Development of supramolecular anticoagulants with on-demand reversibility.

Drugs are administered at a dosing schedule set by their therapeutic index, and termination of action is achieved by clearance and metabolism of the drug. In some cases, such as anticoagulant drugs or immunotherapeutics, it is important to be able to quickly reverse the drug's action. Here, we report a general strategy to achieve on-demand reversibility by designing a supramolecular drug (a noncovalent assembly of two cooperatively interacting drug fragments held together by transient hybridization of peptide nucleic acid (PNA)) that can be reversed with a PNA antidote that outcompetes the hybridization between the fragments.

Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56.

Bacterial AB toxins are secreted key virulence factors that are internalized by target cells through receptor-mediated endocytosis, translocating their enzymatic domain to the cytosol from endosomes (short-trip) or the endoplasmic reticulum (long-trip). To accomplish this, bacterial AB toxins evolved a multidomain structure organized into either a single polypeptide chain or non-covalently associated polypeptide chains. The prototypical short-trip single-chain toxin is characterized by a receptor-binding domain that confers cellular specificity and a translocation domain responsible for pore formation whereby the catalytic domain translocates to the cytosol in an endosomal acidification-dependent way.

Pathogen-specific structural features of Ras1 activation complex: uncovering new antifungal drug targets.

An important feature associated with pathogenicity is its ability to switch between yeast and hyphal forms, a process in which CaRas1 plays a key role. CaRas1 is activated by the guanine nucleotide exchange factor (GEF) CaCdc25, triggering hyphal growth-related signaling pathways through its conserved GTP-binding (G)-domain. An important function in hyphal growth has also been proposed for the long hypervariable region downstream the G-domain, whose unusual content of polyglutamine stretches and Q/N repeats make CaRas1 unique within Ras proteins.

Self-recycling and partially conservative replication of mycobacterial methylmannose polysaccharides.

The steep increase in nontuberculous mycobacteria (NTM) infections makes understanding their unique physiology an urgent health priority. NTM synthesize two polysaccharides proposed to modulate fatty acid metabolism: the ubiquitous 6-O-methylglucose lipopolysaccharide, and the 3-O-methylmannose polysaccharide (MMP) so far detected in rapidly growing mycobacteria. The recent identification of a unique MMP methyltransferase implicated the adjacent genes in MMP biosynthesis.

DisProt in 2022: improved quality and accessibility of protein intrinsic disorder annotation.

The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%.

Synthesis and evaluation of peptidic thrombin inhibitors bearing acid-stable sulfotyrosine analogues.

Tyrosine sulfation is an important post-translational modification of peptides and proteins which underpins and modulates many protein-protein interactions. In order to overcome the inherent instability of the native modification, we report the synthesis of two sulfonate analogues and their incorporation into two thrombin-inhibiting sulfopeptides. The effective mimicry of these sulfonate analogues for native sulfotyrosine was validated in the context of their thrombin inhibitory activity and binding mode, as determined by X-ray crystallography.

Potent Cyclic Peptide Inhibitors of FXIIa Discovered by mRNA Display with Genetic Code Reprogramming.

The contact system comprises a series of serine proteases that mediate procoagulant and proinflammatory activities the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. Inhibition of Factor XIIa (FXIIa), an initiator of the contact system, has been demonstrated to lead to thrombo-protection and anti-inflammatory effects in animal models and serves as a potentially safer target for the development of antithrombotics. Herein, we describe the use of the Randomised Nonstandard Peptide Integrated Discovery (RaPID) mRNA display technology to identify a series of potent and selective cyclic peptide inhibitors of FXIIa.

A Secreted NlpC/P60 Endopeptidase from Photobacterium damselae subsp. Cleaves the Peptidoglycan of Potentially Competing Bacteria.

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-containing peptidase from subsp.

Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods.

Blood feeding arthropods, such as leeches, ticks, flies and mosquitoes, provide a privileged source of peptidic anticoagulant molecules. These primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite I or exosite II. Herein, we describe the rational design of a novel class of trivalent thrombin inhibitors that simultaneously block both exosites as well as the active site.

MIRRAGGE - Minimum Information Required for Reproducible AGGregation Experiments.

Reports on phase separation and amyloid formation for multiple proteins and aggregation-prone peptides are recurrently used to explore the molecular mechanisms associated with several human diseases. The information conveyed by these reports can be used directly in translational investigation, e.g.

Sulfotyrosine-Mediated Recognition of Human Thrombin by a Tsetse Fly Anticoagulant Mimics Physiological Substrates.

Despite possessing only 32 residues, the tsetse thrombin inhibitor (TTI) is among the most potent anticoagulants described, with sub-picomolar inhibitory activity against thrombin. Unexpectedly, TTI isolated from the fly is 2000-fold more active and 180 Da heavier than synthetic and recombinant variants. We predicted the presence of a tyrosine O-sulfate post-translational modification of TTI, prompting us to investigate the effect of the modification on anticoagulant activity.

Major Improvements in Robustness and Efficiency during the Screening of Novel Enzyme Effectors by the 3-Point Kinetics Assay.

The throughput level currently reached by automatic liquid handling and assay monitoring techniques is expected to facilitate the discovery of new modulators of enzyme activity. Judicious and dependable ways to interpret vast amounts of information are, however, required to effectively answer this challenge. Here, the 3-point method of kinetic analysis is proposed as a means to significantly increase the hit success rates and decrease the number of falsely identified compounds (false positives).

interferENZY: A Web-Based Tool for Enzymatic Assay Validation and Standardized Kinetic Analysis.

Enzymatic assays are widely employed to characterize important allosteric and enzyme modulation effects. The high sensitivity of these assays can represent a serious problem if the occurrence of experimental errors surreptitiously affects the reliability of enzyme kinetics results. We have addressed this problem and found that hidden assay interferences can be unveiled by the graphical representation of progress curves in modified reaction coordinates.

Biochemical characterization of biliverdins IXβ/δ generated by a selective heme oxygenase.

The pro-oxidant effect of free heme (Fe2+-protoporphyrin IX) is neutralized by phylogenetically-conserved heme oxygenases (HMOX) that generate carbon monoxide, free ferrous iron, and biliverdin (BV) tetrapyrrole(s), with downstream BV reduction by non-redundant NADPH-dependent BV reductases (BLVRA and BLVRB) that retain isomer-restricted functional activity for bilirubin (BR) generation. Regioselectivity for the heme α-meso carbon resulting in predominant BV IXα generation is a defining characteristic of canonical HMOXs, thereby limiting generation and availability of BVs IXβ, IXδ, and IXγ as BLVRB substrates. We have now exploited the unique capacity of the Pseudomonas aeruginosa (P.

The structural characterization of a glucosylglycerate hydrolase provides insights into the molecular mechanism of mycobacterial recovery from nitrogen starvation.

Bacteria are challenged to adapt to environmental variations in order to survive. Under nutritional stress, several bacteria are able to slow down their metabolism into a nonreplicating state and wait for favourable conditions. It is almost universal that bacteria accumulate carbon stores to survive during this nonreplicating state and to fuel rapid proliferation when the growth-limiting stress disappears.

Rapid assembly and profiling of an anticoagulant sulfoprotein library.

Hematophagous organisms produce a suite of salivary proteins which interact with the host's coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues.

A simple linearization method unveils hidden enzymatic assay interferences.

Enzymes are among the most important drug targets in the pharmaceutical industry. The bioassays used to screen enzyme modulators can be affected by unaccounted interferences such as time-dependent inactivation and inhibition effects. Using procaspase-3, caspase-3, and α-thrombin as model enzymes, we show that some of these effects are not eliminated by merely ignoring the reaction phases that follow initial-rate measurements.

Molecular Fingerprints for a Novel Enzyme Family in with Glucosamine Kinase Activity.

have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases.

Genetic code ambiguity modulates the activity of a C. albicans MAP kinase linked to cell wall remodeling.

The human fungal pathogen Candida albicans ambiguously decodes the universal leucine CUG codon predominantly as serine but also as leucine. C. albicans has a high capacity to survive and proliferate in adverse environments but the rate of leucine incorporation fluctuates in response to different stress conditions.

Host-defense peptides AC12, DK16 and RC11 with immunomodulatory activity isolated from Hypsiboas raniceps skin secretion.

Inflammation is a natural defense mechanism of the immune system; however, when unregulated, it can lead to chronic illness. Glucocorticoids are the most commonly used agents to effectively treat inflammatory conditions, including autoimmune diseases, however these substances can trigger a number of side effects. Thus, viable alternatives to the use of these drugs would be advantageous.

Mosquito-Derived Anophelin Sulfoproteins Are Potent Antithrombotics.

The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the mosquito to fulfill a vital role in blood feeding. A bioinformatic analysis of anophelin sequences revealed the presence of conserved tyrosine residues in an acidic environment that were predicted to be post-translationally sulfated . To test this prediction, insect cell expression of two anophelin proteins, from and , was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites.

and crystallographic studies identify key structural features of biliverdin IXβ reductase inhibitors having nanomolar potency.

Heme cytotoxicity is minimized by a two-step catabolic reaction that generates biliverdin (BV) and bilirubin (BR) tetrapyrroles. The second step is regulated by two non-redundant biliverdin reductases (IXα (BLVRA) and IXβ (BLVRB)), which retain isomeric specificity and NAD(P)H-dependent redox coupling linked to BR's antioxidant function. Defective BLVRB enzymatic activity with antioxidant mishandling has been implicated in metabolic consequences of hematopoietic lineage fate and enhanced platelet counts in humans.

Molecular motion regulates the activity of the Mitochondrial Serine Protease HtrA2.

HtrA2 (high-temperature requirement 2) is a human mitochondrial protease that has a role in apoptosis and Parkinson's disease. The structure of HtrA2 with an intact catalytic triad was determined, revealing a conformational change in the active site loops, involving mainly the regulatory LD loop, which resulted in burial of the catalytic serine relative to the previously reported structure of the proteolytically inactive mutant. Mutations in the loops surrounding the active site that significantly restricted their mobility, reduced proteolytic activity both in vitro and in cells, suggesting that regulation of HtrA2 activity cannot be explained by a simple transition to an activated conformational state with enhanced active site accessibility.