antimicrobial strategies against this pathogen.

Background: We report 2-year persistence of immune response to Takeda's prophylactic purified formalin-inactivated whole Zika virus vaccine candidate (TAK-426) compared with that observed after natural infection., Methods: A randomized, observer-blind, placebo-controlled, dose-selection, phase 1 trial was conducted in 18-49-year-old adults at 9 centers (7 in the United States, 2 in Puerto Rico) from 13 November 2017 to 24 November 2020. Primary objectives were safety, tolerability, and immunogenicity of 3 increasing doses of TAK-426 administered as 2 doses 28 days apart to flavivirus (FV)-naive and FV-primed adults. Here, we report on safety and persistence of immunity up to 2 years after primary vaccination with 10-μg TAK-426, the highest dose, and compare neutralizing antibody responses with those observed after natural infection., Results: TAK-426 at 10-μg had an acceptable safety profile in FV-naive and FV-primed adults up to 24 months after dose 2. Seropositivity for neutralizing antibodies was 100% at 1 year, and 93.8% and 76.2% at 2 years in FV-naive and FV-primed groups, respectively. TAK-426 responses were comparable in magnitude and kinetics with those elicited by natural Zika virus infection., Conclusions: These results support the further clinical development of TAK-426 for both FV-naive and FV-primed populations., Clinical Trials Registration: NCT03343626., Competing Interests: Potential conflicts of interest. C. J. A., F. N., K. J. M., K. B., P. K., M. L., E. W., T. D. P., W. R. B., J. S., and G. D. are employees of Takeda and hold stock/stock options in Takeda. H. H. H., H. P., F. P., and V. M. were employees of Takeda when the study was conducted and may hold stock/stock options in Takeda. E. J. A. reports consultancy for Janssen, Medscape, Pfizer, Sanofi Pasteur, GlaxoSmithKline, and Moderna; safety monitoring boards for Kentucky BioProcessing, Sanofi Pasteur, and WCG and ACI Clinical; institutional clinical research funding from GlaxoSmithKline, Janssen, MedImmune, Merck Sharp & Dohme, Micron, PaxVax, Pfizer, Regeneron, and Sanofi Pasteur; and institutional funding from the National Institutes of Health for clinical research on Moderna and Janssen vaccines. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

A clinical casein kinase 2 inhibitor, CX-4945 (silmitasertib), shows significant affinity toward the DYRK1A and GSK3β kinases, involved in down syndrome phenotypes, Alzheimer's disease, circadian clock regulation, and diabetes. This off-target activity offers an opportunity for studying the effect of the DYRK1A/GSK3β kinase system in disease biology and possible line extension. Motivated by the dual inhibition of these kinases, we solved and analyzed the crystal structures of DYRK1A and GSK3β with CX-4945. We built a quantum-chemistry-based model to rationalize the compound affinity for CK2α, DYRK1A, and GSK3β kinases. Our calculations identified a key element for CK2α's subnanomolar affinity to CX-4945. The methodology is expandable to other kinase selectivity modeling. We show that the inhibitor limits DYRK1A- and GSK3β-mediated cyclin D1 phosphorylation and reduces kinase-mediated NFAT signaling in the cell. Given the CX-4945's clinical and pharmacological profile, this inhibitory activity makes it an interesting candidate with potential for application in additional disease areas.

Trypanosomiases are neglected tropical diseases caused by Trypanosoma (sub)species. Available treatments are limited and have considerable adverse effects and questionable efficacy in the chronic stage of the disease, urgently calling for the identification of new targets and drug candidates. Recently, we have shown that impairment of glycosomal protein import by the inhibition of the PEX5-PEX14 protein-protein interaction (PPI) is lethal to Trypanosoma. Here, we report the development of a novel dibenzo[b,f][1,4]oxazepin-11(10H)-one scaffold for small molecule inhibitors of PEX5-PEX14 PPI. The initial hit was identified by a high throughput screening (HTS) of a library of compounds. A bioisosteric replacement approach allowed to replace the metabolically unstable sulphur atom from the initial dibenzo[b,f][1,4]thiazepin-11(10H)-one HTS hit with oxygen. A crystal structure of the hit compound bound to PEX14 surface facilitated the rational design of the compound series accessible by a straightforward chemistry for the initial structure-activity relationship (SAR) analysis. This guided the design of compounds with trypanocidal activity in cell-based assays providing a promising starting point for the development of new drug candidates to tackle trypanosomiases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Trypanosomiases are life-threatening infections of humans and livestock, and novel effective therapeutic approaches are needed. Trypanosoma compartmentalize glycolysis into specialized organelles termed glycosomes. Most of the trypanosomal glycolytic enzymes harbor a peroxisomal targeting signal-1 (PTS1) which is recognized by the soluble receptor PEX5 to facilitate docking and translocation of the cargo into the glycosomal lumen. Given its pivotal role in the glycosomal protein import, the PEX5-PTS1 interaction represents a potential target to inhibit import of glycolytic enzymes and thus kill the parasite. We developed a fluorescence polarization (FP)-based assay for monitoring the PEX5-PTS1 interaction and performed a High Throughput Screening (HTS) campaign to identify small molecule inhibitors of the interaction. Six of the identified hits passed orthogonal selection criteria and were found to inhibit parasite growth in cell culture. Our results validate PEX5 as a target for small molecule inhibitors and provide scaffolds suitable for further pre-clinical development of novel trypanocidal compounds., (© 2022. The Author(s).)

During RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10-mediated modulation remains unclear in the absence of the nsp14 structure. Here, we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10-bound structures explain the modulatory role of the co-factor protein and reveal the allosteric nsp14 control mechanism essential for drug discovery. Further, the flexibility of the N-terminal lid of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp14 structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Immune checkpoint targeting immunotherapy has revolutionized the treatment of certain cancers in the recent years. Determination of the status of immune checkpoint expression in particular cancers may assist decision making. Here, we describe the development of a single-stranded aptamer-based molecular probe specifically recognizing human PD-L1. Target engaging aptamers are selected by iterative enrichment from a random ssDNA pool and the binding is characterized biochemically. Specificity and dose dependence is demonstrated in vitro in the cell culture using human kidney tumor cells (786-0), human melanoma cells (WM115 and WM266.4) and human glioblastoma LN18 cancer cells. The utility of the probe in vivo is demonstrated using two mouse tumor models, where we show that the probe exhibits excellent potential in imaging. We postulate that further development of the probe may allow universal imaging of different types of tumors depending on their PD-L1 status, which may find utility in cancer diagnosis.

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PL pro ) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PL pro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks., Competing Interests: Declaration of interests ACF and its derivatives and their use against betacoronaviruses are protected by European patent application no. 20214108.1, submitted by the authors of this paper. Disclosure statement: M.J.B. is a current employee of AstraZeneca., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Peroxisomal acyl-CoA oxidase 1a (ACOX1a) catalyzes the first and rate-limiting step of fatty acid oxidation, the conversion of acyl-CoAs to 2-trans-enoyl-CoAs. The dysfunction of human ACOX1a (hACOX1a) leads to deterioration of the nervous system manifesting in myeloneuropathy, hypotonia and convulsions. Crystal structures of hACOX1a in apo- and cofactor (FAD)-bound forms were solved at 2.00 and 2.09 Å resolution, respectively. hACOX1a exists as a homo-dimer with solvation free energy gain (ΔG o ) of -44.7 kcal mol -1 . Two FAD molecules bind at the interface of protein monomers completing the active sites. The substrate binding cleft of hACOX1a is wider compared to human mitochondrial very-long chain specific acyl-CoA dehydrogenase. Mutations (p.G178C, p.M278V and p.N237S) reported to cause dysfunctionality of hACOX1a are analyzed on its 3D-structure to understand structure-function related perturbations and explain the associated phenotypes., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for Spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin., (© 2022. The Author(s).)

Background: A universal adaptor protein, MyD88, orchestrates the innate immune response by propagating signals from toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R). Receptor activation seeds MyD88 dependent formation of a signal amplifying supramolecular organizing center (SMOC)-the myddosome. Alternatively spliced variant MyD88S, lacking the intermediate domain (ID), exhibits a dominant negative effect silencing the immune response, but the mechanistic understanding is limited., Methods: Luciferase reporter assay was used to evaluate functionality of MyD88 variants and mutants. The dimerization potential of MyD88 variants and myddosome nucleation process were monitored by co-immunoprecipitation and confocal microscopy. The ID secondary structure was characterized in silico employing I-TASSER server and in vitro using nuclear magnetic resonance (NMR) and circular dichroism (CD)., Results: We show that MyD88S is recruited to the nucleating SMOC and inhibits its maturation by interfering with incorporation of additional components. Biophysical analysis suggests that important functional role of ID is not supported by a well-defined secondary structure. Mutagenesis identifies Tyr116 as the only essential residue within ID required for myddosome nucleation and signal propagation (NF-κB activation)., Conclusions: Our results argue that the largely unstructured ID of MyD88 is not only a linker separating toll-interleukin-1 receptor (TIR) homology domain and death domain (DD), but contributes intermolecular interactions pivotal in MyD88-dependent signaling. The dominant negative effect of MyD88S relies on quenching the myddosome nucleation and associated signal transduction. Video abstract., (© 2022. The Author(s).)

The rising prevalence of diabetes is threatening global health. It is known not only for the occurrence of severe complications but also for the SARS-Cov-2 pandemic, which shows that it exacerbates susceptibility to infections. Current therapies focus on artificially maintaining insulin homeostasis, and a durable cure has not yet been achieved. We demonstrate that our set of small molecule inhibitors of DYRK1A kinase potently promotes β-cell proliferation, enhances long-term insulin secretion, and balances glucagon level in the organoid model of the human islets. Comparable activity is seen in INS-1E and MIN6 cells, in isolated mice islets, and human iPSC-derived β-cells. Our compounds exert a significantly more pronounced effect compared to harmine, the best-documented molecule enhancing β-cell proliferation. Using a body-like environment of the organoid, we provide a proof-of-concept that small-molecule-induced human β-cell proliferation via DYRK1A inhibition is achievable, which lends a considerable promise for regenerative medicine in T1DM and T2DM treatment.

The clinical success of PD-1/PD-L1 immune checkpoint targeting antibodies in cancer is followed by efforts to develop small molecule inhibitors with better penetration into solid tumors and more favorable pharmacokinetics. Here we report the crystal structure of a macrocyclic peptide inhibitor (peptide 104) in complex with PD-L1. Our structure shows no indication of an unusual bifurcated binding mode demonstrated earlier for another peptide of the same family (peptide 101). The binding mode relies on extensive hydrophobic interactions at the center of the binding surface and an electrostatic patch at the side. An interesting sulfur/π interaction supports the macrocycle-receptor binding. Overall, our results allow a better understanding of forces guiding macrocycle affinity for PD-L1, providing a rationale for future structure-based inhibitor design and rational optimization.

Growing antibiotic resistance of bacteria is a burning problem of human and veterinary medicine. Expansion and introduction of novel microbicidal therapeutics is highly desirable. However, antibiotic treatment disturbs the balance of physiological microbiota by changing its qualitative and/or quantitative composition, resulting in a number of adverse effects that include secondary infections. Although such dysbiosis may be reversed by the treatment with probiotics, a more attractive alternative is the use of antibiotics that target only pathogens, while sparing the commensals. Here, we describe lysostaphin LSp222, an enzyme produced naturally by Staphylococcus pseudintermedius 222. LSp222 is highly effective against S. aureus, including its multi-drug resistant strains. Importantly, the inhibitory concentration for S. epidermidis, the predominant commensal in healthy human skin, is at least two orders of magnitude higher compared to S. aureus. Such significant therapeutic window makes LSp222 a microbiota-friendly antibacterial agent with a potential application in the treatment of S. aureus-driven skin infections., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Glycosomal malate dehydrogenase from Trypanosoma cruzi (tcgMDH) catalyzes the oxidation/reduction of malate/oxaloacetate, a crucial step of the glycolytic process occurring in the glycosome of the human parasite. Inhibition of tcgMDH is considered a druggable trait for the development of trypanocidal drugs. Sequence comparison of MDHs from different organisms revealed a distinct insertion of a prolin rich 9-mer (62-KLPPVPRDP-70) in tcgMDH as compared to other eukaryotic MDHs. Crystal structure of tcgMDH is solved here at 2.6 Å resolution with R work /R free values of 0.206/0.216. The tcgMDH forms homo-dimer with the solvation free energy (ΔG o ) gain of -9.77 kcal/mol. The dimeric form is also confirmed in solution by biochemical assays, chemical-crosslinking and dynamic light scattering. The inserted 9-mer adopts a structure of a solvent accessible loop in the vicinity of NAD + binding site. The distinct sequence and structural feature of tcgMDH, revealed in the present report, provides an anchor point for the development of inhibitors specific for tcgMDH, possible trypanocidal agents of the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

BacSp222 is a multifunctional peptide produced by Staphylococcus pseudintermedius 222. This 50-amino acid long peptide belongs to subclass IId of bacteriocins and forms a four-helix bundle molecule. In addition to bactericidal functions, BacSp222 possesses also features of a virulence factor, manifested in immunomodulatory and cytotoxic activities toward eukaryotic cells. In the present study, we demonstrate that BacSp222 is produced in several post-translationally modified forms, succinylated at the ε-amino group of lysine residues. Such modifications have not been previously described for any bacteriocins. NMR and circular dichroism spectroscopy studies have shown that the modifications do not alter the spatial structure of the peptide. At the same time, succinylation significantly diminishes its bactericidal and cytotoxic potential. We demonstrate that the modification of the bacteriocin is an effect of non-enzymatic reaction with a highly reactive intracellular metabolite, i.e., succinyl-coenzyme A. The production of succinylated forms of the bacteriocin depends on environmental factors and on the access of bacteria to nutrients. Our study indicates that the production of succinylated forms of bacteriocin occurs in response to the changing environment, protects producer cells against the autotoxicity of the excreted peptide, and limits the pathogenicity of the strain.

This work describes the modification of a gold electrode with the BMS-8 compound that interacts with the Programmed Death-Ligand 1 (PD-L1), an immune checkpoint protein. The results show that we can confirm the presence of the sPD-L1 in the concentration range of 10 -18 to 10 -8 M using electrochemical impedance spectroscopy (EIS) with a limit of detection (LOD) of 1.87 × 10 -14 M for PD-L1 (S/N = 3.3) and at a concentration of 10 -14 M via cyclic voltammetry (CV). Additionally, high-resolution X-ray photoelectron spectroscopy (XPS), contact angle, and surface free energy measurements were applied to confirm the functionalization of the electrode. We investigated the selectivity of the electrode for other proteins: Programmed Death-1 (PD-1), cluster of differentiation 160 (CD160), and B- and T-lymphocyte attenuator (BTLA) at concentrations of 10 -8 M. Differentiation between PD-L1 and PD-1 was achieved based on the analysis of the capacitance effect frequency dispersion at the surface of the modified Au electrode with BMS-8 after incubation at various concentrations of PD-L1 and PD-1 proteins in the range of 10 -18 to 10 -8 M. Significant differences were observed in the heterogeneity of PD-L1 and PD-1. The results of the quasi-capacitance studies demonstrate that BMS-8 strongly and specifically interacts with the PD-L1 protein., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus , may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system.

Red palm weevil ( Rhynchophorus ferrugineus Olivier, 1791, Coleoptera: Curculionidae) is a destructive pest of palms, rapidly extending its native geographical range and causing large economic losses worldwide. The present work describes isolation, identification, and bioinformatic analysis of antibacterial proteins and peptides from the immunized hemolymph of this beetle. In total, 17 different bactericidal or bacteriostatic compounds were isolated via a series of high-pressure liquid chromatography steps, and their partial amino acid sequences were determined by N-terminal sequencing or by mass spectrometry. The bioinformatic analysis of the results facilitated identification and description of corresponding nucleotide coding sequences for each peptide and protein, based on the recently published R. ferrugineus transcriptome database. The identified compounds are represented by several well-known bactericidal factors: two peptides similar to defensins, one cecropin-A1-like peptide, and one attacin-B-like protein. Interestingly, we have also identified some unexpected compounds comprising five isoforms of pheromone-binding proteins as well as seven isoforms of odorant-binding proteins. The particular role of these factors in insect response to bacterial infection needs further investigation.

Glycerol is an organic compound that can be utilized as an alternative source of carbon by various organisms. One of the ways to assimilate glycerol by the cell is the phosphorylative catabolic pathway in which its activation is catalyzed by glycerol kinase (GK) and glycerol-3-phosphate (G3P) is formed. To date, several GK crystal structures from bacteria, archaea, and unicellular eukaryotic parasites have been solved. Herein, we present a series of crystal structures of GK from Chaetomium thermophilum (CtGK) in apo and glycerol-bound forms. In addition, we show the feasibility of an ADP-dependent glucokinase (ADPGK)-coupled enzymatic assay to measure the CtGK activity. New structures described in our work provide structural insights into the GK catalyzed reaction in the filamentous fungus and set the foundation for understanding the glycerol metabolism in eukaryotes.