Non-Traditional Antibacterial Screening Approaches for the Identification of Novel Inhibitors of the Glyoxylate Shunt in Gram-Negative Pathogens

Antibacterial compounds that affect bacterial viability have traditionally been identified, confirmed, and characterized in standard laboratory media. The historical success of identifying new antibiotics via this route has justifiably established a traditional means of screening for new antimicrobials. The emergence of multi-drug-resistant (MDR) bacterial pathogens has expedited the need for new antibiotics, though many in the industry have questioned the source(s) of these new compounds. As many pharmaceutical companies'' chemical libraries have been exhaustively screened via the traditional route, we have concluded that all compounds with any antibacterial potential have been identified. While new compound libraries and platforms are being pursued, it also seems prudent to screen the libraries we currently have in hand using alternative screening approaches. One strategy involves screening under conditions that better reflect the environment pathogens experience during an infection, and identifying in vivo essential targets and pathways that are dispensable for growth in standard laboratory media in vitro. Here we describe a novel screening strategy for identifying compounds that inhibit the glyoxylate shunt in Pseudomonas aeruginosa, a pathway that is required for bacterial survival in the pulmonary environment. We demonstrate that these compounds, which were not previously identified using traditional screening approaches, have broad-spectrum antibacterial activity when they are tested under in vivo-relevant conditions. We also show that these compounds have potent activity on both enzymes that comprise the glyoxylate shunt, a feature that was supported by computational homology modeling. By dual-targeting both enzymes in this pathway, we would expect to see a reduced propensity for resistance development to these compounds. Taken together, these data suggest that understanding the in vivo environment that bacterial pathogens must tolerate, and adjusting the antibacterial screening paradigm to reflect those conditions, could identify novel antibiotics for the treatment of serious MDR pathogens.     

High-throughput screening for antimicrobial compounds using a 96-well format bacterial motility absorbance assay

There is a pressing need to develop new antimicrobial drugs because of the increasing resistance of pathogenic bacteria to existing antibiotics. The preliminary development and validation of a novel methodology for the high-throughput screening of antimicrobial compounds and inhibitors of bacterial motility is described. This method uses a bacterial motility swarming agar assay, combined with the use of offset inoculation of the wells in a standard, clear, 96-well plate, to enable rapid screening of compounds for potential antibiotic and antimotility properties with a standard absorbance microplate reader. Thus, the methodology should be compatible with 96-well laboratory automation technology used in drug discovery and chemical biology studies. To validate the screening method, the Genesis Plus structurally diverse library of 960 biologically active compounds was screened against a motile strain of the gram-negative bacterial pathogen Salmonella typhimurium. The average Z' value for the positive and negative motility controls on all 12 compound plates was 0.67 +/- 0.14, and the signal-to-baseline ratio calculated from the positive and negative controls was 5.9 +/- 1.1. A collection of 70 compounds with well-known antimicrobial properties was successfully identified using this assay.     

High-Throughput Screening for Novel Inhibitors of Neisseria gonorrhoeae Penicillin-Binding Protein 2

The increasing prevalence of N. gonorrhoeae strains exhibiting decreased susceptibility to third-generation cephalosporins and the recent isolation of two distinct strains with high-level resistance to cefixime or ceftriaxone heralds the possible demise of β-lactam antibiotics as effective treatments for gonorrhea. To identify new compounds that inhibit penicillin-binding proteins (PBPs), which are proven targets for β-lactam antibiotics, we developed a high-throughput assay that uses fluorescence polarization (FP) to distinguish the fluorescent penicillin, Bocillin-FL, in free or PBP-bound form. This assay was used to screen a 50,000 compound library for potential inhibitors of N. gonorrhoeae PBP 2, and 32 compounds were identified that exhibited >50% inhibition of Bocillin-FL binding to PBP 2. These included a cephalosporin that provided validation of the assay. After elimination of compounds that failed to exhibit concentration-dependent inhibition, the antimicrobial activity of the remaining 24 was tested. Of these, 7 showed antimicrobial activity against susceptible and penicillin- or cephalosporin-resistant strains of N. gonorrhoeae. In molecular docking simulations using the crystal structure of PBP 2, two of these inhibitors docked into the active site of the enzyme and each mediate interactions with the active site serine nucleophile. This study demonstrates the validity of a FP-based assay to find novel inhibitors of PBPs and paves the way for more comprehensive high-throughput screening against highly resistant strains of N. gonorrhoeae. It also provides a set of lead compounds for optimization of anti-gonococcal agents.     

Small molecule microarrays: recent advances and applications

Directed or exploratory drug development programs constantly seek robust screening platforms for the high fidelity identification and validation of potential targets. Small-molecule microarrays (SMMs) have risen to this call by elegantly forging the capability of combinatorial chemistry in producing myriad compounds with the powerful throughput afforded by microarrays. This synergism offers scientists a versatile tool for rapid compound analysis and discovery. Microarrays of small molecules have already been successfully applied in important areas ranging from protein profiling to the discovery of therapeutic leads. Recent interesting developments towards improved immobilization strategies and library creation methods, together with novel advances herein described, have set the stage for SMMs to take on wider and more routine applications in academia and industry. As a rapidly maturing technology, SMMs pave the way forward in high-throughput exploration, both in the identification of biologically significant natural and synthetic small molecules and in harnessing their vast potential towards medicinal and diagnostic applications.     

Marcel Faber Roundtable: Is our antibiotic pipeline unproductive because of starvation, constipation or lack of inspiration?

There are few new antibiotics in the pipeline today. The reasons may include starvation at the front of the pipeline due to inadequate sources of suitable compounds to screen coupled with poorly validated discovery methodologies. A successful antibiotic discovery approach in the past, based upon whole cell antibiotic screening of natural products from actinomycetes and fungi, eventually suffered from constipation in the middle of the pipeline due to rediscovery of known compounds, even though low throughput methodology was employed at the front end. The current lack of productivity may be attributed to the poor choice of strategies to address the discovery of new antibiotics. Recent applications of high throughput in vitro screening of individual antibacterial targets to identify lead compounds from combinatorial chemical libraries, traditional chemical libraries, and partially purified natural product extracts has not produced any significant clinical candidates. The solution to the current dilemma may be to return to natural product whole cell screening. For this approach to work in the current millennium, the process needs to be miniaturized to increase the throughput by orders of magnitude over traditional screening, and the rediscovery of known antibiotics needs to be minimized by methods that can be readily monitored and improved over time.     

Sources of novel antibiotics—aside the common roads

Microbial pathogens are becoming increasingly resistant to available treatments, and new antibiotics are badly needed, but the pipeline of compounds under development is scarce. Furthermore, the majority of antibiotics under development are improved derivatives of marketed compounds, which are at best only partially effective against prevailing resistance mechanisms. In contrast, antibiotics endowed with new mechanisms of action are expected to be highly effective against multi-drug resistant pathogens. In this review, examples are provided of new antibiotics classes in late discovery or clinical development, arising from three different avenues: (1) compounds discovered and never brought to market by large pharmaceutical companies; (2) old compounds reanalyzed and rejuvinated with today’s tools; and (3) newly discovered molecules. For each compound, we will briefly describe original discovery, mechanism of action, any known resistance, antimicrobial profile, and current status of development.     

Comparison of Microscopy and Alamar Blue Reduction in a Larval Based Assay for Schistosome Drug Screening

Background

In view of the current widespread use of and reliance on a single schistosomicide, praziquantel, there is a pressing need to discover and develop alternative drugs for schistosomiasis. One approach to this is to develop High Throughput in vitro whole organism screens (HTS) to identify hits amongst large compound libraries.

Methodology/Principal Findings

We have been carrying out low throughput (24-well plate) in vitro testing based on microscopic evaluation of killing of ex-vivo adult S. mansoni worms using selected compound collections mainly provided through the WHO-TDR Helminth Drug Initiative. To increase throughput, we introduced a similar but higher throughput 96-well primary in vitro assay using the schistosomula stage which can be readily produced in vitro in large quantities. In addition to morphological readout of viability we have investigated using fluorometric determination of the reduction of Alamar blue (AB), a redox indicator of enzyme activity widely used in whole organism screening. A panel of 7 known schistosome active compounds including praziquantel, produced diverse effects on larval morphology within 3 days of culture although only two induced marked larval death within 7 days. The AB assay was very effective in detecting these lethal compounds but proved more inconsistent in detecting compounds which damaged but did not kill. The utility of the AB assay in detecting compounds which cause severe morbidity and/or death of schistosomula was confirmed in testing a panel of compounds previously selected in library screening as having activity against the adult worms. Furthermore, in prospective library screening, the AB assay was able to detect all compounds which induced killing and also the majority of compounds designated as hits based on morphological changes.

Conclusion

We conclude that an HTS combining AB readout and image-based analysis would provide an efficient and stringent primary assay for schistosome drug discovery.     

Identification of novel natural inhibitor for NorM – a multidrug and toxic compound extrusion transporter – an insilico molecular modeling and simulation studies

The emergence of bacterial multidrug resistance is an increasing problem in treatment of infectious diseases. An important cause for the multidrug resistance of bacteria is the expression of multidrug efflux transporters. The multidrug and toxic compound extrusion (MATE) transporters are most recently recognized as unique efflux system for extrusion of antimicrobials and therapeutic drugs due to energy stored in either Na⁺ or H⁺ electrochemical gradient. In the present study, high throughput virtual screening of natural compound collections against NorM – a MATE transporter from Neisseria gonorrhea (NorM-NG) has been carried out followed by flexible docking. The molecular simulation in membrane environment has been performed for understanding the stability and binding energetic of top lead compounds. Results identified a compound from the Indian medicinal plant “Terminalia chebula” which has good binding free energy compared to substrates (rhodamine 6 g, ethidium) and more favorable interactions with the central cavity forming active site residues. The compound has restricted movement in TM7, TM8, and TM1, thus blocking the disruption of Na+ – coordination along with equilibrium state bias towards occlude state of NorM transporter. Thus, this compound blocks the effluxing pathway of antimicrobial drugs and provides as a natural bioactive lead inhibitor against NorM transporter in drug-resistant gonorrhea.     

Forward chemical genetics: progress and obstacles on the path to a new pharmacopoeia

Forward chemical genetics is a new method to systematize the discovery and use of small molecules as tools for basic biological research. This approach requires three basic components: a library of compounds; an assay, in which the library is screened for a cellular or organismal phenotype; and a method to trace an active compound to its biological target. Bioactive compounds have traditionally been isolated from natural product extracts, although 'diversity-oriented synthesis' and commercial compound collections are gaining in prominence. New techniques, such as image-based screening and the cytoblot method, have increased the throughput of phenotypic assays. Strategies are also being developed to streamline target identification using molecular biological approaches.     

A screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit

The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.     

David and Goliath: chemical perturbation of eukaryotes by bacteria

Environmental microbes produce biologically active small molecules that have been mined extensively as antibiotics and a smaller number of drugs that act on eukaryotic cells. It is known that there are additional bioactives to be discovered from this source. While the discovery of new antibiotics is challenged by the frequent discovery of known compounds, we contend that the eukaryote-active compounds may be less saturated. Indeed, despite there being far fewer eukaryotic-active natural products these molecules interact with a far richer diversity of molecular and cellular targets.     

Driving efficiency in a high-throughput metabolic stability assay through a generic high-resolution accurate mass method and automated data mining

Improving analytical throughput is the focus of many quantitative workflows being developed for early drug discovery. For drug candidate screening, it is common practice to use ultra-high performance liquid chromatography (U-HPLC) coupled with triple quadrupole mass spectrometry. This approach certainly results in short analytical run time; however, in assessing the true throughput, all aspects of the workflow needs to be considered, including instrument optimization and the necessity to re-run samples when information is missed. Here we describe a high-throughput metabolic stability assay with a simplified instrument set-up which significantly improves the overall assay efficiency. In addition, as the data is acquired in a non-biased manner, high information content of both the parent compound and metabolites is gathered at the same time to facilitate the decision of which compounds to proceed through the drug discovery pipeline.     

Evaluation of penicillin-based inhibitors of the class A and B beta-lactamases from Bacillus anthracis

Bacillus anthracis contains a class A (Bla1) and class B (Bla2) beta-lactamase, which confer resistance to beta-lactam antibiotics when expressed in Escherichia coli. In an effort to find new beta-lactamase inhibitors, several penicillin derivatives have been evaluated including experimental compounds incorporating a 6-mercaptomethyl group or a 6-pyridylmethylidene group, along with clavulanate and tazobactam, as inhibitors against Bla1 and Bla2. The 6-mercaptomethyl-substituted penicillins showed much greater activity against the zinc-containing Bla2 than Bla1. The compound that incorporated a 6-pyridylmethylidene substituent and a catecholic substituent at the 2' position was the most effective inhibitor of Bla1 with Ki=0.057 microM. Inhibitors containing iron-chelating functional groups have previously been shown to work in combination with antibiotics to inhibit growth of antibiotic-resistant bacteria expressing beta-lactamase. The development of similar compounds, incorporating these types of substituents, may help overcome resistance to currently used antibiotics.     

Small Molecule Screening in Human Induced Pluripotent Stem Cell-derived Terminal Cell Types

A need for better clinical outcomes has heightened interest in the use of physiologically relevant human cells in the drug discovery process. Patient-specific human induced pluripotent stem cells may offer a relevant, robust, scalable, and cost-effective model of human disease physiology. Small molecule high throughput screening in human induced pluripotent stem cell-derived cells with the intent of identifying novel therapeutic compounds is starting to influence the drug discovery process; however, the use of these cells presents many high throughput screening development challenges. This technology has the potential to transform the way drug discovery is performed.     

Identification of plasmepsin inhibitors as selective anti-malarial agents using ligand based drug design

We describe the application of ligand based virtual screening technologies towards the discovery of novel plasmepsin (PM) inhibitors, a family of malarial parasitic aspartyl proteases. Pharmacophore queries were used to screen vendor libraries in search of active and selective compounds. The virtual hits were biologically assessed for activity and selectivity using whole cell Plasmodium falciparum parasites and on target in PM II, PM IV and the closely related human homologue, Cathepsin D assays. Here we report the virtual screening highlights, structures of the hits and their demonstrated biological activity.     

A place for high-throughput electrophysiology in cardiac safety: screening hERG cell lines and novel compounds with the ion works HTTM system

Several commercially available pharmaceutical compounds have been shown to block the IKr current of the cardiac action potential. This effect can cause a prolongation of the electrocardiogram QT interval and a delay in ventricular repolarization. The Food and Drug Administration recommends that all new potential drug candidates be assessed for IKr block to avoid a potentially lethal cardiac arrhythmia known as torsades de pointes. Direct compound interaction with the human ether-a-go-go- related gene (hERG) product, a delayed rectifier potassium channel, has been identified as a molecular mechanism of IKr block. One strategy to identify compounds with hERG liability is to monitor hERG current inhibition using electrophysiology techniques. The authors describe the Ion Works HT instrument as a tool for screening cell lines expressing hERG channels. Based on current amplitude and stability criteria, a cell line was selected and used to perform a 300-compound screen. The screen was able to identify compounds with hERG activity within projects that spanned different therapeutic areas. The cell line selection and optimization, as well as the screening abilities of the Ion Works HT system, provide a powerful means of assessing hERGactive compounds early in the drug discovery pipeline.     

New principles of ion exchange preparative chromatography and their applications for isolation, purification and super purification of antibiotics]

New types of structurally segregated heteronetwork biosorbents with given parameters of heterogeneity and porosity have been developed. Physico-chemical characteristics of the biosorbents on the basis of which one can predict optimal structures of ion exchangers to be used in preparative chromatography of biologically active compounds were studied. A new principle of sequential displacement of ions of organic compounds, in particular antibiotics, adsorbed on selective biosorbents with a high adsorption capacity was developed, which enables purification and superpurification of the desired compound. The method is based on the effect of small thermodynamic shifts in physico-chemical parameters of the elution system, which results in preparative separation of substances with close properties and in purification of the desired compound from microadmixtures. The "small shift effect" is realized in the case of limiting thermodynamic and kinetic parameters of heterogeneous mass exchange and with biosorbents possessing a highly selective adsoprtion capacity.     

Chalcone derivatives and their antibacterial activities: Current development

The increase in antibiotic resistance due to various factors has encouraged the look for novel compounds which are active against multidrug-resistant pathogens. In this framework, chalcone-based compounds showed a diversity of pharmacological properties, and its derivatives possess a high degree of structural diversity, and it is helpful for the discovery of new therapeutic agents. The growing resistance to antibiotics worldwide has endangered their efficacy. This has led to a surging interest in the discovery of new antibacterial agents. Thus, there is an urgent need for new antibacterial drug candidates with increased strength, new targets, low cost, superior pharmacokinetic properties, and minimum side effects. The present review concluded and focuses on the recent developments in the area of medicinal chemistry to explore the diverse chemical structures of potent antibacterial agents and also describes its structure-activity relationships studies. The various synthetic structures leading to this class of neutral protective compound is common and additional structural optimization is promising for potential drug discovery and development.     

Transgenic parasites accelerate drug discovery

Parasitic neglected diseases are in dire need of new drugs either to replace old drugs rendered ineffective because of resistance development, to cover clinical needs that had never been addressed or to tackle other associated problems of existing drugs such as high cost, difficult administration, restricted coverage or toxicity. The availability of transgenic parasites expressing reporter genes facilitates the discovery of new drugs through high throughput screenings, but also by allowing rapid screening in animal models of disease. Taking advantage of these, we propose an alternative pathway of drug development for neglected diseases, going from high throughput screening directly into in vivo testing of the top ranked compounds selected by medicinal chemistry. Rapid assessment animal models allow for identification of compounds with bona fide activity in vivo early in the development chain, constituting a solid basis for further development and saving valuable time and resources.     

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

Background

A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma.

Methods

Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds.

Results

Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo.

Conclusions

This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.