Ruben C. Hartkoorn

Cross-Resistance between Clofazimine and Bedaquiline through Upregulation of MmpL5 in Mycobacterium tuberculosis

ABSTRACT The antileprosy drug clofazimine is also of interest for the treatment of multidrug-resistant tuberculosis. To understand possible resistance mechanisms, clofazimine-resistant Mycobacterium tuberculosis mutants were isolated in vitro, and, unexpectedly, found to be cross-resistant to bedaquiline. Mutations in the transcriptional regulator Rv0678, with concomitant upregulation of the multisubstrate efflux pump, MmpL5, accounted for this cross-resistance. Mutation in Rv0678 should therefore be considered a confounding factor for the treatment of tuberculosis with clofazimine or bedaquiline.

HIV protease inhibitors are substrates for OATP1A2, OATP1B1 and OATP1B3 and lopinavir plasma concentrations are influenced by SLCO1B1 polymorphisms

Objective OATP1B1 and OATP1B3 are major hepatic drug transporters whilst OATP1A2 is mainly located in the brain but is also located in liver and several other organs. These transporters affect the distribution and clearance of many endobiotics and xenobiotics and have been reported to have functional single nucleotide polymorphisms (SNPs). We have assessed the substrate specificities of these transporters for a panel of antiretrovirals and investigated the effects of SNPs within these transporters on the pharmacokinetics of lopinavir. Methods SLCO1A2, SLCO1B1 and SLCO1B3 were cloned, verified and used to generate cRNA for use in the Xenopuslaevis oocyte transport system. Using the oocyte system, antiretrovirals were tested for their substrate specificities. Plasma samples (n=349) from the Liverpool therapeutic drug monitoring registry were genotyped for SNPs in SLCO1A2, SLCO1B1 and SLCO1B3 and associations between SNPs and lopinavir plasma concentrations were analysed. Result Antiretroviral protease inhibitors, but not non-nucleoside reverse transcriptase inhibitors, are substrates for OATP1A2, OATP1B1 and OATP1B3. Furthermore, ritonavir was not an inhibitor of OATP1B1. The 521T>C polymorphism in SLCO1B1 was significantly associated with higher lopinavir plasma concentrations. No associations were observed with functional variants of SLCO1A2 and SLCO1B3. Conclusion These data add to our understanding of the factors that contribute to variability in plasma concentrations of protease inhibitors. Further studies are now required to confirm the association of SLCO1B1 521T>C with lopinavir plasma concentrations and to assess the influence of other polymorphisms in the SLCO family.

Towards a new combination therapy for tuberculosis with next generation benzothiazinones.

The benzothiazinone lead compound, BTZ043, kills Mycobacterium tuberculosis by inhibiting the essential flavo‐enzyme DprE1, decaprenylphosphoryl‐beta‐D‐ribose 2‐epimerase. Here, we synthesized a new series of piperazine‐containing benzothiazinones (PBTZ) and show that, like BTZ043, the preclinical candidate PBTZ169 binds covalently to DprE1. The crystal structure of the DprE1‐PBTZ169 complex reveals formation of a semimercaptal adduct with Cys387 in the active site and explains the irreversible inactivation of the enzyme. Compared to BTZ043, PBTZ169 has improved potency, safety and efficacy in zebrafish and mouse models of tuberculosis (TB). When combined with other TB drugs, PBTZ169 showed additive activity against M. tuberculosis in vitro except with bedaquiline (BDQ) where synergy was observed. A new regimen comprising PBTZ169, BDQ and pyrazinamide was found to be more efficacious than the standard three drug treatment in a murine model of chronic disease. PBTZ169 is thus an attractive drug candidate to treat TB in humans.

Towards a new tuberculosis drug: pyridomycin – nature's isoniazid

Tuberculosis, a global threat to public health, is becoming untreatable due to widespread drug resistance to frontline drugs such as the InhA‐inhibitor isoniazid. Historically, by inhibiting highly vulnerable targets, natural products have been an important source of antibiotics including potent anti‐tuberculosis agents. Here, we describe pyridomycin, a compound produced by Dactylosporangium fulvum with specific cidal activity against mycobacteria. By selecting pyridomycin‐resistant mutants of Mycobacterium tuberculosis, whole‐genome sequencing and genetic validation, we identified the NADH‐dependent enoyl‐ (Acyl‐Carrier‐Protein) reductase InhA as the principal target and demonstrate that pyridomycin inhibits mycolic acid synthesis in M. tuberculosis. Furthermore, biochemical and structural studies show that pyridomycin inhibits InhA directly as a competitive inhibitor of the NADH‐binding site, thereby identifying a new, druggable pocket in InhA. Importantly, the most frequently encountered isoniazid‐resistant clinical isolates remain fully susceptible to pyridomycin, thus opening new avenues for drug development.

Simple Model for Testing Drugs against Nonreplicating Mycobacterium tuberculosis

ABSTRACT Nonreplicating or dormant cells of Mycobacterium tuberculosis constitute a challenge to tuberculosis (TB) therapy because of their tolerance or phenotypic resistance to most drugs. Here, we propose a simple model for testing drugs against nongrowing cells that exploits the 18b strain of M. tuberculosis, a streptomycin (STR)-dependent mutant. Optimal conditions were established that allowed 18b cells to replicate in the presence of STR and to survive, but not multiply, following withdrawal of STR. In the presence of the antibiotic, M. tuberculosis 18b was susceptible to the currently approved TB drugs, isoniazid (INH) and rifampin (RIF), and to the experimental drugs TMC207, PA-824, meropenem (MER), benzothiazinone (BTZ), and moxifloxacin (MOXI). After STR depletion, the strain displayed greatly reduced susceptibility to the cell wall inhibitors INH and BTZ but showed increased susceptibility to RIF and PA-824, while MOXI and MER appeared equipotent under both conditions. The same potency ranking was found against nonreplicating M. tuberculosis 18b after in vivo treatment of chronically infected mice with five of these drugs. Despite the growth arrest, strain 18b retains significant metabolic activity in vitro, remaining positive in the resazurin reduction assay. Upon adaption to a 96-well format, this assay was shown to be suitable for high-throughput screening with strain 18b to find new inhibitors of dormant M. tuberculosis.

Modulation of the intracellular accumulation of saquinavir in peripheral blood mononuclear cells by inhibitors of MRP1, MRP2, P-gp and BCRP.

Background:The efflux transporters P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRP) and breast cancer resistance protein (BCRP) limit the accumulation of antiretrovirals in cell lines but it is more important to know whether the expression of these transporters in peripheral blood mononuclear cells (PBMC) impacts cellular drug concentrations. Objectives:To study the transport and accumulation of saquinavir (SQV) in PBMC and the effects of specific inhibitors of MRP1, MRP2, P-gp and BCRP. Methods:Transport and accumulation of [3H]-SQV was measured in PBMC in the absence or presence of specific and non-specific inhibitors of MRP1, MRP2, P-gp and BCRP. Flow cytometric, western blot and real-time PCR assays were used to examine the relative expression of the drug efflux transporters in the same batches of PBMC. Results:MRP2 is present in PBMC. The expression of P-gp, MRP1, MRP2 (mRNA) and BCRP all displayed batch-to-batch variability. Specific and non-specific inhibitors of MRP1, P-gp and MRP2 significantly increased the baseline accumulation of SQV. Accumulation of SQV was not correlated with the expression of any single transporter. Conclusions:Multiple drug efflux transporters are important in the intracellular accumulation of SQV in PBMC. If drug efflux contributes towards virological failure, then all contributing transporters will need to be inhibited.

Virulence regulator EspR of Mycobacterium tuberculosis is a nucleoid-associated protein.

The principal virulence determinant of Mycobacterium tuberculosis (Mtb), the ESX-1 protein secretion system, is positively controlled at the transcriptional level by EspR. Depletion of EspR reportedly affects a small number of genes, both positively or negatively, including a key ESX-1 component, the espACD operon. EspR is also thought to be an ESX-1 substrate. Using EspR-specific antibodies in ChIP-Seq experiments (chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing) we show that EspR binds to at least 165 loci on the Mtb genome. Included in the EspR regulon are genes encoding not only EspA, but also EspR itself, the ESX-2 and ESX-5 systems, a host of diverse cell wall functions, such as production of the complex lipid PDIM (phenolthiocerol dimycocerosate) and the PE/PPE cell-surface proteins. EspR binding sites are not restricted to promoter regions and can be clustered. This suggests that rather than functioning as a classical regulatory protein EspR acts globally as a nucleoid-associated protein capable of long-range interactions consistent with a recently established structural model. EspR expression was shown to be growth phase-dependent, peaking in the stationary phase. Overexpression in Mtb strain H37Rv revealed that EspR influences target gene expression both positively or negatively leading to growth arrest. At no stage was EspR secreted into the culture filtrate. Thus, rather than serving as a specific activator of a virulence locus, EspR is a novel nucleoid-associated protein, with both architectural and regulatory roles, that impacts cell wall functions and pathogenesis through multiple genes.

In Vitro Combination Studies of Benzothiazinone Lead Compound BTZ043 against Mycobacterium tuberculosis

ABSTRACT Benzothiazinones (BTZ) are a new class of drug candidates to combat tuberculosis that inhibit decaprenyl-phosphoribose epimerase (DprE1), an essential enzyme involved in arabinan biosynthesis. Using the checkerboard method and cell viability assays, we have studied the interaction profiles of BTZ043, the current lead compound, with several antituberculosis drugs or drug candidates against Mycobacterium tuberculosis strain H37Rv, namely, rifampin, isoniazid, ethambutol, TMC207, PA-824, moxifloxacin, meropenem with or without clavulanate, and SQ-109. No antagonism was found between BTZ043 and the tested compounds, and most of the interactions were purely additive. Data from two different approaches clearly indicate that BTZ043 acts synergistically with TMC207, with a fractional inhibitory concentration index of 0.5. TMC207 at a quarter of the MIC (20 ng/ml) used in combination with BTZ043 (1/4 MIC, 0.375 ng/ml) had a stronger bactericidal effect on M. tuberculosis than TMC207 alone at a concentration of 80 ng/ml. This synergy was not observed when the combination was tested on a BTZ-resistant M. tuberculosis mutant, suggesting that DprE1 inhibition is the basis for the interaction. This finding excludes the possibility of synergy occurring through an off-target mechanism. We therefore hypothesize that sub-MICs of BTZ043 weaken the bacterial cell wall and allow improved penetration of TMC207 to its target. Synergy between two new antimycobacterial compounds, such as TMC207 and BTZ043, with novel targets, offers an attractive foundation for a new tuberculosis regimen.

Streptomycin-Starved Mycobacterium tuberculosis 18b, a Drug Discovery Tool for Latent Tuberculosis

ABSTRACT Mycobacterium tuberculosis 18b, a streptomycin (STR)-dependent mutant that enters a viable but nonreplicating state in the absence of STR, has been developed as a simple model for drug testing against dormant bacilli. Here, we further evaluated the STR-starved 18b (SS18b) model both in vitro and in vivo by comparing the behavior of 22 approved and experimental tuberculosis drugs. Using the resazurin reduction microplate assay (REMA), rifampin (RIF), rifapentine (RPT), TMC207, clofazimine (CFM), and linezolid (LIN) were found to be active against SS18b in vitro, and their bactericidal activity was confirmed by determining the number of CFU. A latent 18b infection was established in mice, and some of the above-mentioned drugs were used for treatment, either alone or in combination with RIF. RIF, RPT, TMC207, CFM, and pyrazinamide (PZA) were all active in vivo, while cell wall inhibitors were not. A comparative kinetic study of rifamycin efficacy was then undertaken, and the results indicated that RPT clears latent 18b infection in mice faster than RIF. Intrigued by the opposing responses of live and dormant 18b cells to cell wall inhibitors, we conducted a systematic analysis of 14 such inhibitors using REMA. This uncovered an SS18b signature (CWPRED) that accurately predicted the activities of cell wall inhibitors and performed well in a blind study. CWPRED will be useful for establishing the mode of action of compounds with unknown targets, while the SS18b system should facilitate the discovery of drugs for treating latent tuberculosis.

Intracellular accumulation of efavirenz and nevirapine is independent of P-glycoprotein activity in cultured CD4 T cells and primary human lymphocytes

BACKGROUND Interaction of antiretrovirals with drug transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), breast cancer resistance protein (BCRP) and solute carrier organic anion transporter (SLCO) may influence the emergence of viral mutants by altering intracellular drug concentrations. Here we characterize the effect of transporter expression in a variety of cell types such as control CEM, CEM(VBL) (P-gp-overexpressing), CEM(E1000) (MRP1-overexpressing), MT4, control MDCKII, MDCKII(MDR1) (P-gp-overexpressing) and peripheral blood mononuclear cells (PBMCs) on the uptake of [(14)C]efavirenz and [(3)H]nevirapine. We also investigated the lipophilicity of [(14)C]efavirenz and [(3)H]nevirapine. METHODS The expression of P-gp, MRP1, MRP2, SLCO1A2, 1B1, 1B3, 2B1, 3A1 and 4A1 was assessed by PCR. Inhibitors of P-gp (XR9576, GF120918, dipyridamole) and MRP (MK571, frusemide, dipyridamole), and SLCO substrate or inhibitor (estrone-3-sulphate or montelukast, respectively) were used to study the role of drug transporters in the accumulation of [(14)C]efavirenz and [(3)H]nevirapine. Lipophilicity was measured by the octanol/saline partition coefficient. RESULTS CEM cells, MT4 cells and PBMCs express various SLCO isoforms, with SLCO3A1 detected in all of the cells. XR9576, dipyridamole and GF120918 had no effects on the accumulation of [(14)C]efavirenz, while MK571 and frusemide produced variable effects in the cells. The accumulation of [(14)C]efavirenz was significantly decreased in all the cells by montelukast and estrone-3-sulphate. CONCLUSIONS P-gp expression had no effect on the accumulation of [(14)C]efavirenz and [(3)H]nevirapine. MRP1/2 expression, lipophilicity and SLCO-like transporters (possibly SLCO3A1) may have greater influence on the accumulation of [(14)C]efavirenz than [(3)H]nevirapine.