Tyrosine-O-sulfation is a widespread affinity enhancer among thrombin interactors.

Tyrosine-O-sulfation is a common post-translational modification (PTM) of proteins following the cellular secretory pathway. First described in human fibrinogen, tyrosine-O-sulfation has long been associated with the modulation of protein-protein interactions in several physiological processes. A number of relevant interactions for hemostasis are largely dictated by this PTM, many of which involving the serine proteinase thrombin (FIIa), a central player in the blood-clotting cascade. Tyrosine sulfation is not limited to endogenous FIIa ligands and has also been found in hirudin, a well-known and potent thrombin inhibitor from the medicinal leech, Hirudo medicinalis. The discovery of hirudin led to successful clinical application of analogs of leech-inspired molecules, but also unveiled several other natural thrombin-directed anticoagulant molecules, many of which undergo tyrosine-O-sulfation. The presence of this PTM has been shown to enhance the anticoagulant properties of these peptides from a range of blood-feeding organisms, including ticks, mosquitos and flies. Interestingly, some of these molecules display mechanisms of action that mimic those of thrombin's bona fide substrates.

Biosynthesis of mycobacterial methylmannose polysaccharides requires a unique 1-O-methyltransferase specific for 3-O-methylated mannosides.

Mycobacteria are a wide group of organisms that includes strict pathogens, such as Mycobacterium tuberculosis, as well as environmental species known as nontuberculous mycobacteria (NTM), some of which-namely Mycobacterium avium-are important opportunistic pathogens. In addition to a distinctive cell envelope mediating critical interactions with the host immune system and largely responsible for their formidable resistance to antimicrobials, mycobacteria synthesize rare intracellular polymethylated polysaccharides implicated in the modulation of fatty acid metabolism, thus critical players in cell envelope assembly. These are the 6-O-methylglucose lipopolysaccharides (MGLP) ubiquitously detected across the Mycobacterium genus, and the 3-O-methylmannose polysaccharides (MMP) identified only in NTM. The polymethylated nature of these polysaccharides renders the intervening methyltransferases essential for their optimal function. Although the knowledge of MGLP biogenesis is greater than that of MMP biosynthesis, the methyltransferases of both pathways remain uncharacterized. Here, we report the identification and characterization of a unique S-adenosyl-l-methionine-dependent sugar 1-O-methyltransferase (MeT1) from Mycobacterium hassiacum that specifically blocks the 1-OH position of 3,3'-di-O-methyl-4α-mannobiose, a probable early precursor of MMP, which we chemically synthesized. The high-resolution 3D structure of MeT1 in complex with its exhausted cofactor, S-adenosyl-l-homocysteine, together with mutagenesis studies and molecular docking simulations, unveiled the enzyme's reaction mechanism. The functional and structural properties of this unique sugar methyltransferase further our knowledge of MMP biosynthesis and provide important tools to dissect the role of MMP in NTM physiology and resilience.

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. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure-activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.

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. These engineered hybrids were synthesized using tandem diselenide-selenoester ligation (DSL) and native chemical ligation (NCL) reactions in one-pot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against α-thrombin and were selective over related coagulation proteases. A lead hybrid inhibitor possessed potent anticoagulant activity, blockade of both thrombin generation and platelet aggregation in vitro and efficacy in a murine thrombosis model at 1 mg kg-1 . The rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for a range of other enzymatic targets that possess multiple sites for the disruption of protein-protein interactions, in addition to an active site.

In silico and crystallographic studies identify key structural features of biliverdin IXß 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 IXß (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. We now outline an integrated platform of in silico and crystallographic studies for the identification of an initial class of compounds inhibiting BLVRB with potencies in the nanomolar range. We found that the most potent BLVRB inhibitors contain a tricyclic hydrocarbon core structure similar to the isoalloxazine ring of flavin mononucleotide and that both xanthene- and acridine-based compounds inhibit BLVRB's flavin and dichlorophenolindophenol (DCPIP) reductase functions. Crystallographic studies of ternary complexes with BLVRB-NADP+-xanthene-based compounds confirmed inhibitor binding adjacent to the cofactor nicotinamide and interactions with the Ser-111 side chain. This residue previously has been identified as critical for maintaining the enzymatic active site and cellular reductase functions in hematopoietic cells. Both acridine- and xanthene-based compounds caused selective and concentration-dependent loss of redox coupling in BLVRB-overexpressing promyelocytic HL-60 cells. These results provide promising chemical scaffolds for the development of enhanced BLVRB inhibitors and identify chemical probes to better dissect the role of biliverdins, alternative substrates, and BLVRB function in physiologically relevant cellular contexts.

Functional analyses yield detailed insight into the mechanism of thrombin inhibition by the antihemostatic salivary protein cE5 from Anopheles gambiae.

Saliva of blood-feeding arthropods carries several antihemostatic compounds whose physiological role is to facilitate successful acquisition of blood. The identification of novel natural anticoagulants and the understanding of their mechanism of action may offer opportunities for designing new antithrombotics disrupting blood clotting. We report here an in-depth structural and functional analysis of the anophelin family member cE5, a salivary protein from the major African malaria vector Anopheles gambiae that specifically, tightly, and quickly binds and inhibits thrombin. Using calorimetry, functional assays, and complementary structural techniques, we show that the central region of the protein, encompassing amino acids Asp-31-Arg-62, is the region mainly responsible for α-thrombin binding and inhibition. As previously reported for the Anopheles albimanus orthologue anophelin, cE5 binds both thrombin exosite I with segment Glu-35-Asp-47 and the catalytic site with the region Pro-49-Arg-56, which includes the highly conserved DPGR tetrapeptide. Moreover, the N-terminal Ala-1-Ser-30 region of cE5 (which includes an RGD tripeptide) and the additional C-terminal serine-rich Asn-63-Glu-82 region (absent in orthologues from anophelines of the New World species A. albimanus and Anopheles darlingi) also played some functionally relevant role. Indeed, we observed decreased thrombin binding and inhibitory properties even when using the central cE5 fragment (Asp-31-Arg-62) alone. In summary, these results shed additional light on the mechanism of thrombin binding and inhibition by this family of salivary anticoagulants from anopheline mosquitoes.

Type IV traffic ATPase TrwD as molecular target to inhibit bacterial conjugation.

Bacterial conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit bacterial conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic ATPase, as the molecular target for fatty acid-mediated inhibition of conjugation. Moreover, 2-alkynoic fatty acids, which are also potent inhibitors of bacterial conjugation, are also powerful inhibitors of the ATPase activity of TrwD. Characterization of the kinetic parameters of ATPase inhibition has led us to identify the catalytic mechanism by which fatty acids exert their activity. These results open a new avenue for the rational design of inhibitors of bacterial conjugation in the fight against the dissemination of antibiotic resistance genes.

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. We demonstrate the approach with thrombin-inhibiting anticoagulants, creating very potent and reversible bivalent direct thrombin inhibitors (Ki = 74 pM). The supramolecular inhibitor effectively inhibited thrombus formation in mice in a needle injury thrombosis model, and this activity could be reversed by administration of the PNA antidote. This design is applicable to therapeutic targets where two binding sites can be identified.

Functional interactions of VirB11 traffic ATPases with VirB4 and VirD4 molecular motors in type IV secretion systems.

Pilus biogenesis and substrate transport by type IV secretion systems require energy, which is provided by three molecular motors localized at the base of the secretion channel. One of these motors, VirB11, belongs to the superfamily of traffic ATPases, which includes members of the type II secretion system and the type IV pilus and archaeal flagellar assembly apparatus. Here, we report the functional interactions between TrwD, the VirB11 homolog of the conjugative plasmid R388, and TrwK and TrwB, the motors involved in pilus biogenesis and DNA transport, respectively. Although these interactions remained standing upon replacement of the traffic ATPase by a homolog from a phylogenetically related conjugative system, namely, TraG of plasmid pKM101, this homolog could not replace the TrwD function for DNA transfer. This result suggests that VirB11 works as a switch between pilus biogenesis and DNA transport and reinforces a mechanistic model in which VirB11 proteins act as traffic ATPases by regulating both events in type IV secretion systems.

Regulation of the type IV secretion ATPase TrwD by magnesium: implications for catalytic mechanism of the secretion ATPase superfamily.

TrwD, the VirB11 homologue in conjugative plasmid R388, is a member of the large secretion ATPase superfamily, which includes ATPases from bacterial type II and type IV secretion systems, type IV pilus, and archaeal flagellae assembly. Based on structural studies of the VirB11 homologues in Helicobacter pylori and Brucella suis and the archaeal type II secretion ATPase GspE, a unified mechanism for the secretion ATPase superfamily has been proposed. Here, we have found that the ATP turnover of TrwD is down-regulated by physiological concentrations of magnesium. This regulation is exerted by increasing the affinity for ADP, hence delaying product release. Circular dichroism and limited proteolysis analysis indicate that magnesium induces conformational changes in the protein that promote a more rigid, but less active, form of the enzyme. The results shown here provide new insights into the catalytic mechanism of the secretion ATPase superfamily.

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. We report a wide distribution of this gene cluster in NTM, including slowly growing mycobacteria such as Mycobacterium avium, which we reveal to produce MMP. Using a combination of MMP purification and chemoenzymatic syntheses of intermediates, we identified the biosynthetic mechanism of MMP, relying on two enzymes that we characterized biochemically and structurally: a previously undescribed α-endomannosidase that hydrolyses MMP into defined-sized mannoligosaccharides that prime the elongation of new daughter MMP chains by a rare α-(1→4)-mannosyltransferase. Therefore, MMP biogenesis occurs through a partially conservative replication mechanism, whose disruption affected mycobacterial growth rate at low temperature.

Autoinhibitory regulation of TrwK, an essential VirB4 ATPase in type IV secretion systems.

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. In a previous work we demonstrated that TrwK is able to hydrolyze ATP. Here, based on the structural homology of VirB4 proteins with the DNA-pumping ATPase TrwB coupling protein, we generated a series of variants of TrwK where fragments of the C-terminal domain were sequentially truncated. Surprisingly, the in vitro ATPase activity of these TrwK variants was much higher than that of the wild-type enzyme. Moreover, addition of a synthetic peptide containing the amino acid residues comprising this C-terminal region resulted in the specific inhibition of the TrwK variants lacking such domain. These results indicate that the C-terminal end of TrwK plays an important regulatory role in the functioning of the T4SS.

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.

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. A combination of chemical synthesis and functional assays was used to reveal that sulfation significantly improved the inhibitory activity of TTI against thrombin. Using X-ray crystallography, we show that the N-terminal sulfated segment of TTI binds the basic exosite II of thrombin, establishing interactions similar to those of physiologic substrates, while the C-terminal segment abolishes the catalytic activity of thrombin. This non-canonical mode of inhibition, coupled with its potency and small size, makes TTI an attractive scaffold for the design of novel antithrombotics.

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 via 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. Cyclic peptides were evaluated in vitro, and three lead compounds exhibited significant prolongation of aPTT, a reduction in thrombin generation, and an inhibition of bradykinin formation. We also describe our efforts to identify the critical residues for binding FXIIa through alanine scanning, analogue generation, and via in silico methods to predict the binding mode of our lead cyclic peptide inhibitors.

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. We thus propose a linearization method (LM) for detecting spurious changes of enzymatic activity based on the representation of progress curves in modified coordinates. This method is highly sensitive to signal readout distortions, thereby allowing rigorous selection of valid kinetic data. The method allows the detection of assay interferences even when their occurrence is not suspected a priori. By knowing the assets and liabilities of the bioassay, enzymology results can be reported with enhanced reproducibility and accuracy. Critical analysis of full progress curves is expected to help discriminating experimental artifacts from true mechanisms of enzymatic inhibition.

Mosquito-Derived Anophelin Sulfoproteins Are Potent Antithrombotics.

The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the Anopheles 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 in vivo. To test this prediction, insect cell expression of two anophelin proteins, from Anopheles albimanus and Anopheles gambiae, was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites. Homogeneously sulfated variants of the two proteins were subsequently generated by chemical synthesis via a one-pot ligation-desulfurization strategy. Tyrosine sulfation of the anophelins was shown to significantly enhance the thrombin inhibitory activity, with a doubly sulfated variant of the anophelin from A. albimanus exhibiting a 100-fold increase in potency compared with the unmodified homologue. Sulfated anophelins were also shown to exhibit potent in vivo anticoagulant and antithrombotic activity.

Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors.

Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification-tyrosine sulfation-of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

Towards an integrated model of bacterial conjugation.

Bacterial conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in bacterial conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating expansion of antibiotic resistance.