Synthesis of LuxS inhibitors targeting bacterial cell-cell communication

[reaction: see text] Quorum sensing is a process by which bacteria sense cell density. This cell-cell communication process is mediated by autoinducers. A cross-species messenger, autoinducer-2 (AI-2) is produced from S-ribosyl-L-homocysteine by the LuxS enzyme. A proposed mechanism for LuxS is an aldose-ketose isomerization of S-ribosylhomocysteine followed by a beta-elimination. We report here the synthesis of two substrate analogues, S-anhydroribosyl-L-homocysteine and S-homoribosyl-L-cysteine, which prevent the initial and final step of the mechanism, respectively.     

Systematic synthesis of inhibitors of the two first enzymes of the bacterial heptose biosynthetic pathway: towards antivirulence molecules targeting lipopolysaccharide biosynthesis

L-Heptoses (L-glycero-D-manno-heptopyranoses) are constituents of the inner core of lipolysaccharide (LPS), a molecule playing key roles in the mortality of many infectious diseases as well as in the virulence of many human pathogens. The inhibition of the first enzymes of the bacterial heptose biosynthetic pathway is an almost unexplored field to date although it appears to be a very novel way for the development of antivirulence drugs. We report the synthesis of a series of D-glycero-D-manno-heptopyranose 7-phosphate (H7P) analogues and their inhibition properties against the isomerase GmhA and the the kinase HldE, the two first enzymes of the bacterial heptose biosynthetic pathway. The heptose structures have been modified at the 1-, 2-, 6- and 7-positions to probe the importance of the key structural features of H7P that allow a tight binding to the target enzymes; H7P being the product of GmhA and the substrate of HldE, the second objective was to find structures that could simultaneously inhibit both enzymes. We found that GmhA and HldE were extremely sensitive to structural modifications at the 6- and 7- positions of the heptose scaffold. To our surprise, the epimeric analogue of H7P displaying a D-glucopyranose configuration was found to be the best inhibitor of both enzymes but also the only molecule of this series that could inhibit GmhA (IC(50)=34 μM) and HldE (IC(50)=9.4 μM) in the low micromolar range. Noteworthy, this study describes the first inhibitors of GmhA ever reported, and paves the way to the design of a second generation of molecules targeting the bacterial virulence.     

Targeting bacterial virulence: inhibitors of type III secretion in Yersinia

Agents that target bacterial virulence without detrimental effect on bacterial growth are useful chemical probes for studies of virulence and potential candidates for drug development. Several gram-negative pathogens employ type III secretion to evade the innate immune response of the host. Screening of a chemical library with a luciferase reporter gene assay in viable Yersinia pseudotuberculosis furnished several compounds that inhibit the reporter gene signal expressed from the yopE promoter and effector protein secretion at concentrations with no or modest effect on bacterial growth. The selectivity patterns observed for inhibition of various reporter gene strains indicate that the compounds target the type III secretion machinery at different levels. Identification of this set of inhibitors illustrates the approach of utilizing cell-based assays to identify compounds that affect complex bacterial virulence systems.     

Rational design of inhibitors and activity-based probes targeting Clostridium difficile virulence factor TcdB

Clostridium difficile is a leading cause of nosocomial infections. The major virulence factors of this pathogen are the multi-domain toxins TcdA and TcdB. These toxins contain a cysteine protease domain (CPD) that autoproteolytically releases a cytotoxic effector domain upon binding intracellular inositol hexakisphosphate. Currently, there are no known inhibitors of this protease. Here, we describe the rational design of covalent small molecule inhibitors of TcdB CPD. We identified compounds that inactivate TcdB holotoxin function in cells and solved the structure of inhibitor-bound protease to 2.0??. This structure reveals the molecular basis of CPD substrate recognition and informed the synthesis of activity-based probes for this enzyme. The inhibitors presented will guide the development of therapeutics targeting C. difficile, and the probes will serve as tools for studying the unique activation mechanism of bacterial toxin CPDs.     

A mechanism-based inhibitor targeting the DD-transpeptidase activity of bacterial penicillin-binding proteins

Penicillin-binding proteins (PBPs) are responsible for the final stages of bacterial cell wall assembly. These enzymes are targets of beta-lactam antibiotics. Two of the PBP activities include dd-transpeptidase and DD-carboxypeptidase activities, which carry out the cross-linking of the cell wall and trimming of the peptidoglycan, the major constituent of the cell wall, by an amino acid, respectively. The activity of the latter enzyme moderates the degree of cross-linking of the cell wall, which is carried out by the former. Both these enzymes go through an acyl-enzyme species in the course of their catalytic events. Compound 6, a cephalosporin derivative incorporated with structural features of the peptidoglycan was conceived as an inhibitor specific for DD-transpeptidases. On acylation of the active sites of dd-transpeptidases, the molecule would organize itself in the two active site subsites such that it mimics the two sequestered strands of the bacterial peptidoglycan en route to their cross-linking. Hence, compound 6 is the first inhibitor conceived and designed specifically for inhibition of DD-transpeptidases. The compound was synthesized in 13 steps and was tested with recombinant PBP1b and PBP5 of Escherichia coli, a dd-transpeptidase and a dd-carboxypeptidase, respectively. Compound 6 was a time-dependent and irreversible inhibitor of PBP1b. On the other hand, compound 6 did not interact with PBP5, neither as an inhibitor (reversible or irreversible) nor as a substrate.     

Combating multidrug resistance in bacterial infection by targeting functional proteome with natural products

Antibiotic resistance has become a major clinical and public health problem within the lifetime of most people living today. Development of new therapeutic approaches to prevent antimicrobial chemotherapy from bacterial multidrug resistance has thus been becoming a primary consideration in the medicinal community. In this study, we describe a protocol that is potential for combating multidrug resistance by rational screening of natural medicines to target the bacterial functional proteome. To achieve this, a pipeline of integrating virtual screening and susceptibility testing has been described to identify antibacterial agents from various natural products with diverse structures and high drug-likeness. A number of promising candidates with potent antibacterial activity were identified, from which six available compounds were assayed to determine their susceptibility to four multidrug-resistant strains. Consequently, while most tested candidates showed moderate (20 < MIC < 50 μg/mL) or low (MIC>50 μg/mL) antibacterial activities, two natural products, i.e. pseudopterosin A and ciprofloxacin, were measured to possess strong broad-spectrum potency combating different strains (MIC < 20 μg/mL).     

Cell-based screening: a high throughput flow cytometry platform for identification of cell-specific targeting molecules

Targeting of drugs and genes to specific cell types is an emerging paradigm in the treatment of many medical conditions. However, targeting structures such as peptides are susceptible to rapid inactivation in vivo. To address this problem, novel targeting molecules can now be rapidly synthesized using a combinatorial approach. Methods to screen the large libraries created in this process are often lacking or compatible only with solution-based screening. This report describes a high-throughput cell-based method utilizing flow cytometry, capable of rapidly screening large libraries of molecules simultaneously for biological functionality and stability. In this method, each library molecule is attached to a microsphere exhibiting a unique set of optical properties, or "fingerprint", conferring modularity and multiplex capability. We investigated the multiplex capability of our flow cytometric method to determine its capacity for high-throughput screening. Current instrumentation in our laboratory allows the screening of at least 75 unique compounds in a single well, a number comparable to available solution-based assays. In state-of-the-art configuration, however, this methodology can support the screening of up to 1875 compounds per well, achieving high-throughput potential in a single multiwell plate. We also investigated the binding capability of targeted microspheres to adherent target cells. These microspheres exhibited a 12-fold increase in binding over control, untargeted microspheres. Competitive inhibition experiments with soluble ligand confirmed the specificity of microsphere binding. Overall, the methodology proposed here is capable of quickly and effectively screening large libraries of targeting molecules using instrumentation readily available to the greater research community.     

Design and synthesis of fluorescent pilicides and curlicides: bioactive tools to study bacterial virulence mechanisms

Pilicides and curlicides are compounds that block the formation of the virulence factors pili and curli, respectively. To facilitate studies of the interaction between these compounds and the pili and curli assembly systems, fluorescent pilicides and curlicides have been synthesized. This was achieved by using a strategy based on structure-activity knowledge, in which key pilicide and curlicide substituents on the ring-fused dihydrothiazolo 2-pyridone central fragment were replaced by fluorophores. Several of the resulting fluorescent compounds had improved activities as measured in pili- and curli-dependent biofilm assays. We created fluorescent pilicides and curlicides by introducing coumarin and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores at two positions on the peptidomimetic pilicide and curlicide central fragment. Fluorescence images of the uropathogenic Escherichia coli (UPEC) strain UTI89 grown in the presence of these compounds shows that the compounds are strongly associated with the bacteria with a heterogeneous distribution.     

Self-assembling bacterial pores as components of nanobiosensors for the detection of single peptide molecules

Nano-sized bacterial pores were inserted into a lipid membrane as a nanobiosensor for the detection of single peptide molecules. Due to the intrinsic properties of single-channel conductance, the transit of individual molecules through the pore can be studied. The analysis of both the blockage current and duration is able to provide specific structural information and allows the detection of specific peptides in bulk mixtures. Supported by the National Natural Science Foundation of China (Grant No. 20875030) and the Shuguang Project of Shanghai (Grant No. 07SG36)     

Cellular delivery of dinucleotides by conjugation with small molecules: targeting translation initiation for anticancer applications

Targeting cap-dependent translation initiation is one of the experimental approaches that could lead to the development of novel anti-cancer therapies. Synthetic dinucleoside 5′,5′-triphosphates cap analogs are potent antagonists of eukaryotic translation initiation factor 4E (eIF4E) in vitro and could counteract elevated levels of eIF4E in cancer cells; however, transformation of these compounds into therapeutic agents remains challenging – they do not easily penetrate into cells and are susceptible to enzymatic cleavage. Here, we tested the potential of several small molecule ligands – folic acid, biotin, glucose, and cholesterol – to deliver both hydrolyzable and cleavage-resistant cap analogs into cells. A broad structure–activity relationship (SAR) study using model fluorescent probes and cap–ligand conjugates showed that cholesterol greatly facilitates uptake of cap analogs without disturbing the interactions with eIF4E. The most potent cholesterol conjugate identified showed apoptosis-mediated cytotoxicity towards cancer cells.

Ligand assisted cellular delivery of negatively charged dinucleotides, which are potential antagonists of the protooncogenic protein eIF4E.     

Traffic jam at the bacterial sec translocase: targeting the SecA nanomotor by small-molecule inhibitors

The rapid rise of drug-resistant bacteria is one of the most serious unmet medical needs facing the world. Despite this increasing problem of antibiotic resistance, the number of different antibiotics available for the treatment of serious infections is dwindling. Therefore, there is an urgent need for new antibacterial drugs, preferably with novel modes of action to potentially avoid cross-resistance with existing antibacterial agents. In recent years, increasing attention has been paid to bacterial protein secretion as a potential antibacterial target. Among the different protein secretion pathways that are present in bacterial pathogens, the general protein secretory (Sec) pathway is widely considered as an attractive target for antibacterial therapy. One of the key components of the Sec pathway is the peripheral membrane ATPase SecA, which provides the energy for the translocation of preproteins across the bacterial cytoplasmic membrane. In this review, we will provide an overview of research efforts on the discovery and development of small-molecule SecA inhibitors. Furthermore, recent advances on the structure and function of SecA and their potential impact on antibacterial drug discovery will be discussed.     

De novo ligand design with explicit water molecules: an application to bacterial neuraminidase

Most computer-aided drug design methods ignore the presence of crystallographically-determined water molecules in the binding site of a target protein. In this paper, our de novo ligand design methods are applied to the X-ray crystal structure of bacterial neuraminidase in the presence of some selected water molecules. We have found that, for this particular protein, the complete removal of all bound water molecules leads to difficulties in generating any potential ligands if the unsatisfied hydrogen-bonding sitepoints left by removing these water molecules are to be satisfied by a ligand. As more of the crystallographically determined water molecules are allowed in the binding site, it becomes much easier to generate ligands in larger numbers and with wider chemical diversity. This example shows that, in some cases, bound water molecules can be more accessible for hydrogen bonding to an incoming ligand than the actual protein binding sitepoints associated with them. From the point of view of de novo ligand design, water molecules can thus act as versatile amphiprotic hydrogen-bonding sitepoints and reduce the conformational constraints of a particular binding site.     

An electrochemical sensor concept for the detection of bacterial virulence factors from Staphylococcus aureus and Pseudomonas aeruginosa

This work presents an electrochemical sensor concept for bacterial toxin detection using biomimetic lipid vesicles containing potassium ferricyanide. The toxin mediated release of redox couples from the vesicles was quantified by measuring the redox current on screen printed electrodes, and the detection limits to rhamnolipid, delta toxin and bacterial supernatants from clinical wound pathogens were examined. Overall detection limits of both redox and carboxyfluorescein containing vesicles were one order of magnitude lower than the cytotoxic dose of studied toxins to T lymphocytes.     

Small molecules targeting the NEDD8·NAE protein–protein interaction

Ubiquitination is a major controller of protein homeostasis in cells. Some ubiquitination pathways are modulated by a NEDDylation cascade, that also features E1 – 3 enzymes. The E1 enzyme in the NEDDylation cascade involves a protein–protein interaction (PPI) between NEDD8 (similar to ubiquitin) and NAE (NEDD8 Activating Enzyme). A small molecule inhibitor of the ATP binding site in NAE is in clinical trials. We hypothesized a similar effect could be induced by disrupting the NEDD8·NAE PPI, though, to the best of our knowledge, no small molecules have been reported to disrupt this to date. In the research described here, Exploring Key Orientations (EKO) was used to evaluate several chemotype designs for their potential to disrupt NEDD8·NAE; specifically, for their biases towards orientation of side-chains in similar ways to protein segments at the interface. One chemotype design was selected, and a targeted library of 24 compounds was made around this theme via solid phase synthesis. An entry level hit for disrupting NEDD8·NAE was identified from this library on the basis of its ability to bind NAE (K_i of 6.4 ± 0.3 μM from fluorescence polarization), inhibit NEDDylation, suppress formation of the corresponding E1 – 3 complexes as monitored by cell-based immunoblotting, and cytotoxicity to K562 leukemia cells via early stage apoptosis. The cell-based immunoblot assay also showed the compound caused NEDD8 to accumulate in cells, presumably due to inhibition of the downstream pathways involving the E1 enzyme. The affinity and cellular activities of the hit compound are modest, but is interesting as first in class for this mode of inhibition of NEDDylation, and as another illustration of the way EKO can be used to evaluate user-defined chemotypes as potential inhibitors of PPIs.

Discovery of the first NEDDylation inhibitor, that targets the NEDD8·NAE protein–protein interaction, was acheived using the Exploring Key Orientations (EKO) approach.     

Theoretical analysis of bacterial efflux pumps inhibitors: Strategies in-search of competent molecules and develop next

Multi-drug resistance (MDR) bacteria pose a significant threat to our ability to effectively treat infections due to the development of several antibiotic resistant mechanisms. A major component in the development of the MDR phenotype in MDR bacteria is over expression of different-type of efflux pumps, which actively pump out antibacterial agents and biocides from the periplasm to the outside of the cell. Consequently, bacterial efflux pumps are an important target for developing novel antibacterial treatments. Potent efflux pump inhibitors (EPIs) could be used as adjunctive therapies that would increase the potency of existing antibiotics and decrease the emergence of MDR bacteria. Several potent inhibitors of efflux pumps have been reported which has been summarized here. All the natural and synthetic EPIs were optimized with Gaussian and Avogadro software. The optimized structures were docked with each class of efflux pumps and their bonding parameters were computed. The theoretical analyses were performed with density functional theory (DFT). Overall, computational study revealed a good trend of electrophilicity and ionization potential of the EPIs, the obtained average values are within in the range of 0.001414 AU ± 0.00032 and 0.208821 AU ± 0.015545, respectively. Interestingly, cathinone interacts with most of the efflux pumps among the tested inhibitors. The electrophilicity and ionization potential of cathinone are 0.00198 and 0.2388 AU, respectively. The study opens a new road for designing future-generation target-specific efflux pump inhibitors, as well as one molecule with multiple inhibition abilities.     

Microwave-assisted synthesis of small molecules targeting the infectious diseases tuberculosis, HIV/AIDS, malaria and hepatitis C

The unique properties of microwave in situ heating offer unparalleled opportunities for medicinal chemists to speed up lead optimisation processes in early drug discovery. The technology is ideal for small-scale discovery chemistry because it allows full reaction control, short reaction times, high safety and rapid feedback. To illustrate these advantages, we herein describe applications and approaches in the synthesis of small molecules to combat four of the most prevalent infectious diseases; tuberculosis, HIV/AIDS, malaria and hepatitis C, using dedicated microwave instrumentation.     

Selection of peptides targeting helix 31 of bacterial 16S ribosomal RNA by screening M13 phage-display libraries

Ribosomal RNA is the catalytic portion of ribosomes, and undergoes a variety of conformational changes during translation. Structural changes in ribosomal RNA can be facilitated by the presence of modified nucleotides. Helix 31 of bacterial 16S ribosomal RNA harbors two modified nucleotides, m2G966 and m?C967, that are highly conserved among bacteria, though the degree and nature of the modifications in this region are different in eukaryotes. Contacts between helix 31 and the P-site tRNA, initiation factors, and ribosomal proteins highlight the importance of this region in translation. In this work, a heptapeptide M13 phage-display library was screened for ligands that target the wild-type, naturally modified bacterial helix 31. Several peptides, including TYLPWPA, CVRPFAL, TLWDLIP, FVRPFPL, ATPLWLK, and DIRTQRE, were found to be prevalent after several rounds of screening. Several of the peptides exhibited moderate affinity (in the high nM to low μM range) to modified helix 31 in biophysical assays, including surface plasmon resonance (SPR), and were also shown to bind 30S ribosomal subunits. These peptides also inhibited protein synthesis in cell-free translation assays.