Mariangela Biava

MmpL3 is the cellular target of the antitubercular pyrrole derivative BM212

ABSTRACT The 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates and Mycobacterium tuberculosis residing within macrophages as well as against Mycobacterium avium and other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. Spontaneous Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in the mmpL3 gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterial membrane protein, large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [14C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.

1,5-Diaryl-2-ethyl pyrrole derivatives as antimycobacterial agents: Design, synthesis, and microbiological evaluation

During the search of novel antitubercular drugs related to BM 212, new diarylpyrroles were designed and synthesized on the basis of a structure-activity relationship analysis of many pyrroles previously described by us. Among them, 1-(4-fluorophenyl)-2-ethyl-3-(thiomorpholin-4-yl)methyl-5-(4-methylphenyl)-1H-pyrrole (2b) proved to be particularly active, with a minimum inhibitory concentration (MIC, expressed as microg/mL) and a protection index (PI) better than or comparable to those of reference compounds. Also the remaining compounds were very active, although their MIC and PI were in general lower than those of their parent 2-methyl analogues.

Importance of the thiomorpholine introduction in new pyrrole derivatives as antimycobacterial agents analogues of BM 212

During the course of our investigations in the field of azole antimicrobial agents, we have identified BM 212, a pyrrole derivative with good in vitro activity against mycobacteria and candidae. These findings prompted us to prepare new pyrrole derivatives 1-10 in the hope of increasing the activity. The microbiological data showed interesting in vitro activity against Mycobacterium tuberculosis and atypical mycobacteria.

New pyrrole derivatives as antimycobacterial agents analogs of BM212

During the course of our investigations in the field of azole antimicrobial agents, we have identified BM212 a pyrrole derivative with good in vitro activity against mycobacteria and candidae. These findings prompted us to prepare new pyrrole derivatives 2-6 in the hope of increasing the activity. The microbiological data showed interesting in vitro activity against Mycobacterium tuberculosis and atypical mycobacteria.

1,5-Diphenylpyrrole Derivatives as Antimycobacterial Agents. Probing the Influence on Antimycobacterial Activity of Lipophilic Substituents at the Phenyl Rings

Synthesis and biological evaluation of new derivatives of 1,5-bis(4-chlorophenyl)-2-methyl-3-(4-methylpiperazin-1-yl)methyl-1H-pyrrole (BM 212, 16) are reported. Variously substituted phenyl rings with different substitution pattern and lipophilicity were added to the pyrrole nucleus to evaluate their influence on the activity toward Mycobacterium tuberculosis (MTB) and atypical mycobacteria. The most active derivatives showed activity between 0.125-0.5 microg/mL (better than 16 and streptomycin) and protection index (64-256) higher than 16 (4) and similar to isoniazid and streptomycin (128).

Improved BM212 MmpL3 Inhibitor Analogue Shows Efficacy in Acute Murine Model of Tuberculosis Infection

1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved in vitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED99 of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery.

Fyn Kinase in Brain Diseases and Cancer: The Search for Inhibitors

Fyn is a non-receptor tyrosine kinase belonging to the Src family kinases. It has been shown to play important roles in neuronal functions, including myelination and oligodendrocytes formation, and in inflammatory processes. It has also demonstrated its involvement in signaling pathways that lead to severe brain pathologies, such as Alzheimers and Parkinsons diseases. Moreover, Fyn is upregulated in some malignancies. Experimental studies demonstrated that Fyn inhibition could be useful in the disruption of metabolic processes involved in cancer neurodegenerative diseases. Unfortunately, no specific Fyn inhibitor has been discovered till today, active compounds on other members of Src family or on different tyrosine kinases have also been reported. However, multitargeted inhibitors might be endowed with therapeutic potential. Indeed, as increasingly reported, also a not completely selective inhibitor of a specific protein could be therapeutically useful, affecting a number of cell pathways involved especially in cancer development. In this review, we report some examples of small molecule tyrosine kinase inhibitors for which data on Fyn inhibition, both in enzymatic and in cell assays, have been reported, with the aim of giving information as starting point for the researchers working in this field.

Synthesis, biological evaluation, and enzyme docking simulations of 1,5-diarylpyrrole-3-alkoxyethyl ethers as selective cyclooxygenase-2 inhibitors endowed with anti-inflammatory and antinociceptive activity.

A series of substituted 1,5-diarylpyrrole-3-alkoxyethyl ethers (6, 7, and 8) has been synthesized with the aim to assess if in the previously reported 1,5-diarylpyrrole derivatives (5) the replacement of the acetic ester moiety with an alkoxyethyl group still led to new, highly selective and potent COX-2 inhibitors. In the in vitro cell culture assay, all the compounds proved to be potent and selective COX-2 inhibitors. In the human whole blood (HWB) assay, compound 8a had a comparable COX-2 selectivity to valdecoxib, while it was more selective than celecoxib but less selective than rofecoxib. The potential anti-inflammatory and antinociceptive activities of compounds 7a, 8a, and 8d were evaluated in vivo, where they showed a very good activity against both carrageenan-induced hyperalgesia and edema in the rat paw test. In the abdominal constriction test compound 7a, 8a, and 8d were able to reduce the number of writhes in a statistically significant manner. Furthermore, the affinity data of these compounds have been rationalized through enzyme docking simulations in terms of interactions with a crystallographic model of the COX-2 binding site by means of the software package Autodock 3.0.5, GRID 21, and MacroModel 8.5 using the complex between COX-2 and SC-558 (1b), refined at a 3 A resolution (Brookhaven Protein Data Bank entry: 6cox ).

Research on antibacterial and antifungal agents. XI: Synthesis and antimicrobial activity of N-heteroaryl benzylamines and their Schiff bases

Summary The synthesis and antimicrobial activity of new N -heteroaryl benzylamines and their Schiff bases are reported. Antifungal data were compared with those obtained with miconazole, ketoconazole, enilconazole and imazalil sulfate, which showed that some of the tested compounds possessed moderate activity against strains of Candida albicans , Candida sp and good activity against isolates of plant pathogenic fungi. In contrast the synthesized compounds showed poor antibacterial activity, except for 3j which exhibited a better activity than nalidixic acid used as positive control. The results obtained are discussed on the basis of structure-activity relationships.

Novel Ester and Acid Derivatives of the 1,5-Diarylpyrrole Scaffold as Anti-Inflammatory and Analgesic Agents. Synthesis and in Vitro and in Vivo Biological Evaluation

A new generation of selective cyclooxygenase-2 (COX-2) inhibitors (coxibs) was developed to circumvent the major side effects of cyclooxygenase-1 (COX-1) and COX-2 inhibitors (stomach ulceration and nephrotoxicity). As a consequence, coxibs are extremely valuable in treating acute and chronic inflammatory conditions. However, the use of coxibs, such as rofecoxib (Vioxx), was discontinued because of the high risk of cardiovascular adverse events. More recent clinical findings highlighted how the cardiovascular toxicity of coxibs could be mitigated by an appropriate COX-1 versus COX-2 selectivity. We previously reported a set of substituted 1,5-diarylpyrrole derivatives, selective for COX-2. Here, we describe the synthesis of new 1,5-diarylpyrroles along with their inhibitory effects in vitro, ex vivo, and in vivo toward COX isoenzymes and their analgesic activity. Isopropyl-2-methyl-5-[4-(methylsulfonyl)phenyl]-1-phenyl-1H-pyrrole-3-acetate (10a), a representative member of the series, was selected for pharmacokinetic and metabolic studies.