Discovery of inhibitors of Pseudomonas aeruginosa virulence through the search for natural-like compounds with a dual role as inducers and substrates of efflux pumps

Multidrug efflux pumps are ancient elements encoded in every genome, from bacteria to humans. In bacteria, in addition to antibiotics, efflux pumps extrude a wide range of substrates, including quorum sensing signals, bacterial metabolites, or plant-produced compounds. This indicates that their original functions may differ from their recently acquired role in the extrusion of antibiotics during human infection. Concerning plant-produced compounds, some of them are substrates and inducers of the same efflux pump, suggesting a coordinated plant/bacteria coevolution. Herein we analyse the ability of 1243 compounds from a Natural Product-Like library to induce the expression of P. aeruginosa mexCD-oprJ or mexAB-oprM efflux pumps' encoding genes. We further characterized natural-like compounds that do not trigger antibiotic resistance in P. aeruginosa and that act as virulence inhibitors, choosing those that were not only inducers but substrates of the same efflux pump. Four compounds impair swarming motility, exotoxin secretion through the Type 3 Secretion System (T3SS) and the ability to kill Caenorhabditis elegans, which might be explained by the downregulation of genes encoding flagellum and T3SS. Our results emphasize the possibility of discovering new anti-virulence drugs by screening natural or natural-like libraries for compounds that behave as both, inducers and substrates of efflux pumps.     

In search of partners: linking extracellular proteases to substrates

Proteases function as molecular switches in signalling circuits at the cell surface and in the extracellular milieu. In light of the many proteases that are encoded by the genome, and the even larger number of bioactive substrates, it is crucial to identify which proteases cleave a particular substrate and which substrates individual proteases cleave. Elucidating the substrate degradomes of proteases will help us to understand the function of proteases in development and disease and to validate proteases as drug targets.     

Inhibitors of multidrug resistance (MDR) have affinity for MDR substrates

Multidrug-resistance (MDR) occurs in many bacterial species and tumour cells. MDR functions by membrane proteins which export drugs from cells, resulting in a low ineffective concentration of the drug. We have shown by molecular modelling that inhibitors of MDR have affinity for substrates of MDR transporters. This affinity may facilitate drug entry into cells and a large inhibitor-drug complex may be a poorer substrate for the MDR mechanism. This complex would effectively 'cloak' the drug rendering it unavailable for efflux.     

Bacterial efflux systems and efflux pumps inhibitors

It is now well established that bacterial resistance to antibiotics has become a serious problem of public health that concerns almost all antibacterial agents and that manifests in all fields of their application. Among the three main mechanisms involved in bacterial resistance (target modification, antibiotic inactivation or default of its accumulation within the cell), efflux pumps, responsible for the extrusion of the antibiotic outside the cell, have recently received a particular attention. Actually, these systems, classified into five families, can confer resistance to a specific class of antibiotics or to a large number of drugs, thus conferring a multi-drug resistance (MDR) phenotype to bacteria. To face this issue, it is urgent to find new molecules active against resistant bacteria. Among the strategies employed, the search for inhibitors of resistance mechanisms seems to be attractive because such molecules could restore antibiotic activity. In the case of efflux systems, efflux pump inhibitors (EPIs) are expected to block the pumps and such EPIs, if active against MDR pumps, would be of great interest. This review will focus on the families of bacterial efflux systems conferring drug resistance, and on the EPIs that have been identified to restore antibiotic activity.     

Granzymes (lymphocyte serine proteases): characterization with natural and synthetic substrates and inhibitors

Natural killer (NK) and cytotoxic T-lymphocytes (CTLs) kill cells within an organism to defend it against viral infections and the growth of tumors. One mechanism of killing involves exocytosis of lymphocyte granules which causes pores to form in the membranes of the attacked cells, fragments nuclear DNA and leads to cell death. The cytotoxic granules contain perforin, a pore-forming protein, and a family of at least 11 serine proteases termed granzymes. Both perforin and granzymes are involved in the lytic activity. Although the biological functions of most granzymes remain to be resolved, granzyme B clearly promotes DNA fragmentation and is directly involved in cell death. Potential natural substrates for Gr B include procaspases and other proteins involved in cell death. Activated caspases are involved in apoptosis. The search continues for natural substrates for the other granzymes. The first granzyme crystal structure remains to be resolved, but in the interim, molecular models of granzymes have provided valuable structural information about their substrate binding sites. The information has been useful to predict the amino acid sequences that immediately flank each side of the scissile peptide bond of peptide and protein substrates. Synthetic substrates, such as peptide thioesters, nitroanilides and aminomethylcoumarins, have also been used to study the substrate specificity of granzymes. The different granzymes have one of four primary substrate specificities: tryptase (cleaving after Arg or Lys), Asp-ase (cleaving after Asp), Met-ase (cleaving after Met or Leu), and chymase (cleaving after Phe, Tyr, or Trp). Natural serpins and synthetic inhibitors (including isocoumarins, peptide chloromethyl ketones, and peptide phosphonates) inhibit granzymes. Studies of substrate and inhibitor kinetics are providing valuable information to identify the most likely natural granzyme substrates and provide tools for the study of key reactions in the cytolytic mechanism.     

Gamma-secretase: substrates and inhibitors

The amyloid β-protein (Aβ) deposited in Alzheimer’s disease (AD), the most common form of dementia in the elderly, is a secreted proteolytic product of the amyloid β-protein precursor (APP). Generation of Aβ from the APP requires two sequential proteolytic events, β-secretase cleavage to generate the amino terminus, followed by γ-secretase cleavage to generate the carboxyl terminus. Because this process is a central event in the pathogenesis of AD, γ-secretase is believed to be an excellent therapeutic target. γ-Secretase activity has been demonstrated to be membrane-associated, with the cleavage site primarily determined by the location of the substrate with respect to the membrane. It has also been shown that this unusual proteolytic activity not only occurs for APP, but also for proteins involved in morphogenic processes or cell proliferation and differentiation such as Notch and ErbB4. Thus far, all γ-secretase substrates are involved in some form of nuclear signaling. These recent findings have important implications for the development of pharmacological interventions that target γ-secretase.     

Substrate specificity analysis of microbial transglutaminase using proteinaceous protease inhibitors as natural model substrates

The substrate specificity of microbial transglutaminase (MTG) from Streptomyces mobaraensis (formerly categorized Streptoverticillium) was studied using a Streptomyces proteinaceous protease inhibitor, STI2, as a model amine-donor substrate. Chemical modification and mutational analysis to address the substrate requirements for MTG were carried out around the putative reactive site region of STI2 on the basis of the highly refined tertiary structure and the solvent accessibility index of Streptomyces subtilisin inhibitor, SSI, a homolog of STI2. The results suggest that the P1 reactive center site (position 70 of STI2) for protease subtilisin BPN' or trypsin may be the prime Lys residue that can be recognized by MTG, when succinylated beta-casein was used as a partner Gln-substrate. It is characteristic in that the same primary enzyme contact region of STI2 is shared by both enzymes, MTG and proteases. For quantitative analysis of the TG reaction, we established an ELISA-based monitoring assay system using an anti-SSI polyclonal antibody highly cross-reactive with STI2. Site-specific STI2 mutants were prepared by an Escherichia coli expression-secretion vector system and subjected to the assay system. We reached several conclusions concerning the nature of the flanking amino acid residues affecting the MTG reactivity of the substrate Lys residue: (i) site-specific mutations from Asn to Lys or Arg at position 69 preceding the amine-donor 70Lys, led to enhanced substrate reactivity; (ii) amino acid replacement at 67Ile with Ser led to higher substrate reactivity, (iii) additive effects were obtained by a combination of the positive mutations at positions 67 and 69 as described above, and (iv) Gly at position 65 might be essential for MTG reaction. Moreover, the substrate specificity of guinea pig liver tissue transglutaminase (GTG) was compared with that of MTG using STI2 and its mutants. In contrast to MTG, replacement of Gly by Asp at position 65 was the most favorable for substrate reactivity. Also, 70Lys appeared not to be a prime amine-donor site for GTG-mediated cross-linking, suggesting a difference in substrate recognition between MTG and GTG.     

Allosteric changes in thrombin's activity produced by peptides corresponding to segments of natural inhibitors and substrates.

Acidic synthetic peptides corresponding to segments of several nonhomologous proteins (hirudin, residues 54-65; heparin cofactor II, residues 54-75; and fibrinogen, residues 410-427 of the gamma B-chain) inhibit thrombin's cleavage of fibrinogen without blocking the enzyme's active site. Here, we examined effects of these peptides on thrombin's cleavage of protein C and small peptides. Activation of protein C by thrombin in the absence of calcium was inhibited by all of the peptides. Maximal inhibition was 60%, and no greater inhibition was produced by higher peptide concentrations. This differed from progressive inhibition of protein C activation by increasing peptide concentrations in the presence of thrombomodulin and calcium. Potencies of the peptides were in the order hirudin-(54-65) greater than heparin cofactor II-(54-75) greater than gamma B-chain-(410-427). Sulfation of the tyrosine residue in hirudin-(54-65) increased its potency about 10-fold, similar to changes in anticlotting activity. The peptides were activators rather than inhibitors of the cleavage of small chromogenic substrates. In the presence of the peptides, the affinity of thrombin for the substrates S-2366 (pyro-Glu-Pro-Arg-4-nitroanilide), Chromozyme TH (tosyl-Gly-Pro-Arg-4-nitroanilide), and S-2251 (D-Val-Leu-Lys-4-nitroanilide) increased 1.5-2-fold with little change in the Vmax of substrate cleavage. Potencies of peptides in these allosteric effects on thrombin was in the same order as for their other effects. The similar actions of these nonhomologous peptides, which are believed to bind to thrombin's anion-binding exosite, suggest that binding of any peptide to this site exerts the same allosteric effect on thrombin's active site. Interactions of these peptides with thrombin may serve as models for regulation of thrombin's interactions with natural substrates and inhibitors.     

Selectivity profiling of DegP substrates and inhibitors

Protein quality control factors are involved in many key physiological processes and severe human diseases that are based on misfolding or amyloid formation. Prokaryotic representatives are often virulence factors of pathogenic bacteria. Therefore, protein quality control factors represent a novel class of drug targets. The bacterial serine protease DegP, belonging to the widely conserved family of HtrA proteases, exhibits unusual structural and functional plasticity that could be exploited by small molecule modulators. However, only one weak synthetic peptide substrate and no inhibitors are available to date. We report the identification of a potent heptameric pNA-substrate and chloromethyl ketone based inhibitors of DegP. In addition, specificity profiling resulted in the identification of one strong inhibitor and a potent substrate for subtilisin as well as a number of specific elastase substrates and inhibitors.     

The use of natural product scaffolds as leads in the search for trypanothione reductase inhibitors

Twenty-three heterocyclic compounds were evaluated for their potential as trypanothione reductase inhibitors. As a result, the harmaline, 10-thiaisoalloxazine, and aspidospermine frameworks were identified as the basis of inhibitors of Trypanosoma cruzi trypanothione reductase. Two new compounds showed moderately strong, linear competitive inhibition, namely N,N-dimethyl-N-[3-(7-methoxy-1-methyl-3,4-dihydro-9H-beta-carbolin-9-yl)propyl]amine (15) and 1,3-bis[3-(dimethylamino)propyl]-1,5-dihydro-2H-pyrimido[4,5-b][1,4]benzothiazine-2,4(3H)-dione (21), with K(i) values of 35.1+/-3.5microM and 26.9+/-1.9microM, respectively. Aspidospermine (25) inhibited T. cruzi TryR with a K(i) of 64.6+/-6.2microM. None of the compounds inhibited glutathione reductase. Their toxicity toward promastigotes of Leishmania amazonensis was assessed.     

In search of potent and selective inhibitors of neuronal nitric oxide synthase with more simple structures

In certain neurodegenerative diseases damaging levels of nitric oxide (NO) are produced by neuronal nitric oxide synthase (nNOS). It, therefore, is important to develop inhibitors selective for nNOS that do not interfere with other NOS isoforms, especially endothelial NOS (eNOS), which is critical for proper functioning of the cardiovascular system. While we have been successful in developing potent and isoform-selective inhibitors, such as lead compounds 1 and 2, the ease of synthesis and bioavailability have been problematic. Here we describe a new series of compounds including crystal structures of NOS-inhibitor complexes that integrate the advantages of easy synthesis and good biological properties compared to the lead compounds. These results provide the basis for additional structure–activity relationship (SAR) studies to guide further improvement of isozyme selective inhibitors.     

MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential

MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.     

Evaluating limited specificity of drug pumps reduced relative resistance in human MDR phenotypes.

In the parallel paper, we developed a property to characterize drug efflux pumps, i.e. the reduced relative resistance (RRR). Using this RRR, we here investigate whether the observed diversity in human multidrug resistance (MDR) phenotypes might be due to variable levels of P-glycoprotein encoded by MDR1. We analyzed resistance phenotypes of various human cell lines in which either one, or both, classical human multidrug resistance genes, MDR1 and MDR3, are overexpressed. In addition, RRR values were calculated for MDR phenotypes presented in the literature. The results suggest that more than a single mechanism is required to account for the observed phenotypic diversity of classical multidrug resistance. This diversity is only partly due to differences in plasma membrane permeabilities between cell line families. It is discussed whether the alternative MDR phenotypes might be MDR1 phenotypes modified by other factors that do not themselves cause MDR. The method we here apply may also be useful for other nonspecific enzymes or pumps.     

Detergents as intrinsic P-glycoprotein substrates and inhibitors

We assessed the interaction of three electrically neutral detergents (Triton X-100, C₁₂EO₈, and Tween 80) with P-glycoprotein (ABCB1, MDR1) and identified the molecular elements responsible for this interaction. To this purpose we titrated P-glycoprotein in inside-out plasma membrane vesicles of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) with the detergents below their critical micelle concentration, CMC. The P-glycoprotein ATPase measured as a function of the detergent concentration yielded bell-shaped activity curves which were evaluated with a two-site binding model. The lipid-water partition coefficient and the transporter-water binding constant of the detergents were measured independently. Knowledge of these two parameters allowed assessment of the free energy of detergent binding to P-glycoprotein in the lipid membrane, ΔG_tl⁰, that reflects the direct detergent-transporter affinity. It increased as the number of ethoxyl groups increased, suggesting that these hydrogen bond acceptor groups are the key elements for the detergent-transporter interaction in the lipid membrane. The free energy of binding to P-glycoprotein per ethoxyl group (EO) was determined as approximately ΔG_EO⁰ = − 1.6 kJ/mol. The present findings moreover document that, depending on the concentration applied, detergents are intrinsic substrates for, or inhibitors of P-glycoprotein.     

Novel substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase

Peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) catalyzes the formation of alpha-amidated peptides from their glycine-extended precursors, thus playing a key role in the processing of peptide neurohormones. We now report that PAM readily catalyzes three alternate monooxygenase reactions--sulfoxidation, amine N-dealkylation, and O-dealkylation. Thus, (4-nitrobenzyl)thioacetic acid is converted to the analogous sulfoxide, N-(4-nitrobenzyl)glycine is converted to 4-nitrobenzylamine and glyoxylate, and [(4-nitrobenzyl)oxy]acetic acid is converted to 4-nitrobenzyl alcohol and glyoxylate. All these new activities display the characteristics expected for the normal PAM-catalyzed reductive oxygenation pathway and produce an equimolar amount of glyoxylate together with the heteroatom-containing dealkylation products. The ester [(4-methoxybenzoyl)oxy]acetic acid is not a PAM substrate, but is instead a good competitive inhibitor (KI = 0.48 mM). In addition, we report that the olefinic substrate analogues trans-benzoylacrylic acid and 4-phenyl-3-butenoic acid are potent time-dependent inactivators of PAM, with inactivation exhibiting the characteristics expected for mechanism-based inhibition. Monoethyl fumarate is also a time-dependent inactivator of PAM. Finally, we introduce several small non-peptide substrates for PAM by demonstrating that PAM catalyzes the transformation of hippuric acid and several ring-substituted derivatives to the corresponding benzamides and glyoxylic acid, with the most facile substrate of this class being 4-nitrohippuric acid. These compounds are the smallest amide substrates yet reported for PAM, and it is thus apparent that only the minimal structure of an acylglycine is required for PAM-catalyzed oxygenative amidation.