Diógenes Santiago Santos

The resumption of consumption - : A review on tuberculosis

Among all infectious diseases that afflict humans, tuberculosis (TB) remains the deadliest. At present, epidemiologists estimate that one-third of the world population is infected with tubercle bacilli, which is responsible for 8 to 10 million new cases of TB and 3 million deaths annually throughout the world. Approximately 95% of new cases and 98% of deaths occur in developing nations, generally due to the few resources available to ensure proper treatment and where human immunodeficiency virus (HIV) infections are common. In 1882, Dr Robert Koch identified an acid-fast bacterium, Mycobacterium tuberculosis, as the causative agent of TB. Thirty-nine years later, BCG vaccine was introduced for human use, and became the most widely used prophylactic strategy to fight TB in the world. The discovery of the properties of first-line antimycobacterial drugs in the past century yielded effective chemotherapies, which considerably decreased TB mortality rates worldwide. The later introduction of some additional drugs to the arsenal used to treat TB seemed to provide an adequate number of effective antimicrobial agents. The modern, standard short-course therapy for TB recommended by the World Health Organization is based on a four-drug regimen that must be strictly followed to prevent drug resistance acquisition, and relies on direct observation of patient compliance to ensure effective treatment. Mycobacteria show a high degree of intrinsic resistance to most antibiotics and chemotherapeutic agents due to the low permeability of its cell wall. Nevertheless, the cell wall barrier alone cannot produce significant levels of drug resistance. M. tuberculosis mutants resistant to any single drug are naturally present in any large bacterial population, irrespective of exposure to drugs. The frequency of mutants resistant to rifampicin and isoniazid, the two principal antimycobacterial drugs currently in use, is relatively high and, therefore, the large extra-cellular population of actively metabolizing and rapidly growing tubercle bacilli in cavitary lesions will contain organisms which are resistant to a single drug. Consequently, monotherapy or improperly administered two-drug therapies will select for drug-resistant mutants that may lead to drug resistance in the entire bacterial population. Thereby, despite the availability of effective chemotherapy and the moderately protective vaccine, new anti-TB agents are urgently needed to decrease the global incidence of TB. The resumption of TB, mainly caused by the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains and HIV epidemics, led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. The latter should be effective to combat both drug-susceptible and MDR/XDR-TB.

Drugs that Inhibit Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

Tuberculosis resurged in the late 1980s and now kills more than 2 million people a year. The reemergence of tuberculosis as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, and the proliferation of multidrug- resistant (MDR) strains have created much scientific interest in developing new antimycobacterial agents to both treat Mycobacterium tuberculosis strains resistant to existing drugs, and shorten the duration of short-course treatment to improve patient compliance. Bacterial cell-wall biosynthesis is a proven target for new antibacterial drugs. Mycolic acids, which are key components of the mycobacterial cell wall, are α-alkyl, β-hydroxy fatty acids, with a species-dependent saturated “short” arm of 20-26 carbon atoms and a “long” meromycolic acid arm of 50-60 carbon atoms. The latter arm is functionalized at regular intervals by cyclopropyl, α-methyl ketone, or α-methyl methylethers groups. The mycolic acid biosynthetic pathway has been proposed to involve five distinct stages: (i) synthesis of C20 to C26 straight-chain saturated fatty acids to provide the α-alkyl branch; (ii) synthesis of the meromycolic acid chain to provide the main carbon backbone, (iii) modification of this backbone to introduce other functional groups; (iv) the final Claisen-type condensation step followed by reduction; and (v) various mycolyltransferase processes to cellular lipids. The drugs shown to inhibit mycolic acid biosynthesis are isoniazid, ethionamide, isoxyl, thiolactomycin, and triclosan. In addition, pyrazinamide was shown to inhibit fatty acid synthase type I which, in turn, provides precursors for fatty acid elongation to long-chain mycolic acids by fatty acid synthase II. Here we review the biosynthesis of mycolic acids and the mechanism of action of antimicrobial agents that act upon this pathway. In addition, we describe molecular modeling studies on InhA, the bona-fide target for isoniazid, which should improve our understanding of the amino acid residues involved in the enzymes mechanism of action and, accordingly, provide a rational approach to the design of new drugs.

THE USE OF BIODIVERSITY AS SOURCE OF NEW CHEMICAL ENTITIES AGAINST DEFINED MOLECULAR TARGETS FOR TREATMENT OF MALARIA, TUBERCULOSIS, AND T-CELL MEDIATED DISEASES – A REVIEW

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

Mycobacterial shikimate pathway enzymes as targets for drug design.

The aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis, is responsible for millions of deaths annually. The increasing prevalence of the disease, the emergence of multidrug-resistant strains, and the devastating effect of human immunodeficiency virus co-infection have led to an urgent need for the development of new and more efficient antimycobacterial drugs. Since the shikimate pathway is present and essential in algae, higher plants, bacteria, and fungi, but absent from mammals, the gene products of the common pathway might represent attractive targets for the development of new antimycobacterial agents. In this review we describe studies on shikimate pathway enzymes, including enzyme kinetics and structural data. We have focused on mycobacterial shikimate pathway enzymes as potential targets for the development of new anti-TB agents.

Purine Salvage Pathway in Mycobacterium tuberculosis

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, infects one-third of the world population. TB remains the leading cause of mortality due to a single bacterial pathogen. The worldwide increase in incidence of M. tuberculosis has been attributed to the high proliferation rates of multi and extensively drug-resistant strains, and to co-infection with the human immunodeficiency virus. There is thus a continuous requirement for studies on mycobacterial metabolism to identify promising targets for the development of new agents to combat TB. Singular characteristics of this pathogen, such as functional and structural features of enzymes involved in fundamental metabolic pathways, can be evaluated to identify possible targets for drug development. Enzymes involved in the pyrimidine salvage pathway might be attractive targets for rational drug design against TB, since this pathway is vital for all bacterial cells, and is composed of enzymes considerably different from those present in humans. Moreover, the enzymes of the pyrimidine salvage pathway might have an important role in the mycobacterial latent state, since M. tuberculosis has to recycle bases and/or nucleosides to survive in the hostile environment imposed by the host. The present review describes the enzymes of M. tuberculosis pyrimidine salvage pathway as attractive targets for the development of new antimycobacterial agents. Enzyme functional and structural data have been included to provide a broader knowledge on which to base the search for compounds with selective biological activity.

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

BackgroundBiopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.ResultsHere we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.ConclusionThe recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (P(i)) and continuous assay of reactions that generate P(i) such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P(i) detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P(i) detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1L cell culture, with a specific activity value of 80 Umg(-1).

Crystal structure of human purine nucleoside phosphorylase at 2.3A resolution.

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. In human, PNP is the only route for degradation of deoxyguanosine and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and its low resolution structure has been used for drug design. Here we report the structure of human PNP solved to 2.3A resolution using synchrotron radiation and cryocrystallographic techniques. This structure allowed a more precise analysis of the active site, generating a more reliable model for substrate binding. The higher resolution data allowed the identification of water molecules in the active site, which suggests binding partners for potential ligands. Furthermore, the present structure may be used in the new structure-based design of PNP inhibitors.

Crystal structure of human purine nucleoside phosphorylase complexed with acyclovir

In human, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. This work reports the first crystallographic study of human PNP complexed with acyclovir (HsPNP:Acy). Acyclovir is a potent clinically useful inhibitor of replicant herpes simplex virus that also inhibits human PNP but with a relatively lower inhibitory activity (K(i)=90 microM). Analysis of the structural differences among the HsPNP:Acy complex, PNP apoenzyme, and HsPNP:Immucillin-H provides explanation for inhibitor binding, refines the purine-binding site, and can be used for future inhibitor design.

Functional Characterization by Genetic Complementation of aroB-Encoded Dehydroquinate Synthase from Mycobacterium tuberculosis H37Rv and Its Heterologous Expression and Purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.