Multitargeting Compounds: A Promising Strategy to Overcome Multi-Drug Resistant Tuberculosis

Tuberculosis is still an urgent global health problem, mainly due to the spread of multi-drug resistant M. tuberculosis strains, which lead to the need of new more efficient drugs. A strategy to overcome the problem of the resistance insurgence could be the polypharmacology approach, to develop single molecules that act on different targets. Polypharmacology could have features that make it an approach more effective than the classical polypharmacy, in which different drugs with high affinity for one target are taken together. Firstly, for a compound that has multiple targets, the probability of development of resistance should be considerably reduced. Moreover, such compounds should have higher efficacy, and could show synergic effects. Lastly, the use of a single molecule should be conceivably associated with a lower risk of side effects, and problems of drug-drug interaction. Indeed, the multitargeting approach for the development of novel antitubercular drugs have gained great interest in recent years. This review article aims to provide an overview of the most recent and promising multitargeting antitubercular drug candidates

The Role of One- and Two-Dimensional Electrophoretic Techniques in Proteomics of the Lung

The current chapter was designed to keep the reader informed about the present status of pulmonary proteome. Taken together, the results documented here demonstrate that, after a decade of activity, proteomics of pulmonary diseases is catching up with its promise. The constantly growing number of reports in this area supports the view of this approach as one of the decisive methodological tools for the identification/characterization of disease-associated proteins. In terms of experimental procedures, the basic options available for proteomic investigations consist in the identification of proteins through the use of gel-based or gel-free techniques followed by MS. Obviously, the question arises of whether sophisticated technologies (such as the non-gel-based proteomic procedures) may currently be more fruitful, in terms of candidate protein marker identification, than “conventional” (read electrokinetic) approaches. In light of the versatility and high degree of reproducibility shown by these new potent strategies, a positive answer is perhaps not surprising. Nevertheless, as documented in this chapter, despite being less sophisticated than competing ones, gel-based techniques still represent a widely used procedure able to generate a reliable protein “fingerprint” and to produce qualitative and quantitative information on the protein patterns of a variety of human fluids

Biochemical Signatures of Doppel Protein in Human Astrocytomas to Support Prediction in Tumor Malignancy

Doppel (Dpl) is a membrane-bound glycoprotein mainly expressed in the testis of adult healthy people. It is generally absent in the central nervous system, but its coding gene sequence is ectopically expressed in astrocytoma specimens and in derived cell lines. In this paper, we investigated the expression and the biochemical features of Dpl in a panel of 49 astrocytoma specimens of different WHO malignancy grades. As a result, Dpl was expressed in the majority of the investigated specimens (86%), also including low grade samples. Importantly, Dpl exhibited different cellular localizations and altered glycan moieties composition, depending on the tumor grade. Most low-grade astrocytomas (83%) showed a membrane-bound Dpl, like human healthy testis tissue, whereas the majority of high-grade astrocytomas (75%) displayed a cytosolic Dpl. Deglycosylation studies with N-glycosidase F and/or neuraminidase highlighted defective glycan moieties and an unexpected loss of sialic acid. To find associations between glial tumor progression and Dpl biochemical features, predictive bioinformatics approaches were produced. In particular, Decision tree and Nomogram analysis showed well-defined Dpl-based criteria that separately clustered low-and high-grade astrocytomas. Taken together, these findings show that in astrocytomas, Dpl undergoes different molecular processes that might constitute additional helpful tools to characterize the glial tumor progression

Discovery of new diketopiperazines inhibiting Burkholderia cenocepacia quorum sensing in vitro and in vivo

Burkholderia cenocepacia, an opportunistic respiratory pathogen particularly relevant for cystic fibrosis patients, is difficult to eradicate due to its high level of resistance to most clinically relevant antimicrobials. Consequently, the discovery of new antimicrobials as well as molecules capable of inhibiting its virulence is mandatory. In this regard quorum sensing (QS) represents a good target for anti-virulence therapies, as it has been linked to biofilm formation and is important for the production of several virulence factors, including proteases and siderophores. Here, we report the discovery of new diketopiperazine inhibitors of the B. cenocepacia acyl homoserine lactone synthase CepI, and report their anti-virulence properties. Out of ten different compounds assayed against recombinant CepI, four were effective inhibitors, with IC50 values in the micromolar range. The best compounds interfered with protease and siderophore production, as well as with biofilm formation, and showed good in vivo activity in a Caenorhabditis elegans infection model. These molecules were also tested in human cells and showed very low toxicity. Therefore, they could be considered for in vivo combined treatments with established or novel antimicrobials, to improve the current therapeutic strategies against B. cenocepacia

The DprE1 enzyme, one of the most vulnerable targets of Mycobacterium tuberculosis

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Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms.

A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer

Mycobacterium tuberculosis Phosphoribosylpyrophosphate Synthetase: Biochemical Features of a Crucial Enzyme for Mycobacterial Cell Wall Biosynthesis

The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase) is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg2+ or Mn2+ and inhibited by Ca2+ and Cu2+ but, differently from other characterized PRPPases, it showed a better affinity for the Mn2+ and Cu2+ ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design

New Chromane-Based Derivatives as Inhibitors of Mycobacterium tuberculosis Salicylate Synthase (MbtI): Preliminary Biological Evaluation and Molecular Modeling Studies

Tuberculosis is the leading cause of death from a single infectious agent worldwide; therefore, the need for new antitubercular drugs is desperate. The recently validated target salicylate synthase MbtI is the first enzyme involved in the biosynthesis of mycobactins, compounds able to chelate iron, an essential cofactor for the survival of Mycobacterium tuberculosis in the host. Here, we report on the synthesis and biological evaluation of chromane-based compounds as new potential inhibitors of MbtI. Our approach successfully allowed the identification of a novel lead compound (1), endowed with a promising activity against this enzyme (IC50 = 55 μM). Molecular modeling studies were performed in order to evaluate the binding mode of 1 and rationalize the preliminary structure-activity relationships, thus providing crucial information to carry out further optimization studies

Structure and Function of Human Erythrocyte Pyruvate Kinase MOLECULAR BASIS OF NONSPHEROCYTIC HEMOLYTIC ANEMIA

Deficiency of human erythrocyte isozyme (RPK) is, together with glucose-6-phosphate dehydrogenase deficiency, the most common cause of the nonspherocytic hemolytic anemia. To provide a molecular framework to the disease, we have solved the 2.7 A resolution crystal structure of human RPK in complex with fructose 1,6-bisphosphate, the allosteric activator, and phosphoglycolate, a substrate analogue, and we have functionally and structurally characterized eight mutants (G332S, G364D, T384M, D390N, R479H, R486W, R504L, and R532W) found in RPK-deficient patients. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. The mutations affect to a different extent thermostability, catalytic efficiency, and regulatory properties. These studies are the first to correlate the clinical symptoms with the molecular properties of the mutant enzymes. Mutations greatly impairing thermostability and/or activity are associated with severe anemia. Some mutant proteins exhibit moderate changes in the kinetic parameters, which are sufficient to cause mild to severe anemia, underlining the crucial role of RPK for erythrocyte metabolism. Prediction of the effects of mutations is difficult because there is no relation between the nature and location of the replaced amino acid and the type of molecular perturbation. Characterization of mutant proteins may serve as a valuable tool to assist with diagnosis and genetic counseling

Various Evolutionary Trajectories Lead to Loss of the Tobramycin-Potentiating Activity of the Quorum-Sensing Inhibitor Baicalin Hydrate in Burkholderia cenocepacia Biofilms

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant bacteria. As potentiators do not interfere with essential processes, it has been hypothesized that they are less likely to induce resistance. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop reduced susceptibility toward one such adjuvant, baicalin hydrate (BH). Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to TOB and TOB + BH compared to the start population, and the potentiating effect of BH toward TOB was lost. Whole-genome sequencing was performed to probe which changes were involved in the reduced effect of BH, and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter). No changes in the MIC or MBC of TOB or changes in the number of persister cells were observed. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that B. cenocepacia J2315 biofilms rapidly lose susceptibility toward the antibiotic-potentiating activity of BH and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as mechanisms