RNase P Inhibitors Identified as Aggregators.

RNase P is an essential enzyme responsible for tRNA 5'-end maturation. In most bacteria, the enzyme is a ribonucleoprotein consisting of a catalytic RNA subunit and a small protein cofactor termed RnpA. Several studies have reported small-molecule inhibitors directed against bacterial RNase P that were identified by high-throughput screenings. Using the bacterial RNase P enzymes from Thermotoga maritima, Bacillus subtilis, and Staphylococcus aureus as model systems, we found that such compounds, including RNPA2000 (and its derivatives), iriginol hexaacetate, and purpurin, induce the formation of insoluble aggregates of RnpA rather than acting as specific inhibitors. In the case of RNPA2000, aggregation was induced by Mg2+ ions. These findings were deduced from solubility analyses by microscopy and high-performance liquid chromatography (HPLC), RnpA-inhibitor co-pulldown experiments, detergent addition, and RnpA titrations in enzyme activity assays. Finally, we used a B. subtilis RNase P depletion strain, whose lethal phenotype could be rescued by a protein-only RNase P of plant origin, for inhibition zone analyses on agar plates. These cell-based experiments argued against RNase P-specific inhibition of bacterial growth by RNPA2000. We were also unable to confirm the previously reported nonspecific RNase activity of S. aureus RnpA itself. Our results indicate that high-throughput screenings searching for bacterial RNase P inhibitors are prone to the identification of "false positives" that are also termed pan-assay interference compounds (PAINS).

Acylated 1H-1,2,4-Triazol-5-amines Targeting Human Coagulation Factor XIIa and Thrombin: Conventional and Microscale Synthesis, Anticoagulant Properties, and Mechanism of Action.

We herein report the conventional and microscale parallel synthesis of selective inhibitors of human blood coagulation factor XIIa and thrombin exhibiting a 1,2,4-triazol-5-amine scaffold. Structural variations of this scaffold allowed identifying derivative 21i, a potent 29 nM inhibitor of FXIIa, with improved selectivity over other tested serine proteases and also finding compound 21m with 27 nM inhibitory activity toward thrombin. For the first time, acylated 1,2,4-triazol-5-amines were proved to have anticoagulant properties and the ability to affect thrombin- and cancer-cell-induced platelet aggregation. Performed mass spectrometric analysis and molecular modeling allowed us to discover previously unknown interactions between the synthesized inhibitors and the active site of FXIIa, which uncovered the mechanistic details of FXIIa inhibition. Synthesized compounds represent a promising starting point for the development of novel antithrombotic drugs or chemical tools for studying the role of FXIIa and thrombin in physiological and pathological processes.

Fibrinolysis Inhibitors: Potential Drugs for the Treatment and Prevention of Bleeding.

Hyperfibrinolytic situations can lead to life-threatening bleeding, especially during cardiac surgery. The approved antifibrinolytic agents such as tranexamic acid, ε-aminocaproic acid, 4-aminomethylbenzoic acid, and aprotinin were developed in the 1960s without the structural insight of their respective targets. Crystal structures of the main antifibrinolytic targets, the lysine binding sites on plasminogen's kringle domains, and plasmin's serine protease domain greatly contributed to the structure-based drug design of novel inhibitor classes. Two series of ligands targeting the lysine binding sites have been recently described, which are more potent than the most-widely used antifibrinolytic agent, tranexamic acid. Furthermore, four types of promising active site inhibitors of plasmin have been developed: tranexamic acid conjugates targeting the S1 pocket and primed sites, substrate-analogue linear homopiperidylalanine-containing 4-amidinobenzylamide derivatives, macrocyclic inhibitors addressing nonprimed binding regions, and bicyclic 14-mer SFTI-1 analogues blocking both, primed and nonprimed binding sites of plasmin. Furthermore, several allosteric plasmin inhibitors based on heparin mimetics have been developed.