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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.

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.

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. Antibiotic resistance is an emergent threat to human health. Bacterial conjugation is the main mechanism for the wide spread dissemination of antibiotic resistance genes. Here, we found that conjugative traffic ATPases are the molecular target for the inhibition of conjugation by unsaturated fatty acids. Identification of this molecular target will provide us with a new tool for the rational design of more potent and efficient drugs to stop the transmission of antibiotic resistance genes.

Antibiotic resistance has become a major health issue. Unfortunately, the development of new antibiotics is being very slow and expensive and we are running out of weapons to fi ght against the appearance of multi-resistant bugs. Bacterial conjugation is the main mechanism for the wide spread dissemination of antibiotic resistance genes and, therefore, the search of specific inhibitors of conjugation (COINs) could become a brand new strategy in this warfare. In the search of potential COINs, previous studies have reported that unsaturated fatty acids (uFAs) were able to inhibit conjugation. Based on these studies we have looked for the molecular targets of these compounds and we have found that the Type IV secretion ATPase TrwD is inhibited by linoleic acid and 2-alkynoic fatty acids, such as the 2-hexadecynoic acid. These two uFAs compounds were the most effective inhibitors in R388 plasmid conjugation. The inhibitory effect is specific for the traffic ATPase TrwD, as the remaining ATPases of the Type IV secretion system are unaffected by both uFAs and 2-AFAs. We have characterized the inhibition mechanism and we have found that in both cases it is a non-competitive inhibition, as the affinities for the substrate (ATP) and product (ADP) are not altered in the presence of the fatty acids. Altogether, these results could open new avenues in the development of new strategies to prevent the dissemination of antibiotic resistance genes.

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. © 2013, American Society for Microbiology.

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. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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.