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 [ 14 C]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.T he rise of multidrug-resistant (MDR) and extensively drugresistant (XDR) Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), makes the validation of new antitubercular agents a major global priority. Since tubercular drug resistance is chromosomally encoded (17), chemotherapeutic agents directed against new cellular targets are likely to be effective against both drug-sensitive and drug-resistant M. tuberculosis strains (5,12,13,18). Target identification and validation are usually achieved by either genetic or chemical approaches. The former has the advantage of identifying a likely cellular target a priori but yields no information with regard to the druggability of the target and the access of the drug to the target (a particular problem in mycobacteria [23]). It is therefore not surprising that no current antitubercular agents have been identified through rational drug design (23). Alternatively, the identification of a cellular target candidate through chemical screening has the advantage of knowing that the compound can bind and affect the cellular target in vivo. The identification of the target for an active compound allows the rational modification of lead candidates through medicinal chemistry while ensuring that the compound retains activity against its primary target. However, finding which proteins are inhibited by a compound can be quite challenging.We randomly screened a library of compounds to identify structures of interest for further development. Several azole compounds containing imidazole, pyrrole, toluidine, or methanamine groups were tested for antimycobacterial activity. Among them, 1-{[1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl]methyl}-4-methylpiperazine (BM212) (Fig. 1) proved to be active against multidrug-resistant clinical isolates, against M. tuberculosis residing within macrophages, and against Mycobacterium avium as well as other nontuberculous mycobacteria (7). The identification of BM21...

The LIM kinases 1 and 2 (LIMK1 and LIMK2) are dual specificity (serine/threonine and tyrosine) kinases. Although they show significant structural similarity, LIMK1 and LIMK2 show different expression, subcellular localization, and functions. They are involved in many cellular functions, such as migration, cycle, and neuronal differentiation and also have a role in pathological processes, such as cancer cell invasion and metastatis, as well as in neurodevelopmental disorders (namely, the William's syndrome). LIM kinases have a relevant number of known partners that are able to induce or limit the ability of LIMK1 and LIMK2 to phosphorylate and inactivate their major substrate, cofilin. On the contrary, only a limited number of small molecules that interact with the two proteins to modulate their kinase activity have been identified. In this review, the most important partners of LIM kinases and their modulating activity toward LIMKs are described. The small compounds identified as LIMK1 and LIMK2 modulators are also reported, as well as their role as possible therapeutic agents for LIMK-induced diseases.

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.

This review mainly focuses on the physiological function of 4-hydroxyphenylpyruvate dioxygenase (HPPD), as well as on the development and application of HPPD inhibitors of several structural classes. Among them, one illustrative example is represented by compounds belonging to the class of triketone compounds. They were discovered by serendipitous observations on weed growth and were developed as bleaching herbicides. Informed reasoning on nitisinone (NTBC, 14), a triketone that failed to reach the final steps of the herbicidal design and development process, allowed it to become a curative agent for type I tyrosinemia (T1T) and to enter clinical trials for alkaptonuria. These results boosted the research of new compounds able to interfere with HPPD activity to be used for the treatment of the tyrosine metabolism-related diseases.

The HIV-1 nucleocapsid protein-7 (NCp7) is a highly basic, small zinc-binding protein involved in both deoxyribonucleic (DNA) and ribonucleic (RNA) acids annealing and in viral particle maturation including genome encapsidation, with an additional chaperoning activity toward reverse transcriptase by promoting the two obligatory strand transfers during reverse transcription. Because of its interaction with highly conserved sequences of the HIV-1 genome, NCp7 is being considered a new potential drug target, resistant to mutation, for antiviral activity. The high flexibility of this protein has, however, limited the identification of structural determinants involved in the interaction with stranded sequences of DNA and RNA. Here, we provide a quantum mechanics (density functional theory) study of the zinc-binding motifs and a molecular dynamics simulation of the protein in complex with RNA and DNA, starting from available nuclear magnetic resonance (NMR) structures. Results show that the interaction between the NCp7 and the viral genome is probably based on electrostatic interactions due to a cluster of basic residues, which is reinforced by the exploitation of nonelectrostatic contacts that further stabilize the complexes. Moreover, a possible mechanism for DNA destabilization that involves amino acids T24 and R26 is also hypothesized. Finally, a network of hydrophobic and hydrogen-bond interactions for the stabilization of complexes with DNA and, especially, with RNA is described here for the first time. The complexes between NCp7 and both DNA and RNA, resulting from computer simulations, showed structural properties that are in agreement with most of the currently available molecular biology evidence and could be considered as reliable models (better than NMR structures currently available) for subsequent structure-based ligand design approaches.