Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H + ) and sodium ions (Na + ). The sodium-powered stators, like those in the PomAPomB stator complex of Vibrio spp, can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium-motility inhibitors have been described since the discovery of phenamil. In this study, we characterised two possible motility inhibitors HM2-16F and BB2-50F from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free swimming bacteria and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na + motors comparable to phenamil at matching concentrations, and could also stop rotation of tethered cells driven by H + motors. Bead measurements in presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na + and H + stator types, and in pathogenic and non-pathogenic strains. Importance: Here we characterised two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. Our two compounds were shown to inhibit flagellar motility at 10 μM across multiple strains, from non-pathogenic E. coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (EHEC/UPEC) and lastly in sodium-powered Vibrio alginolyticus . Broad anti-motility compounds such as these are important tools in our efforts control virulence of pathogens in health and agricultural settings.

Urokinase plasminogen activator as an anti-metastases target: Inhibitor design principles, recent amiloride derivatives and issues with human/mouse species selectivity

The urokinase plasminogen activator (uPA) is a widely studied anticancer drug target with multiple classes of inhibitors reported to date. Many of these inhibitors contain amidine or guanidine groups, while others lacking these show improved oral bioavailability. Most of the X-ray co-crystal structures of small molecule uPA inhibitors show a key salt bridge between the side-chain carboxylate of Asp189 in the S1 pocket of uPA. This review summarises the different classes of uPA inhibitors, their binding interactions and experimentally measured inhibitory potencies and highlights species selectivity issues with recent 6-substituted amiloride and 5‐N,N-(hexamethylene)amiloride (HMA) derivatives.

Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomAPomB stator complex of Vibrio spp, can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium-motility inhibitors have been described since the discovery of phenamil. In this study, we discovered two motility inhibitors HM2-16F and BB2-50F from a small library of previously reported amiloride derivatives. Using a tethered cell assay, we showed that both HM2-16F and BB2-50F had inhibition comparable to that of phenamils on Na+ driven motors at matching concentrations, with an additional ability to inhibit rotation in H+ driven motors. The two compounds did not exhibit adverse effects on bacterial growth at the motility-inhibiting concentration of 10 uM, however toxicity was seen for BB2-50F at 100 uM. We performed higher resolution measurements to examine rotation inhibition at moderate (1 um polystyrene bead) and low loads (60 nm gold bead) and in both the presence and absence of stators. These measurements suggested that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F showed reversible inhibition of motility across a range of loads, in both Na+ and H+ stator types, and in pathogenic and non-pathogenic strains.

Degradation of Pharmaceuticals Through Sequential Photon Absorption and Photoionization – the Case of Amiloride Derivatives

Photodegradation of pharmaceutical and agrochemical compounds is an important concern for health and the environment. Amiloride derivatives undergo clean photosubstitution in protic solvents. We have studied this apparent photo-SNAr reaction with a range of experimental and computational techniques. Available evidence points to a mechanism starting with photoexcitation followed by absorption of a second photon to eject an electron to give a radical cation intermediate. Subsequent substitution reaction with the protic solvent is assisted by a general base, possibly strengthened by the proximal solvated electron. Final recombination with the solvated electron generates the observed product. Quantum chemical computations reveal that excited state antiaromaticity is relieved when an electron is ejected from the photoexcited molecule by the second photon, leading to a weakly aromatic radical cation. The mechanism indicated here could have wide applicability to photoinduced degradation of similar heteroaromatic compounds in the environment as well as in protic solvents. There are also strong similarities to a class of increasingly popular synthetic photoredox methods.

Degradation of Pharmaceuticals Through Sequential Photon Absorption and Photoionization – the Case of Amiloride Derivatives

Photodegradation of pharmaceutical and agrochemical compounds is an important concern for health and the environment. Amiloride derivatives undergo clean photosubstitution in protic solvents. We have studied this apparent photo-SNAr reaction with a range of experimental and computational techniques. Available evidence points to a mechanism starting with photoexcitation followed by absorption of a second photon to eject an electron to give a radical cation intermediate. Subsequent substitution reaction with the protic solvent is assisted by a general base, possibly strengthened by the proximal solvated electron. Final recombination with the solvated electron generates the observed product. Quantum chemical computations reveal that excited state antiaromaticity is relieved when an electron is ejected from the photoexcited molecule by the second photon, leading to a weakly aromatic radical cation. The mechanism indicated here could have wide applicability to photoinduced degradation of similar heteroaromatic compounds in the environment as well as in protic solvents. There are also strong similarities to a class of increasingly popular synthetic photoredox methods.

Degradation of Pharmaceuticals Through Sequential Photon Absorption and Photoionization – the Case of Amiloride Derivatives

Photodegradation of pharmaceutical and agrochemical compounds is an important concern for health and the environment. Amiloride derivatives undergo clean photosubstitution in protic solvents. We have studied this apparent photo-SNAr reaction with a range of experimental and computational techniques. Available evidence points to a mechanism starting with photoexcitation followed by absorption of a second photon to eject an electron to give a radical cation intermediate. Subsequent substitution reaction with the protic solvent is assisted by a general base, possibly strengthened by the proximal solvated electron. Final recombination with the solvated electron generates the observed product. Quantum chemical computations reveal that excited state antiaromaticity is relieved when an electron is ejected from the photoexcited molecule by the second photon, leading to a weakly aromatic radical cation. The mechanism indicated here could have wide applicability to photoinduced degradation of similar heteroaromatic compounds in the environment as well as in protic solvents. There are also strong similarities to a class of increasingly popular synthetic photoredox methods.