Sandhya Bansal

Contribution of mutations in DNA gyrase and topoisomerase IV genes to ciprofloxacin resistance in Escherichia coli clinical isolates

DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) are the two essential type II topoisomerases in Escherichia coli. These enzymes act via inhibition of DNA replication. Mutations in the quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC and parE genes from clinical isolates of E. coli were determined by DNA sequencing of 54 ciprofloxacin-resistant clinical isolates from a hospital in Delhi, India. The majority of the E. coli isolates were shown to carry mutations in gyrA, parC and parE. Ciprofloxacin resistance due to accumulation of such a high number of mutations in the QRDR regions of gyrA at positions Ser83 and Asp87 and parC at position Ser80 as well as outside of the QRDR region of parE at Ser458 and Glu460 confers high-level resistance of ciprofloxacin in clinical isolates. The high frequency of occurrence of mutations in the parE gene (44.4% strains) is alarming, as topoisomerase IV is a secondary target of quinolones.

Design, Synthesis, and Mechanistic Investigations of Bile Acid–Tamoxifen Conjugates for Breast Cancer Therapy

We have synthesized two series of bile acid tamoxifen conjugates using three bile acids lithocholic acid (LCA), deoxycholic acid (DCA), and cholic acid (CA). These bile acid-tamoxifen conjugates possess 1, 2, and 3 tamoxifen molecules attached to hydroxyl groups of bile acids having free acid and amine functionalities at the tail region of bile acids. The in vitro anticancer activities of these bile acid-tamoxifen conjugates show that the free amine headgroup based cholic acid-tamoxifen conjugate (CA-Tam3-Am) is the most potent anticancer conjugate as compared to the parent drug tamoxifen and other acid and amine headgroup based bile acid-tamoxifen conjugates. The cholic acid-tamoxifen conjugate (CA-Tam3-Am) bearing three tamoxifen molecules shows enhanced anticancer activities in both estrogen receptor +ve and estrogen receptor -ve breast cancer cell lines. The enhanced anticancer activity of CA-Tam3-Am is due to more favorable irreversible electrostatic interactions followed by intercalation of these conjugates in hydrophobic core of membrane lipids causing increase in membrane fluidity. Annexin-FITC based FACS analysis showed that cells undergo apoptosis, and cell cycle analysis showed the arrest of cells in sub G0 phase. ROS assays showed a high amount of generation of ROS independent of ER status of the cell line indicating changes in mitochondrial membrane fluidity upon the uptake of the conjugate that further leads to the release of cytochrome c, a direct and indirect regulator of ROS. The mechanistic studies for apoptosis using PCR and western analysis showed apoptotsis by intrinsic and extrinsic pathways in ER +ve MCF-7 cells and by only an intrinsic pathway in ER -ve cells. In vivo studies in the 4T1 tumor model showed that CA-Tam3-Am is more potent than tamoxifen. These studies showed that bile acids provide a new scaffold for high drug loading and that their anticancer activities strongly depend on charge and hydrophobicity of lipid-drug conjugates.

Deciphering the role of charge, hydration, and hydrophobicity for cytotoxic activities and membrane interactions of bile acid based facial amphiphiles

We synthesized four cationic bile acid based facial amphiphiles featuring trimethyl ammonium head groups. We evaluated the role of these amphiphiles for cytotoxic activities against colon cancer cells and their membrane interactions by varying charge, hydration and hydrophobicity. The singly charged cationic Lithocholic acid based amphiphile (LCA-TMA1) is most cytotoxic, whereas the triply charged cationic Cholic acid based amphiphile (CA-TMA3) is least cytotoxic. Light microscopy and Annexin-FITC assay revealed that these facial amphiphiles caused late apoptosis. In addition, we studied the interactions of these amphiphiles with model membrane systems by Prodan-based hydration, DPH-based anisotropy, and differential scanning calorimetry. LCA-TMA1 is most hydrophobic with a hard charge causing efficient dehydration and maximum perturbations of membranes thereby facilitating translocation and high cytotoxicity against colon cancer cells. In contrast, the highly hydrated and multiple charged CA-TMA3 caused least membrane perturbations leading to low translocation and less cytotoxicity. As expected, Chenodeoxycholic acid and Deoxycholic acid based amphiphiles (CDCA-TMA2, DCA-TMA2) featuring two charged head groups showed intermediate behavior. Thus, we deciphered that charge, hydration, and hydrophobicity of these amphiphiles govern membrane interactions, translocation, and resulting cytoxicity against colon cancer cells.

Cell Penetrating Synthetic Antimicrobial Peptides (SAMPs) Exhibiting Potent and Selective Killing of Mycobacterium by Targeting Its DNA

Naturally occurring antimicrobial peptides (AMPs) are powerful defence tools to tackle pathogenic microbes. However, limited natural production and high synthetic costs in addition to poor selectivity limit large-scale use of AMPs in clinical settings. Here, we present a series of synthetic AMPs (SAMPs) that exhibit highly selective and potent killing of Mycobacterium (minimum inhibitory concentration <20 μg mL(-1)) over E. coli or mammalian cells. These SAMPs are active against rapidly multiplying as well as growth saturated Mycobacterium cultures. These SAMPs are not membrane-lytic in nature, and are readily internalized by Mycobacterium and mammalian cells; whereas in E. coli, the lipopolysaccharide layer inhibits their cellular uptake, and hence, their antibacterial action. Upon internalization, these SAMPs interact with the unprotected genomic DNA of mycobacteria, and impede DNA-dependent processes, leading to bacterial cell death.

3,4-Dimethoxyphenyl bis-benzimidazole, a novel DNA topoisomerase inhibitor that preferentially targets Escherichia coli topoisomerase I

OBJECTIVES Antibiotic resistance in bacterial pathogens is a serious clinical problem. Novel targets are needed to combat increasing drug resistance in Escherichia coli. Our objective is to demonstrate that 2-(3,4-dimethoxyphenyl)-5-[5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2yl]-1H-benzimidazole (DMA) inhibits E. coli DNA topoisomerase I more strongly than human topoisomerase I. In addition, DMA is non-toxic to mammalian cells at antibiotic dosage level. METHODS In the present study, we have established DMA as an antibacterial compound by determining MICs, post-antibiotic effects (PAEs) and MBCs for different standard as well as clinical strains of E. coli. We have described the differential catalytic inhibitory mechanism of bis-benzimidazole, DMA, for human and E. coli topoisomerase I and topoisomerase II by performing different assays, including relaxation assays, cleavage-religation assays, DNA unwinding assays, ethidium bromide displacement assays, decatenation assays and DNA gyrase supercoiling assays. RESULTS DMA significantly inhibited bacterial growth at a very low concentration, but did not affect human cell viability at higher concentrations. Activity assays showed that it preferentially targeted E. coli topoisomerase I over human topoisomerase I, topoisomerase II and gyrase. Cleavage-religation assays confirmed DMA as a poison inhibitor of E. coli topoisomerase I. This study illuminates new properties of DMA, which may be further modified to develop an efficient topoisomerase inhibitor that is selective towards bacterial topoisomerase I. CONCLUSIONS This is the first report of a bis-benzimidazole acting as an E. coli topoisomerase I inhibitor. DMA is a safe, non-cytotoxic molecule to human cells at concentrations that are needed for antibacterial activity.

Synthesis, structure–activity relationship, and mechanistic investigation of lithocholic acid amphiphiles for colon cancer therapy

We report a structure-activity relationship of lithocholic acid amphiphiles for their anticancer activities against colon cancer. We synthesized ten cationic amphiphiles differing in nature of cationic charged head groups using lithocholic acid. We observed that anticancer activities of these amphiphiles against colon cancer cell lines are contingent on nature of charged head group. Lithocholic acid based amphiphile possessing piperidine head group (LCA-PIP1 ) is ~10 times more cytotoxic as compared to its precursor. Biochemical studies revealed that enhanced activity of LCA-PIP1 as compared to lithocholic acid is due to greater activation of apoptosis.LCA-PIP1 induces sub G0 arrest and causes cleavage of caspases. A single dose of lithocholic acid-piperidine derivative is enough to reduce the tumor burden by 75% in tumor xenograft model.

Bile acid amphiphiles with tunable head groups as highly selective antitubercular agents

Tuberculosis faces major challenges for its cure due to (a) long treatment period, (b) emergence of drug resistance bacteria, and (c) poor patient compliance. Disrupting the membrane integrity of mycobacteria as a therapeutic strategy has not been explored well as the rigid, waxy, and hydrophobic nature of mycobacterial lipids does not allow binding and penetration of charged amtimicrobial amphiphiles and peptides. Here, we present a new concept that fine-tuning of the charged head group modulates the specificity of amphiphiles against bacterial membranes. We show that hard-charged amphiphiles interact with mycobacterial trehalose dimycolates and penetrate through rigid mycobacterial membranes. In contrast, soft-charged amphiphiles specifically inhibit the growth of both E. coli and S. aureus via electrostatic interactions. These subtle variations between interactions of amphiphiles and bacterial membranes could be explored further to design more specific and potent antimycobacterial agents.

Nature of the charged head group dictates the anticancer potential of lithocholic acid-tamoxifen conjugates for breast cancer therapy

Modulation of existing drugs is required to achieve enhanced activity for cancer therapy by lowering their effective dose. Strategies for introduction of cationic charge and hydrophobicity have been proposed and explored to enhance the therapeutic effects of anticancer drugs. In this manuscript, we planned modulation of tamoxifen and synthesized eight tamoxifen (Tam) conjugated lithocholic acid (LCA) amphiphiles with variable cationic charged head groups. We determined the anticancer potential of these amphiphiles against different breast cancer cell lines. Activity of these amphiphiles is contingent on nature of the charged head group, as hard-charged amphiphiles exhibit strong membrane interactions and enhanced anticancer activity compared to soft-charged amphiphiles. In-depth mechanistic studies concluded that conjugation of a dimethyl amino pyridine (DMAP) charged head group in case of LCA-Tam-DMAP enhances therapeutic effect of Tam in breast cancer cells, and makes it highly effective even against ER negative cells. Amphiphilic character of these lipid-drug conjugates can be further explored for engineering nanotherapeutics for targeting tumors. Therefore, fine-tuning the interactions of drugs with cell membranes can help in engineering future lipid-drug conjugates for effective cancer therapy.

Topoisomerases: Resistance versus Sensitivity, How Far We Can Go?

DNA topoisomerases are ubiquitously present remarkable molecular machines that help in altering topology of DNA in living cells. The crucial role played by these nucleases during DNA replication, transcription, and recombination vis‐à‐vis less sequence similarity among different species makes topoisomerases unique and attractive targets for different anticancer and antibacterial drugs. However, druggability of topoisomerases by the existing class of molecules is increasingly becoming questationable due to resistance development predominated by mutations in the corresponding genes. The current scenario facing a decline in the development of new molecules further comprises an important factor that may challenge topoisomerase‐targeting therapy. Thus, it is imperative to wisely use the existing inhibitors lest with this rapid rate of losing grip over the target we may not go too far. Furthermore, it is important not only to design new molecules but also to develop new approaches that may avoid obstacles in therapies due to multiple resistance mechanisms. This review provides a succinct account of different classes of topoisomerase inhibitors, focuses on resistance acquired by mutations in topoisomerases, and discusses the various approaches to increase the efficacy of topoisomerase inhibitors. In a later section, we also suggest the possibility of using bisbenzimidazoles along with efflux pump inhibitors for synergistic bactericidal effects.