Publications by authors named "Maria Rosalia Pasca"

58 Publications

A Coumarin-Based Analogue of Thiacetazone as Dual Covalent Inhibitor and Potential Fluorescent Label of HadA in .

ACS Infect Dis 2021 03 22;7(3):552-565. Epub 2021 Feb 22.

ITAV, Université de Toulouse, CNRS USR3505, UPS, 1 place Pierre Potier, 31106 Toulouse Cedex 1, France.

A novel coumarin-based molecule, designed as a fluorescent surrogate of a thiacetazone-derived antitubercular agent, was quickly and easily synthesized from readily available starting materials. This small molecule, coined , exhibited a combination of appropriate physicochemical and biological properties, including resistance toward hydrolysis and excellent antitubercular efficiency similar to that of well-known thiacetazone derivatives, as well as efficient covalent labeling of HadA, a relevant therapeutic target to combat . More remarkably, was successfully implemented as an imaging probe that is capable of labeling in a selective manner, with an enrichment at the level of the poles, thus giving relevant insights about the polar localization of HadA in the mycobacteria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.0c00325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8022203PMC
March 2021

Gut Microbial Signatures in Sporadic and Hereditary Colorectal Cancer.

Int J Mol Sci 2021 Jan 28;22(3). Epub 2021 Jan 28.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy.

Colorectal cancer (CRC) is the fourth most common cause of cancer-related death and the third most common cancer in the world. Depending on the origin of the mutation, colorectal carcinomas are classified as sporadic or hereditary. Cancers derived from mutations appearing during life, affecting individual cells and their descendants, are called sporadic and account for almost 95% of the CRCs. Less than 5% of CRC cases result from constitutional mutations conferring a very high risk of developing cancer. Screening for hereditary-related cancers is offered to individuals at risk for hereditary CRC, who have either not undergone genetic evaluation or have uncertain genetic test results. In this review, we briefly summarize the main findings on the correlation between sporadic CRC and the gut microbiota, and we specifically focus on the few evidences about the role that gut microorganisms have on the development of CRC hereditary syndromes. The characterization of a gut microbiota associated with an increased risk of developing CRC could have a profound impact for prevention purposes. We also discuss the potential role of the gut microbiota as therapeutic treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22031312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865401PMC
January 2021

Design and Synthesis of Pyrano[3,2-]indolones Showing Antimycobacterial Activity.

ACS Infect Dis 2021 01 22;7(1):88-100. Epub 2020 Dec 22.

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russian Federation.

Latent infection presents one of the largest challenges for tuberculosis control and novel antimycobacterial drug development. A series of pyrano[3,2-]indolone-based compounds was designed and synthesized via an original eight-step scheme. The synthesized compounds were evaluated for their activity against strains H37Rv and streptomycin-starved 18b (SS18b), representing models for replicating and nonreplicating mycobacteria, respectively. Compound exhibited good activity with MIC values of 0.3 and 0.4 μg/mL against H37Rv and SS18b, respectively, as well as low toxicity, acceptable intracellular activity, and satisfactory metabolic stability and was selected as the lead compound for further studies. An analysis of -resistant mutants disclosed a cross-resistance with pretomanid and altered relative amounts of different forms of cofactor F in these strains. Complementation experiments showed that F-dependent glucose-6-phosphate dehydrogenase and the synthesis of mature F were important for activity. Overall these studies revealed to be a prodrug that is activated by an unknown F-dependent enzyme in mycobacteria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.0c00622DOI Listing
January 2021

Nitric oxide-releasing compounds for the treatment of lung infections.

Drug Discov Today 2021 Feb 9;26(2):542-550. Epub 2020 Nov 9.

Department of Biology and Biotechnology 'Lazzaro Spallanzani', University of Pavia, via Ferrata 9, 27100, Pavia, Italy. Electronic address:

The spread of acquired drug resistance and of microorganisms naturally resistant to antibiotics is a major threat to global health, leading to an urgent need for novel antimicrobial compounds. Exogenous nitric oxide (NO) represents an attractive and promising antimicrobial approach, showing both bactericidal and biofilm dispersal activities. Numerous studies have been performed to develop NO donor scaffolds, including small molecules, macromolecular compounds, nanoparticles (NPs), and polymeric materials. This approach has resulted in successful outcomes, with some NO-releasing compounds entering clinical practice. In this review, we highlight the importance of this strategy, with a focus on lung infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.drudis.2020.10.027DOI Listing
February 2021

Study of Bedaquiline Resistance in Multi-Drug Resistant Clinical Isolates.

Front Microbiol 2020 17;11:559469. Epub 2020 Sep 17.

Laboratory of Molecular Microbiology, Department of Biology and Biotechnology Lazzaro Spallanzani, University of Pavia, Pavia, Italy.

Tuberculosis (TB) is one of the major causes of death related to antimicrobial resistance worldwide because of the spread of multi- and extensively drug resistant (multi-drug resistant (MDR) and extensively drug-resistant (XDR), respectively) clinical isolates. To fight MDR and XDR tuberculosis, three new antitubercular drugs, bedaquiline (BDQ), delamanid, and pretomanid were approved for use in clinical setting. Unfortunately, BDQ quickly acquired two main mechanisms of resistance, consisting in mutations in either gene, encoding the target, or in , coding for the repressor of the MmpS5-MmpL5 efflux pump. To better understand the spreading of BDQ resistance in MDR- and XDR-TB, studies could be a valuable tool. To this aim, in this work an generation of mutants resistant to BDQ was performed starting from two MDR clinical isolates as parental cultures. The two MDR clinical isolates were firstly characterized by whole genome sequencing, finding the main mutations responsible for their MDR phenotype. Furthermore, several BDQ resistant mutants were isolated by both MDR strains, harboring mutations in both and genes. These BDQ resistant mutants were further characterized by studying their growth rate that could be related to their spreading in clinical settings. Finally, we also constructed a data sheet including the mutations associated with BDQ resistance that could be useful for the early detection of BDQ-resistance in MDR/XDR patients with the purpose of a better management of antibiotic resistance in clinical settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2020.559469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527418PMC
September 2020

2-Aminooxazole as a Novel Privileged Scaffold in Antitubercular Medicinal Chemistry.

ACS Med Chem Lett 2020 Jul 8;11(7):1435-1441. Epub 2020 Jun 8.

P4T group and Food and Drug Department, University of Parma, 43124 Parma, Italy.

To obtain effective eradication of numerous infectious diseases such as tuberculosis, it is important to supply the medicinal chemistry arsenal with novel chemical agents. Isosterism and bioisosterism are widely known concepts in the field of early drug discovery, and in several cases, rational isosteric replacements have contributed to improved efficacy and physicochemical characteristics throughout the hit-to-lead optimization process. However, sometimes the synthesis of isosteres might not be as straightforward as that of the parent compounds, and therefore, novel synthetic strategies must be elaborated. In this regard, we herein report the evaluation of a series of N-substituted 4-phenyl-2-aminooxazoles that, despite being isosteres of a widely used nucleus such as the 2-aminothiazole, have been only seldom explored. After elaboration of a convenient synthetic strategy, a small set of 2-aminothiazoles and their 2-aminooxazole counterparts were compared with regard to antitubercular activity and physicochemical characteristics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsmedchemlett.0c00173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357219PMC
July 2020

Rv0579 Is Involved in the Resistance to the TP053 Antitubercular Prodrug.

Front Microbiol 2020 25;11:292. Epub 2020 Feb 25.

Department of Biology and Biotechnology "Lazzaro Spallanzani," University of Pavia, Pavia, Italy.

Tuberculosis remains one of the leading causes of death from a single pathogen globally. It is estimated that 1/4 of the world's population harbors latent tuberculosis, but only a 5-10% of patients will develop active disease. During latent infection, can persist unaffected by drugs for years in a non-replicating state with low metabolic activity. The rate of the successful tuberculosis treatment is curbed by the presence of these non-replicating bacilli that can resuscitate after decades and also by the spread of drug-resistant strains. International agencies, including the World Health Organization, urge the international community to combat this global health emergency. The thienopyrimidine TP053 is a promising new antitubercular lead compound highly active against both replicating and non-replicating cells, with an MIC of 0.125 μg/ml. TP053 is a prodrug activated by the reduced form of the mycothiol-dependent reductase Mrx2, encoded by gene. After its activation, TP053 releases nitric oxide and a highly reactive metabolite, explaining its activity also against non-replicating cells. In this work, a new mechanism of TP053 resistance was discovered. spontaneous mutants resistant to TP053 were isolated harboring the mutation L240V in Rv0579, a protein with unknown function, but without mutation in gene. Recombineering method demonstrated that this mutation is linked to TP053 resistance. To better characterize Rv0579, the protein was recombinantly produced in and a direct interaction between the Mrx2 activated TP053 and Rv0579 was shown by an innovative target-fishing experiment based on click chemistry. Thanks to achieved results, a possible contribution of Rv0579 in RNA metabolism was hypothesized, linked to toxin anti-toxin system. Overall, these data confirm the role of Rv0579 in TP053 resistance and consequently in the metabolism of this prodrug.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2020.00292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052010PMC
February 2020

First triclosan-based macrocyclic inhibitors of InhA enzyme.

Bioorg Chem 2020 01 6;95:103498. Epub 2019 Dec 6.

LSPCMIB, UMR-CNRS 5068, Université Paul Sabatier-Toulouse III, 118 Route de Narbonne, 31062 Toulouse Cedex 9, France. Electronic address:

Two macrocyclic derivatives based on the triclosan frame were designed and synthesized as inhibitors of Mycobacterium tuberculosis InhA enzyme. One of the two molecules M02 displayed promising inhibitory activity against InhA enzyme with an IC of 4.7 μM. Molecular docking studies of these two compounds were performed and confirmed that M02 was more efficient as inhibitor of InhA activity. These molecules are the first macrocyclic direct inhibitors of InhA enzyme able to bind into the substrate pocket. Furthermore, these biaryl ether compounds exhibited antitubercular activities comparable to that of triclosan against M. tuberculosis H37Rv strain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bioorg.2019.103498DOI Listing
January 2020

, an Emerging and Worrisome Pathogen among Cystic Fibrosis Patients.

Int J Mol Sci 2019 Nov 22;20(23). Epub 2019 Nov 22.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy.

Nontuberculous mycobacteria (NTM) have recently emerged as important pathogens among cystic fibrosis (CF) patients worldwide. is becoming the most worrisome NTM in this cohort of patients and recent findings clarified why this pathogen is so prone to this disease. drug therapy takes up to 2 years and its failure causes an accelerated lung function decline. The colonization of lung alveoli begins with smooth strains producing glycopeptidolipids and biofilm, whilst in the invasive infection, "rough" mutants are responsible for the production of trehalose dimycolate, and consequently, cording formation. Human-to-human transmission was demonstrated among geographically separated CF patients by whole-genome sequencing of clinical isolates worldwide. Using a infected CF zebrafish model, it was demonstrated that (cystic fibrosis transmembrane conductance regulator) dysfunction seems to have a specific role in the immune control of infections only. This pathogen is also intrinsically resistant to many drugs, thanks to its physiology and to the acquisition of new mechanisms of drug resistance. Few new compounds or drug formulations active against are present in preclinical and clinical development, but recently alternative strategies have been investigated, such as phage therapy and the use of β-lactamase inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms20235868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928860PMC
November 2019

New Insights into the Mechanism of Action of the Thienopyrimidine Antitubercular Prodrug TP053.

ACS Infect Dis 2020 02 2;6(2):313-323. Epub 2019 Dec 2.

Department of Biology and Biotechnology "Lazzaro Spallanzani" , University of Pavia , via Ferrata 9 , Pavia 27100 , Italy.

The thienopyrimidine TP053 is an antitubercular prodrug active against both replicating and nonreplicating () cells, which requires activation by the mycothiol-dependent nitroreductase Mrx2. The investigation of the mechanism of action of TP053 revealed that Mrx2 releases nitric oxide from this drug both in the enzyme assays with purified Mrx2 and in mycobacterial cultures, which can explain its activity against nonreplicating bacilli, similar to pretomanid activated by the nitroreductase Ddn. In addition, we identified a highly reactive metabolite, 2-(4-mercapto-6-(methylamino)-2-phenylpyrimidin-5-yl)ethan-1-ol, which can contribute to the antimycobacterial effects on replicating cells as well as on nonreplicating cells. In summary, we explain the mechanism of action of TP053 on both replicating and nonreplicating and report a novel activity for Mrx2, which in addition to Ddn, represents another example of nitroreductase releasing nitric oxide from its substrate. These findings are particularly relevant in the context of drugs targeting nonreplicating , which is shown to be killed by increased levels of nitric oxide.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.9b00388DOI Listing
February 2020

Gut Microbiota Analysis in Postoperative Lynch Syndrome Patients.

Front Microbiol 2019 30;10:1746. Epub 2019 Jul 30.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.

Lynch syndrome (LS) is a dominantly inherited condition with incomplete penetrance, characterized by high predisposition to colorectal cancer (CRC), endometrial and ovarian cancers, as well as to other tumors. LS is associated with constitutive DNA mismatch repair (MMR) gene defects, and carriers of the same pathogenic variants can show great phenotypic heterogeneity in terms of cancer spectrum. In the last years, human gut microbiota got a foothold among risk factors responsible for the onset and evolution of sporadic CRC, but its possible involvement in the modulation of LS patients' phenotype still needs to be investigated. In this pilot study, we performed 16S rRNA gene sequencing of bacterial DNA extracted from fecal samples of 10 postoperative LS female patients who had developed colonic lesions (L-CRC) or gynecological cancers (L-GC). Our preliminary data show no differences between microbial communities of L-CRC and L-GC patients, but they plant the seed of the possible existence of a fecal microbiota pattern associated with LS genetic background, with , , , , and species being the most significantly over-represented in LS patients (comprising both L-CRC and L-GC groups) compared to healthy subjects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2019.01746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682596PMC
July 2019

The EU approved antimalarial pyronaridine shows antitubercular activity and synergy with rifampicin, targeting RNA polymerase.

Tuberculosis (Edinb) 2018 09 11;112:98-109. Epub 2018 Aug 11.

Collaborations Pharmaceuticals, Inc., 840 Main Campus Drive, Lab 3510, Raleigh, NC 27606, USA; Collaborative Drug Discovery, 1633 Bayshore Highway, Suite 342, Burlingame, CA 94403, USA. Electronic address:

The search for compounds with biological activity for many diseases is turning increasingly to drug repurposing. In this study, we have focused on the European Union-approved antimalarial pyronaridine which was found to have in vitro activity against Mycobacterium tuberculosis (MIC 5 μg/mL). In macromolecular synthesis assays, pyronaridine resulted in a severe decrease in incorporation of C-uracil and C-leucine similar to the effect of rifampicin, a known inhibitor of M. tuberculosis RNA polymerase. Surprisingly, the co-administration of pyronaridine (2.5 μg/ml) and rifampicin resulted in in vitro synergy with an MIC 0.0019-0.0009 μg/mL. This was mirrored in a THP-1 macrophage infection model, with a 16-fold MIC reduction for rifampicin when the two compounds were co-administered versus rifampicin alone. Docking pyronaridine in M. tuberculosis RNA polymerase suggested the potential for it to bind outside of the RNA polymerase rifampicin binding pocket. Pyronaridine was also found to have activity against a M. tuberculosis clinical isolate resistant to rifampicin, and when combined with rifampicin (10% MIC) was able to inhibit M. tuberculosis RNA polymerase in vitro. All these findings, and in particular the synergistic behavior with the antitubercular rifampicin, inhibition of RNA polymerase in combination in vitro and its current use as a treatment for malaria, may suggest that pyronaridine could also be used as an adjunct for treatment against M. tuberculosis infection. Future studies will test potential for in vivo synergy, clinical utility and attempt to develop pyronaridine analogs with improved potency against M. tuberculosis RNA polymerase when combined with rifampicin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tube.2018.08.004DOI Listing
September 2018

Shifts of Faecal Microbiota During Sporadic Colorectal Carcinogenesis.

Sci Rep 2018 07 9;8(1):10329. Epub 2018 Jul 9.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.

Gut microbiota has been implicated in the etiopathogenesis of colorectal cancer. The development of colorectal cancer is a multistep process by which healthy epithelium slowly develops into preneoplastic lesions, which in turn progress into malignant carcinomas over time. In particular, sporadic colorectal cancers can arise from adenomas (about 85% of cases) or serrated polyps through the "adenoma-carcinoma" or the "serrated polyp-carcinoma" sequences, respectively. In this study, we performed 16 S rRNA gene sequencing of bacterial DNA extracted from faecal samples to compare the microbiota of healthy subjects and patients with different preneoplastic and neoplastic lesions. We identified putative microbial biomarkers associated with stage-specific progression of colorectal cancer. In particular, bacteria belonging to the Firmicutes and Actinobacteria phyla, as well as members of the Lachnospiraceae family, proved to be specific of the faecal microbiota of patients with preneoplastic lesions, including adenomas and hyperplastic polyps. On the other hand, two families of the Proteobacteria phylum, Alcaligeneaceae and Enterobacteriaceae, with Sutterella and Escherichia/Shigella being the most representative genera, appeared to be associated with malignancy. These findings, once confirmed on larger cohorts of patients, can represent an important step towards the development of more effective diagnostic strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-28671-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6037773PMC
July 2018

A multitarget approach to drug discovery inhibiting Mycobacterium tuberculosis PyrG and PanK.

Sci Rep 2018 02 16;8(1):3187. Epub 2018 Feb 16.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.

Mycobacterium tuberculosis, the etiological agent of the infectious disease tuberculosis, kills approximately 1.5 million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a "double-tool" in order to find additional hit compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-21614-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816626PMC
February 2018

Mechanochemical Synthesis and Biological Evaluation of Novel Isoniazid Derivatives with Potent Antitubercular Activity.

Molecules 2017 Sep 1;22(9). Epub 2017 Sep 1.

Department of Chemistry, Université de Toulouse, UPS, CNRS UMR 5068, LSPCMIB, 118 Route de Narbonne, 31062 Toulouse, France.

A series of isoniazid derivatives bearing a phenolic or heteroaromatic coupled frame were obtained by mechanochemical means. Their pH stability and their structural (conformer/isomer) analysis were checked. The activity of prepared derivatives against cell growth was evaluated. Some compounds such as phenolic hydrazine and almost all heteroaromatic ones, especially , and , are more active than isoniazid, and their activity against some MDR clinical isolates was determined. Compounds and present a selectivity index >1400 evaluated on MRC5 human fibroblast cells. The mechanism of action of selected hydrazones was demonstrated to block mycolic acid synthesis due to InhA inhibition inside the mycobacterial cell.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules22091457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6151834PMC
September 2017

A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors.

ACS Infect Dis 2017 06 11;3(6):428-437. Epub 2017 May 11.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia , via Ferrata 9, 27100 Pavia, Italy.

Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with K values ranging from 3 to 20 μM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labeling experiments demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a M. tuberculosis pyrG conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.7b00006DOI Listing
June 2017

New and Old Hot Drug Targets in Tuberculosis.

Curr Med Chem 2016 ;23(33):3813-3846

Department of Biology and Biotechnology Lazzaro Spallanzani, University of Pavia, via Ferrata 1, Pavia, Italy.

Tuberculosis is an infectious disease caused by the bacillus Mycobacterium tuberculosis. The World Health Organization publishes global tuberculosis reports annually in order to provide the latest information in the surveillance of drug resistance. Given the alarming rise of resistance to antitubercular drugs worldwide, finding new cellular targets and developing new analogues or new compounds with greater potency against already known targets are both important aspects in fighting drug-sensitive and drug-resistant M. tuberculosis strains. In this context, the introduction of the phenotypic screens as an efficient tool for the identification of active compounds for tuberculosis drug discovery has improved the possibility to find new effective targets. With this review we describe the state of art of the currently well validated antitubercular drug targets as well as the advances in discovery of new ones. The main targets will be discussed starting from the oldest such as the enoyl reductase InhA which is constantly repurposed with new inhibitors, through the well assessed targets like the gyrase, the ATP synthetase or the RNA polymerase, up to the hot promiscuous targets decaprenylphosphoryl-Dribose oxidase DprE1 and the mycolic acid transporter MmpL3, or the newly validated and promising targets like the CTP synthetase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1389557516666160831164925DOI Listing
February 2017

New prodrugs against tuberculosis.

Drug Discov Today 2017 03 17;22(3):519-525. Epub 2016 Sep 17.

Department of Biology and Biotechnology 'Lazzaro Spallanzani', University of Pavia, via Ferrata 1, 27100 Pavia, Italy. Electronic address:

The term 'prodrug' was first introduced by Albert in 1958. Generally, prodrugs can be utilized for improving active drug solubility and bioavailability, increasing drug permeability and absorption, modifying the distribution profile, preventing fast metabolism and excretion, and reducing toxicity. Previously, the prodrug approach was a final resort during the drug discovery process only after all other approaches had been exhausted. However, this strategy is now considered during the early stages of the drug development process. Most antitubercular agents are defined as 'prodrugs', including isoniazid and ethionamide. Thus, the prodrug approach could provide novel targets for the rational design of more effective treatments for tuberculosis (TB).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.drudis.2016.09.006DOI Listing
March 2017

Pyrrolidinone and pyrrolidine derivatives: Evaluation as inhibitors of InhA and Mycobacterium tuberculosis.

Eur J Med Chem 2016 Nov 16;123:462-475. Epub 2016 Jul 16.

CNRS, Laboratoire de Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique, LSPCMIB, UMR-5068, 118 Route de Narbonne, F-31062, Toulouse cedex 9, France; Université de Toulouse, UPS, Laboratoire de Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique, LSPCMIB, 118 route de Narbonne, F-31062, Toulouse cedex 9, France. Electronic address:

A series of GEQ analogues bearing pyrrolidinone or pyrrolidine cores were synthesized and evaluated against InhA, essential target for Mycobacterium tuberculosis (M.tb) survival. The compounds were also evaluated against M.tb H37Rv growth. Interestingly, some of the compounds, not efficient as InhA inhibitors, are active against M.tb with MICs up to 1.4 μM. In particular, compound 4b was screened with different M.tb mutated strains in order to identify the cellular target, but without success, suggesting a new possible mode of action.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2016.07.028DOI Listing
November 2016

Evaluation of the inhibitory activity of (aza)isoindolinone-type compounds: toward in vitro InhA action, Mycobacterium tuberculosis growth and mycolic acid biosynthesis.

Chem Biol Drug Des 2016 11 16;88(5):740-755. Epub 2016 Jul 16.

Laboratoire de Chimie de Coordination (LCC), Centre National de la Recherche Scientifique (CNRS), Toulouse, Cedex 4, France.

Inhibitors of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) are considered as potential promising therapeutics for the treatment of tuberculosis. Previously, we reported that azaisoindolinone-type compounds displayed, in vitro, inhibitory activity toward InhA. Herein, we describe chemical modifications of azaisoindolinone scaffold, the synthesis of 15 new compounds and their evaluations toward the in vitro InhA activity. Based on these results, a structure-InhA inhibitory activity relationship analysis and a molecular docking study, using the conformation of InhA found in the 2H7M crystal structure, were carried out to predict a possible mode of interaction of the best (aza)isoindolinone-type inhibitors with InhA in vitro. Then, the work was extended toward evaluations of these compounds against Mycobacterium tuberculosis (Mtb) growth, and finally, some of them were also investigated in respect of their ability to inhibit mycolic acid biosynthesis inside mycobacteria. Although, some azaisoindolinones were able to inhibit InhA activity and Mtb growth in vitro, they did not inhibit the mycolic acid biosynthesis inside Mtb.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/cbdd.12804DOI Listing
November 2016

Iron Acquisition Pathways as Targets for Antitubercular Drugs.

Curr Med Chem 2016 ;23(35):4009-4026

Dipartimento di Scienze Farmaceutiche, Faculty of Scienze del Farmaco, via L. Mangiagalli, 25-20133 Milano, Italy.

Tuberculosis nowadays ranks as the second leading cause of death from an infectious disease worldwide. In the last twenty years, this disease has again started to spread mainly for the appearance of multi-drug resistant forms. Therefore, new targets are needed to address the growing emergence of bacterial resistance and for antitubercular drug development. Efficient iron acquisition is crucial for the pathogenesis of Mycobacterium tuberculosis, because it serves as cofactor in many essential biological processes, including DNA biosynthesis and cellular respiration. Bacteria acquire iron chelating non-heme iron from the host using the siderophore mycobactins and carboxymycobactins and by the uptake of heme iron released by damaged red blood cells, through several acquisition systems. Drug discovery focused its efforts on the inhibition of MbtI and MbtA, which are are two enzymes involved in the mycobactin biosynthetic pathway. In particular, MbtI inhibitors have been studied in vitro, while MbtA inhibitors showed activity also in infected mice. Another class of compounds, MmpL3 inhibitors, showed antitubercular activity in vitro and in vivo, but their mechanism of action seems to be off-target. Some compounds inhibiting 4'-phosphopantetheinyl transferase were discovered but not tested on in vivo assays. The available data reported in this study based on inhibitors and gene deletion studies, suggest that targeting iron acquisition systems could be considered a promising antitubercular strategy. Due to their redundancy, the relative importance of each pathway for Mycobacterium tuberculosis survival has still to be determined. Thus, in vivo studies with new, potent and specific inhibitors are needed to highlight target selection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/0929867323666160607223747DOI Listing
February 2017

The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053.

J Biol Chem 2015 Dec 6;290(52):31077-89. Epub 2015 Nov 6.

From the Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC,UPV/EHU), Barrio Sarriena s/n, Leioa, Bizkaia 48940, Spain, the Departamento de Bioquímica, Universidad del País Vasco, Leioa, Bizkaia 48940, Spain, the IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain,

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys(19)-Pro(20)-Trp(21)-Cys(22) motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a "closed" compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an "open" conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M115.677039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692232PMC
December 2015

Design, synthesis and evaluation of new GEQ derivatives as inhibitors of InhA enzyme and Mycobacterium tuberculosis growth.

Eur J Med Chem 2015 Aug 20;101:218-35. Epub 2015 Jun 20.

Laboratoire de Synthèse et Physicochimie de Molécules d'Intérêt Biologique (SPCMIB), Centre National de la Recherche Scientifique (CNRS), 118 Route de Narbonne, 31062, Toulouse, Cedex 09, France; Université de Toulouse, Université Paul Sabatier, LSPCMIB, F-31077, Toulouse, France.

A series of fluorene-based derivatives was synthesized and evaluated for inhibiting both InhA and Mycobacterium tuberculosis growth. These compounds were inspired by the previously reported Genz-10850 molecule, a good InhA inhibitor, but with a poor activity against M. tuberculosis growth. Structure-activity relationships were performed by introducing the following chemical modifications: 1) the piperazine ring; 2) the amide group; 3) the aryl moiety; and 4) the fluorene moiety. Among these new derivatives, one of them was more effective against both the InhA activity and mycobacterial growth, compared to the hit compound. Docking studies were also performed to rationalize activities of these derivatives. Furthermore, we showed for the first time that efflux pump inhibitors potentiated the efficacy of Genz-10850 (GEQ) derivatives against M. tuberculosis growth, demonstrating that these compounds could be substrates of some efflux pumps.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2015.06.035DOI Listing
August 2015

Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG.

Chem Biol 2015 Jul 18;22(7):917-27. Epub 2015 Jun 18.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy. Electronic address:

To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2015.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521081PMC
July 2015

2-Carboxyquinoxalines kill mycobacterium tuberculosis through noncovalent inhibition of DprE1.

ACS Chem Biol 2015 Mar 9;10(3):705-14. Epub 2014 Dec 9.

†More Medicines for Tuberculosis (MM4TB) Consortium (www.mm4tb.org).

Phenotypic screening of a quinoxaline library against replicating Mycobacterium tuberculosis led to the identification of lead compound Ty38c (3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylic acid). With an MIC99 and MBC of 3.1 μM, Ty38c is bactericidal and active against intracellular bacteria. To investigate its mechanism of action, we isolated mutants resistant to Ty38c and sequenced their genomes. Mutations were found in rv3405c, coding for the transcriptional repressor of the divergently expressed rv3406 gene. Biochemical studies clearly showed that Rv3406 decarboxylates Ty38c into its inactive keto metabolite. The actual target was then identified by isolating Ty38c-resistant mutants of an M. tuberculosis strain lacking rv3406. Here, mutations were found in dprE1, encoding the decaprenylphosphoryl-d-ribose oxidase DprE1, essential for biogenesis of the mycobacterial cell wall. Genetics, biochemical validation, and X-ray crystallography revealed Ty38c to be a noncovalent, noncompetitive DprE1 inhibitor. Structure-activity relationship studies generated a family of DprE1 inhibitors with a range of IC50's and bactericidal activity. Co-crystal structures of DprE1 in complex with eight different quinoxaline analogs provided a high-resolution interaction map of the active site of this extremely vulnerable target in M. tuberculosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cb5007163DOI Listing
March 2015

Differential roles of RND efflux pumps in antimicrobial drug resistance of sessile and planktonic Burkholderia cenocepacia cells.

Antimicrob Agents Chemother 2014 Dec 29;58(12):7424-9. Epub 2014 Sep 29.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium

Burkholderia cenocepacia is notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance in B. cenocepacia can be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps in B. cenocepacia strain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia J2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strain B. cenocepacia J2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.03800-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249551PMC
December 2014

Trends in discovery of new drugs for tuberculosis therapy.

J Antibiot (Tokyo) 2014 Sep 6;67(9):655-9. Epub 2014 Aug 6.

Department of Biology and Biotechnology 'Lazzaro Spallanzani', University of Pavia, Pavia, Italy.

After the introduction of isoniazid and rifampicin, the second one discovered in the Lepetit Research Laboratories (Milan, Italy), under the supervision of Professor Piero Sensi, tuberculosis (TB) was considered an illness of the past. Unfortunately, this infectious disease is still a global health fear, due to the multidrug-resistant Mycobacterium tuberculosis and extensively circulating drug-resistant strains, as well as the unrecognized TB transmission, especially in regions with high HIV incidence. In the last few years, new antitubercular molecules appeared on the horizon both in preclinical and clinical stage of evaluation. In this review, we focus on a few of them and on their mechanism of action. Two new promising drug targets, DprE1 and MmpL3, are also discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ja.2014.109DOI Listing
September 2014

Rv2466c mediates the activation of TP053 to kill replicating and non-replicating Mycobacterium tuberculosis.

ACS Chem Biol 2014 Jul 13;9(7):1567-75. Epub 2014 Jun 13.

Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC, UPV/EHU), Leioa, Bizkaia 48940, Spain.

The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis highlights the need to discover new antitubercular agents. Here we describe the synthesis and characterization of a new series of thienopyrimidine (TP) compounds that kill both replicating and non-replicating M. tuberculosis. The strategy to determine the mechanism of action of these TP derivatives was to generate resistant mutants to the most effective compound TP053 and to isolate the genetic mutation responsible for this phenotype. The only non-synonymous mutation found was a g83c transition in the Rv2466c gene, resulting in the replacement of tryptophan 28 by a serine. The Rv2466c overexpression increased the sensitivity of M. tuberculosis wild-type and resistant mutant strains to TP053, indicating that TP053 is a prodrug activated by Rv2466c. Biochemical studies performed with purified Rv2466c demonstrated that only the reduced form of Rv2466c can activate TP053. The 1.7 Å resolution crystal structure of the reduced form of Rv2466c, a protein whose expression is transcriptionally regulated during the oxidative stress response, revealed a unique homodimer in which a β-strand is swapped between the thioredoxin domains of each subunit. A pronounced groove harboring the unusual active-site motif CPWC might account for the uncommon reactivity profile of the protein. The mutation of Trp28Ser clearly predicts structural defects in the thioredoxin fold, including the destabilization of the dimerization core and the CPWC motif, likely impairing the activity of Rv2466c against TP053. Altogether our experimental data provide insights into the molecular mechanism underlying the anti-mycobacterial activity of TP-based compounds, paving the way for future drug development programmes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cb500149mDOI Listing
July 2014

4-aminoquinolone piperidine amides: noncovalent inhibitors of DprE1 with long residence time and potent antimycobacterial activity.

J Med Chem 2014 Jun 11;57(12):5419-34. Epub 2014 Jun 11.

Department of Medicinal Chemistry, ‡Department of Biosciences, and §DMPK (Drug Metabolism and Pharmacokinetics) and Animal Sciences, AstraZeneca India Pvt. Ltd. , Bellary Road, Hebbal, Bangalore 560024, India.

4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of ∼100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm5005978DOI Listing
June 2014

Synthesis of 3-heteryl substituted pyrrolidine-2,5-diones via catalytic Michael reaction and evaluation of their inhibitory activity against InhA and Mycobacterium tuberculosis.

Eur J Med Chem 2014 Jan 4;71:46-52. Epub 2013 Nov 4.

Université de Toulouse, UPS, Laboratoire de Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique, LSPCMIB, 118 route de Narbonne, F-31062 Toulouse cedex 9, France; CNRS, Laboratoire de Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique, LSPCMIB, UMR-5068, 118 Route de Narbonne, F-31062 Toulouse cedex 9, France. Electronic address:

In the present paper, we report the synthesis via catalytic Michael reaction and biological results of a series of 3-heteryl substituted pyrrolidine-2,5-dione derivatives as moderate inhibitors against Mycobacterium tuberculosis H37Rv growth. Some of them present also inhibition activities against InhA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2013.10.069DOI Listing
January 2014