The evolution of antibacterial chemotherapy: targeting RecA to sabotage antibiotic tolerance and resistance mechanisms

Antibiotic resistant bacteria are rendering the current supply of available antibacterial drugs ineffective at an alarming rate and there is a dearth of novel drug targets for the treatment of bacterial infectious diseases. New strategies are required to combat pathogenic bacteria and in this context RecA has emerged as an intriguing candidate for inhibition studies. In the bacterial kingdom, the RecA protein is a highly conserved recombinase enzyme that mediates DNA repair and horizontal gene transfer and across all species it almost uniformly regulates the SOS response to DNA damage. Recent evidence suggests that these RecA-controlled processes are responsible for an increased tolerance to antibiotic chemotherapy and they up-regulate pathways which ultimately lead to full-fledged antibiotic resistance. We propose targeting RecA with small molecules to sabotage the molecular mechanisms which are believed to cause antibiotic chemotherapy to fail. Towards the goal of validating RecA as an important and novel target for the chemotherapeutic treatment of bacterial infectious diseases we have studied the interaction of metal-dithiols, nucleotide analogs and drug-like small molecules with the RecA protein. Upon activation RecA binds ssDNA and performs ATP hydrolysis, therefore we observed either a reduction of RecA-ssDNA binding or ATP hydrolysis in the presence of potential inhibitors using fast and efficient screening assays. As the size and complexity of the compound libraries increased in our studies, the methods we employed to identify inhibitors evolved to meet the demand they imposed. In all, more than 64,000 compounds were assayed against RecA and we identified several lead structures which were active against RecA in Escherichia coli cell cultures. We demonstrate that cell-permeable inhibitors of RecA are capable of abrogating the SOS response and potentiate the toxicity of bactericidal antibiotics, e.g. ciprofloxacin. The results of this study suggests that RecA may serve as a novel antibacterial drug target not belonging to any class of currently prescribed antibiotics, and which has not previously been examined in this regard

Directed molecular screening for RecA ATPase inhibitors

The roles of bacterial RecA in the evolution and transmission of antibiotic resistance genes make it an attractive target for inhibition by small molecules. We report two complementary fluorescence-based ATPase assays that were used to screen for inhibitors of RecA. We elected to employ the ADP-linked variation of the assay, with a Z′ factor of 0.83 in 96-well microplates, to assess whether 18 select compounds could inhibit ATP hydrolyis by RecA. The compounds represented five sets of related inhibitor scaffolds, each of which had the potential to cross-inhibit RecA. Although nucleotide analogs, known inhibitors of GHL ATPases, and known protein kinase inhibitors were not active against RecA, we found that three suramin-like agents substantially inhibited RecA's ATPase activity

A complementary pair of rapid molecular screening assays for RecA activities

The bacterial RecA protein has been implicated in the evolution of antibiotic resistance in pathogens, which is an escalating problem worldwide. The discovery of small molecules that can selectively modulate RecA’s activities can be exploited to tease apart its roles in the de novo development and transmission of antibiotic resistance genes. Toward the goal of discovering small-molecule ligands that can prevent either assembly of an active RecA-DNA filament or its subsequent ATP-dependent motor activities, we report the design and initial validation of a pair of rapid and robust screening assays suitable for the identification of inhbitors of RecA activities. One assay is based on established methods for monitoring ATPase enzyme activity and the second is a novel assay for RecA-DNA filament assembly using fluorescence polarization. Taken together, the assay results reveal complementary sets of agents that can either selectively suppress only the ATP-driven motor activities of the RecA-DNA filament or prevent assembly of active RecA-DNA filaments altogether. The screening assays can be readily configured for use in future automated HTS projects to discover potent inhibitors that may be developed into novel adjuvants for antibiotic chemotherapy that moderate the development and transmission of antibiotic resistance genes and increase the antibiotic therapeutic index

Biophysical Probes Reveal a “Compromise” Nature of the Methyl-lysine Binding Pocket in L3MBTL1

Histone lysine methylation (Kme) encodes essential information modulating many biological processes including gene expression and transcriptional regulation. However, the atomic-level recognition mechanisms of methylated histones by their respective adaptor proteins are still elusive. For instance, it is unclear how L3MBTL1, a methyl-lysine histone code reader, recognizes equally well both mono- and di-methyl marks, but ignores unmodified and trimethylated lysine residues. We made use of Molecular Dynamics (MD) and Free Energy Perturbation (FEP) techniques in order to investigate the energetics and dynamics of the methyllysine recognition. Isothermal Titration Calorimetry (ITC) was employed to experimentally validate the computational findings. Both computational and experimental methods were applied to a set of designed “biophysical” probes that mimic the shape of a single lysine residue and reproduce the binding affinities of cognate histone peptides. Our results suggest that, besides forming favorable interactions, the L3MBTL1 binding pocket energetically penalizes both methylation states and has most probably evolved as a “compromise” that non-optimally fit to both mono- and di-methyl-lysine marks

Novel Inhibitors of E. coli RecA ATPase Activity

The bacterial RecA protein has been implicated as a bacterial drug target not as an antimicrobial target, but as an adjuvant target with the potential to suppress the mechanism by which bacteria gain drug resistance. In order to identify small molecules that inhibit RecA/ssDNA nucleoprotein filament formation, we have adapted the phosphomolybdate-blue ATPase assay for high throughput screening to determine RecA ATPase activity against a library of 33,600 compounds, which is a selected representation of diverse structure of 350,000. Four distinct chemotypes were represented among the 40 validated hits. SAR and further chemical synthesis is underway to optimize this set of inhibitors to be used as antimicrobial adjuvant agents

Identification of Non-Peptide Malignant Brain Tumor (MBT) Repeat Antagonists by Virtual Screening of Commercially Available Compounds

The Malignant Brain Tumor (MBT) repeat is an important epigenetic-code “reader” and is functionally associated with differentiation, gene silencing and tumor suppression1–3. Small molecule probes of MBT domains should enable a systematic study of MBT-containing proteins, and potentially reveal novel druggable targets. We designed and applied a virtual screening strategy, which identified potential MBT antagonists in a large database of commercially available compounds. A small set of virtual hits was purchased and submitted to experimental testing. Nineteen of the purchased compounds showed a specific dose-dependent protein binding and will provide critical structure-activity information for subsequent lead generation and optimization

Accessing Protein Methyltransferase and Demethylase Enzymology Using Microfluidic Capillary Electrophoresis

The discovery of small molecules targeting the > 80 enzymes that add (methyltransferases) or remove (demethylases) methyl marks from lysine and arginine residues, most notably present in histone tails, may yield unprecedented chemotherapeutic agents and facilitate regenerative medicine. To better enable chemical exploration of these proteins, we have developed a novel and highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes and the kinetics of their inhibition. This technology separates small biomolecules, i.e., peptides, based on their charge-to-mass ratio. Methylation, however, does not alter the charge of peptide substrates. To overcome this limitation, we have employed a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules

Small-molecule ligands of methyl-lysine binding proteins: Optimization of selectivity for L3MBTL3

Proteins which bind methylated lysines (“readers” of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first co-crystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design

Resistance gene expression determines the in vitro chemosensitivity of non-small cell lung cancer (NSCLC)

Background NSCLC exhibits considerable heterogeneity in its sensitivity to chemotherapy and similar heterogeneity is noted in vitro in a variety of model systems. This study has tested the hypothesis that the molecular basis of the observed in vitro chemosensitivity of NSCLC lies within the known resistance mechanisms inherent to these patients' tumors. Methods The chemosensitivity of a series of 49 NSCLC tumors was assessed using the ATP-based tumor chemosensitivity assay (ATP-TCA) and compared with quantitative expression of resistance genes measured by RT-PCR in a Taqman Array™ following extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissue. Results There was considerable heterogeneity between tumors within the ATP-TCA, and while this showed no direct correlation with individual gene expression, there was strong correlation of multi-gene signatures for many of the single agents and combinations tested. For instance, docetaxel activity showed some dependence on the expression of drug pumps, while cisplatin activity showed some dependence on DNA repair enzyme expression. Activity of both drugs was influenced more strongly still by the expression of anti- and pro-apoptotic genes by the tumor for both docetaxel and cisplatin. The doublet combinations of cisplatin with gemcitabine and cisplatin with docetaxel showed gene expression signatures incorporating resistance mechanisms for both agents. Conclusion Genes predicted to be involved in known mechanisms drug sensitivity and resistance correlate well with in vitro chemosensitivity and may allow the definition of predictive signatures to guide individualized chemotherapy in lung cancer