Design,Synthesis and Biological Evaluation of Bengamide Analogues as ClpP Activators

To combat multidrug‐resistant Gram‐positive bacteria, new antimicrobials particularly those with novel mechanism of action are badly needed. Different with conventional antibiotics which are typical inhibitors, small‐molecule activators of bacterial ClpP represent a new class of antibiotics. No ClpP activator has been developed for clinical trial. Herein, we conducted a screening on our library of bengamide‐like ring‐opened analogues and found that L472‐2 possesses a low minimum inhibitory concentration (MIC) against S.aureus and shows no activity for ClpP activation in vitro, but it displayed reduced antibacterial activity against S. aureus with clpP deletion. In order to obtain bengamide analogues that activate ClpP in vitro as well as possess antibacterial activity, we perform further structural modifications starting from L472‐2 . Compound 37 remains the antimicrobial activity and activation of ClpP protein in vitro, which could be viewed as a new chemical scaffold for ClpP activators and worthy of further investigation.     

Reversible Inhibitors Arrest ClpP in a Defined Conformational State that Can Be Revoked by ClpX Association

Caseinolytic protease P (ClpP) is an important regulator of Staphylococcus aureus pathogenesis. A high‐throughput screening for inhibitors of ClpP peptidase activity led to the identification of the first non‐covalent binder for this enzyme class. Co‐crystallization of the small molecule with S. aureus ClpP revealed a novel binding mode: Because of the rotation of the conserved residue proline 125, ClpP is locked in a defined conformational state, which results in distortion of the catalytic triad and inhibition of the peptidase activity. Based on these structural insights, the molecule was optimized by rational design and virtual screening, resulting in derivatives exceeding the potency of previous ClpP inhibitors. Strikingly, the conformational lock is overturned by binding of ClpX, an associated chaperone that enables proteolysis by substrate unfolding in the ClpXP complex. Thus, regulation of inhibitor binding by associated chaperones is an unexpected mechanism important for ClpP drug development.     

Rapid detection of Staphylococcus aureus by a combination of monoclonal antibody-coated latex and capillary electrophoresis

The rapid detection of pathogenic bacteria is extremely important in biotechnology and clinical diagnosis. CE has been utilized in the field of bacterial analysis for many years, but to some extent, simultaneous separation and identification of certain microbes from complex samples by CE coupled with UV detector is still a challenge. In this paper, we propose a new strategy for rapid separation and identification of Staphylococcus aureus (S. aureus) in bacterial mixtures by means of specific mAb-coated latex coupled with CZE. An appropriate set of conditions that selectively isolated S. aureus from the microorganisms Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were established. S. aureus could be differentiated from the others by unique peaks in the electropherograms. The validity was also confirmed by LIF with antibodies specific to both the latex and the microbial cells. The LOD is as low as 9.0 x 10(5) colony forming unit/mL. We have also utilized this technology to identify S. aureus in a stool sample coming from a healthy volunteer spiked successfully with S. aureus. This CZE-UV technique can be applied to rapid diagnosis of enteritis caused by S. aureus or other bacterial control-related fields needing rapid identification of target pathogens from microbial mixtures. In theory, this method is suitable for the detection of any bacterium as long as corresponding bacterium-specific antibody-coated latex is available.     

Photoactivated antimicrobial activity of carbon nanotube-porphyrin conjugates

We report the design of antimicrobial nanocomposite films based on conjugates of multiwalled carbon nanotubes (MWNT) and protoporphyrin IX (PPIX) that are highly effective against Staphylococcus aureus (S. aureus) upon irradiation with visible light. S. aureus infections can lead to life-threatening situations, especially when caused by antibiotic-resistant strains. While the light-activated antimicrobial activity of porphyrins against such pathogens is well-known, a facile way to incorporate porphyrins into coatings may lead to their more effective use. To that end, we decided to synthesize and characterize MWNT-PPIX conjugates which combine the biocidal capacity of porphyrins with the mechanical strength of MWNTs. The conjugates could effectively deactivate S. aureus cells in solution upon irradiation with visible light. We also designed large area nanocomposite films comprised of the MWNT-PPIX conjugates that showed potent antimicrobial activity. These MWNT-PPIX conjugates represent a facile strategy for the design of antimicrobial and antifouling coatings.     

Co-opting the cell wall in fighting methicillin-resistant Staphylococcus aureus: potent inhibition of PBP 2a by two anti-MRSA beta-lactam antibiotics

Methicillin-resistant Staphylococcus aureus (MRSA) is a global bacterial scourge that has become resistant to many classes of antibiotics, and treatment options for MRSA infections are limited. The cause of MRSA resistance to all commercially available beta-lactam antibiotics is the acquisition of the gene mecA, which encodes penicillin-binding protein 2a (PBP 2a). PBP 2a is a transpeptidase, which in contrast to the other transpeptidases of S. aureus does not experience inhibition by beta-lactam antibiotics. The lack of inhibition is due to a closed conformation for the active site for PBP 2a, which opens up only in the course of the catalytic function of the protein. Here we show that two new anti-MRSA antibiotics now undergoing clinical trials, ceftaroline and ME1036, are able to inhibit PBP 2a effectively, a process that is enhanced in the presence of a cell wall structural surrogate. It is likely that in the course of bacterial growth the occupancy of the allosteric site for the cell wall is co-opted by these antibiotics, and under these conditions the second-order rate constant for the encounter of the antibiotic and PBP 2a approaches the clinically useful value of 10(4)-10(5) M-1 s-1. These compounds are potent inhibitors of PBP 2a as well as PBPs from other species, and have potential as therapeutic agents for treatment of serious infections by MRSA and other resistant bacterial pathogens.     

Highly Stable,Amide‐Bridged Autoinducing Peptide Analogues that Strongly Inhibit the AgrC Quorum Sensing Receptor in Staphylococcus aureus

Blocking quorum sensing (QS) pathways has attracted considerable interest as an approach to suppress virulence in bacterial pathogens. Toward this goal, we recently developed analogues of a native autoinducing peptide (AIP‐III) signal that can inhibit AgrC‐type QS receptors and attenuate virulence phenotypes in Staphylococcus aureus. Application of these compounds is limited, however, as they contain hydrolytically unstable thioester linkages and have only low aqueous solubilities. Herein, we report amide‐linked AIP analogues with greatly enhanced hydrolytic stabilities and solubilities relative to our prior analogues, whilst maintaining strong potencies as AgrC receptor inhibitors in S. aureus. These compounds represent powerful tools for the study of QS.     

Highly Stable,Amide‐Bridged Autoinducing Peptide Analogues that Strongly Inhibit the AgrC Quorum Sensing Receptor in Staphylococcus aureus

Blocking quorum sensing (QS) pathways has attracted considerable interest as an approach to suppress virulence in bacterial pathogens. Toward this goal, we recently developed analogues of a native autoinducing peptide (AIP‐III) signal that can inhibit AgrC‐type QS receptors and attenuate virulence phenotypes in Staphylococcus aureus. Application of these compounds is limited, however, as they contain hydrolytically unstable thioester linkages and have only low aqueous solubilities. Herein, we report amide‐linked AIP analogues with greatly enhanced hydrolytic stabilities and solubilities relative to our prior analogues, whilst maintaining strong potencies as AgrC receptor inhibitors in S. aureus. These compounds represent powerful tools for the study of QS.     

Assessing the Molecular Targets and Mode of Action of Furanone C-30 on Pseudomonas aeruginosa Quorum Sensing

Quorum sensing (QS), a sophisticated system of bacterial communication that depends on population density, is employed by many pathogenic bacteria to regulate virulence. In view of the current reality of antibiotic resistance, it is expected that interfering with QS can address bacterial pathogenicity without stimulating the incidence of resistance. Thus, harnessing QS inhibitors has been considered a promising approach to overriding bacterial infections and combating antibiotic resistance that has become a major threat to public healthcare around the globe. Pseudomonas aeruginosa is one of the most frequent multidrug-resistant bacteria that utilize QS to control virulence. Many natural compounds, including furanones, have demonstrated strong inhibitory effects on several pathogens via blocking or attenuating QS. While the natural furanones show no activity against P. aeruginosa, furanone C-30, a brominated derivative of natural furanone compounds, has been reported to be a potent inhibitor of the QS system of the notorious opportunistic pathogen. In the present study, we assess the molecular targets and mode of action of furanone C-30 on P. aeruginosa QS system. Our results suggest that furanone C-30 binds to LasR at the ligand-binding site but fails to establish interactions with the residues crucial for the protein’s productive conformational changes and folding, thus rendering the protein dysfunctional. We also show that furanone C-30 inhibits RhlR, independent of LasR, suggesting a complex mechanism for the agent beyond what is known to date.     

Tailored Peptide Phenyl Esters Block ClpXP Proteolysis by an Unusual Breakdown into a Heptamer–Hexamer Assembly

The proteolytic complex ClpXP is fundamental to bacterial homeostasis and pathogenesis. Because of its conformational flexibility, the development of potent ClpXP inhibitors is challenging, and novel tools to decipher its intricate regulation are urgently needed. Herein, we present amino acid based phenyl esters as molecular probes to study the activity and oligomerization of the ClpXP complex of S. aureus. Systematic screening of (R)‐ and (S)‐amino acids led to compounds showing potent inhibition, as well as stimulation of ClpXP‐mediated proteolysis. Substoichiometric binding of probes arrested ClpXP in an unprecedented heptamer–hexamer assembly, in which the two heptameric ClpP rings are dissociated from each other. At the same time, the affinity between ClpX and ClpP increased, leading to inhibition of both enzymes. This conformational arrest is beneficial for the consolidated shutdown of ClpXP, as well as for the study of the oligomeric state during its catalytic cycle.     

Identification of the Binding Site of Brucella VirB8 Interaction Inhibitors

Secretion systems translocate virulence factors of many bacterial pathogens, enabling their survival inside the host organism. Consequently, inhibition strongly attenuates pathogenicity and can be considered a target for novel antimicrobial drugs. The type IV secretion system (T4SS) of the intracellular pathogen Brucella is a prerequisite for its virulence, and in this work we targeted the interactions of the?essential assembly factor protein, VirB8, using small-molecule inhibitors. High-throughput screening identified several potent and specific inhibitors, and the target-binding site of these inhibitors was identified by X-ray crystallography, in?silico docking, and analysis of the derivates of the inhibitor B8I-2. VirB8 interaction inhibitors bind to a surface groove opposite to the dimerization interface, and by varying the binding-site residues, we were able to determine which residues are required for inhibitor activity. E115 and K182 were found to be especially important, and changes at R114, Y229, and L151 also reduced inhibitor efficiency.     

Infection control by antibody disruption of bacterial quorum sensing signaling

Quorum sensing (QS) is the process through which bacteria communicate utilizing small diffusible molecules termed autoinducers. It has been demonstrated that QS controls a plethora of microbial processes including the expression of virulence factors. Here we report an immunopharmacotherapeutic approach for the attenuation of QS in the Gram-positive human pathogen Staphylococcus aureus. An anti-autoinducer monoclonal antibody, AP4-24H11, was elicited against a rationally designed hapten, and efficiently inhibited QS in vitro through the sequestration of the autoinducing peptide (AIP)-4 produced by S. aureus RN4850. Importantly, AP4-24H11 suppressed S. aureus pathogenicity in an abscess formation mouse model in vivo and provided complete protection against a lethal S. aureus challenge. These findings provide a strong foundation for further investigations of immunopharmacotherapy for the treatment of bacterial infections in which QS controls the expression of virulence factors.     

Discovery of kibdelomycin, a potent new class of bacterial type II topoisomerase inhibitor by chemical-genetic profiling in Staphylococcus aureus

Bacterial resistance to known therapeutics has led to an urgent need for new chemical classes of antibacterial agents. To address this we have applied?a Staphylococcus aureus fitness test strategy to natural products screening. Here we report the discovery of kibdelomycin, a novel class of antibiotics produced by a new member of the genus Kibdelosporangium. Kibdelomycin exhibits broad-spectrum, gram-positive antibacterial activity and is a potent inhibitor of DNA synthesis. We demonstrate through chemical genetic fitness test profiling and biochemical enzyme assays that kibdelomycin is a structurally new class of bacterial type II topoisomerase inhibitor preferentially inhibiting the ATPase activity of DNA gyrase and topoisomerase IV. Kibdelomycin is thus the first truly novel bacterial type II topoisomerase inhibitor with potent antibacterial activity discovered from natural product sources in more than six decades.     

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.     

Polycyclic Polyprenylated Acylphloroglucinols: An Emerging Class of Non‐Peptide‐Based MRSA‐ and VRE‐Active Antibiotics

In the past 20 years, peptide‐based antibiotics, such as vancomycin, teicoplanin, and daptomycin, have often been considered as second‐line antibiotics. However, in recent years, an increasing number of reports on vancomycin resistance in pathogens appeared, which forces researchers to find novel lead structures for potent new antibiotics. Herein, we report the total synthesis of a defined endo‐type B PPAP library and their antibiotic activity against multiresistant S. aureus and various vancomycin‐resistant Enterococci . Four new compounds that combine high activities and low cytotoxicity were identified, indicating that the PPAP core might become a new non‐peptide‐based lead structure in antibiotic research.     

Secretory immunoglobulin a from health y human mothers' milk catalyzes nucleic acid hydrolysis

The human milk secretory immune system is known to be the first line of protection for the newborn infant against various pathogens. Secretory IgA (sIgA), the typical immunoglobulin found in secretions, can fight infections through many mechanisms. Using different methods, we have shown that sIgA from the milk of healthy women possesses DNAse and RNAse activities. The catalytic center is localized in the light chain of catalytic sIgA, while the DNA-binding center is predominantly formed by its heavy chain. The enzymic properties and substrate specificity of catalytic sIgA distinguish it from other known DNases and RNases. It is reasonable to assume, that the milk DNA- and RNA-hydrolyzing antibodies are capable not only of neutralizing viral and bacterial nucleic acids by binding these antigens as well as by hydrolyzing them. The DNA-hydrolyzing activity of Abs raises the possibility that these catalytic Abs may provide protective functions for the newborn through the hydrolysis of viral and bacterial nucleic acids.     

Antagonism of chemical genetic interaction networks resensitize MRSA to β-lactam antibiotics

Antibiotic drug resistance among hospital and community acquired methicillin resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current therapeutics. We describe a chemical genetic strategy using antisense interference to broadly identify new drug targets that potentiate the effects of existing antibiotics against both etiological classes of MRSA infection. Further, we describe the resulting chemical genetic interaction networks and highlight the prominent and overlapping target sets that restore MRSA susceptibility to penicillin, cephalosporins, and carbapenems. Pharmacological validation of this approach is the potent synergy between a known inhibitor to a member of this genetic potentiation network (GlmS) and a broad set of β-lactam antibiotics against methicillin resistant Staphylococci. Developing drug-like leads to these targets may serve as rational and effective combination agents when paired with existing β-lactam antibiotics to restore their efficacy against MRSA.     

Inhibition of Quorum-Sensing Regulator from Pseudomonas aeruginosa Using a Flavone Derivative

Quorum sensing (QS) is a cell-to-cell communication process that controls bacterial collective behaviors. The QS network regulates and coordinates bacterial virulence factor expression, antibiotic resistance and biofilm formation. Therefore, inhibition of the QS system is an effective strategy to suppress the bacterial virulence. Herein, we identify a phosphate ester derivative of chrysin as a potent QS inhibitor of the human pathogen Pseudomonas aeruginosa (P. aeruginosa) using a designed luciferase reporter assay. In vitro biochemical analysis shows that the chrysin derivative binds to the bacterial QS regulator LasR and abrogates its DNA-binding capability. In particular, the derivative exhibits higher anti-virulence activity compared to the parent molecule. All the results reveal the potential application of flavone derivative as an anti-virulence compound to combat the infectious diseases caused by P. aeruginosa.     

Combining Topology and Sequence Design for the Discovery of Potent Antimicrobial Peptide Dendrimers against Multidrug‐Resistant Pseudomonas aeruginosa

Multidrug‐resistant opportunistic bacteria, such as Pseudomonas aeruginosa, represent a major public health threat. Antimicrobial peptides (AMPs) and related peptidomimetic systems offer an attractive opportunity to control these pathogens. AMP dendrimers (AMPDs) with high activity against multidrug‐resistant clinical isolates of P. aeruginosa and Acinetobacter baumannii were now identified by a systematic survey of the peptide sequences within the branches of a distinct type of third‐generation peptide dendrimers. Combined topology and peptide sequence design as illustrated here represents a new and general strategy to discover new antimicrobial agents to fight multidrug‐resistant bacterial pathogens.