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

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.

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

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 Ki 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.

New and Old Hot Drug Targets in Tuberculosis.

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.

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

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.

Estudio transversal bioquímico clínico en pacientes con artritis reumatoidea y su relación con el tabaquismo / Clinical laboratory transversal study in patients with rheumatoid arthritis and its relationship with smoking habits / Estudo transversal bioquímico clínico em pacientes com artrite reumatóide e sua relação com o tabagismo

En una población de pacientes con artritis reumatoidea (AR) se estudió la relación de esta patología con el tabaquismo, su perfil hormonal, la concentración de interleuquina 6 (IL 6) y la coexistencia de fibromialgia. Casi en el 50% de los pacientes, los valores de IL 6 fueron seis veces superiores al valor considerado normal. El hábito del tabaco, la edad y el tipo de tratamiento no influyeron en los valores de IL 6. En el subgrupo de pacientes con fibromialgia se observaron tanto altos niveles de IL 6 como bajos niveles de dehidroepiandrosterona (DHEA).

The relation between rheumatoid arthritis and smoking habits, hormonal status, interleuquin-6 levels and fibromyalgia in a population of patients was analyzed. Almost 50% showed IL6 values six times higher than the normal level. Smoking habits, age and the type of treatment did not influence IL-6 concentrations. Patients with fibromyalgia had high levels of IL6 as well as low dehydroepiandrosterone values.

Estuda-se numa população de pacientes com artrite reumatóide a relação desta patologia com o tabagismo, seu perfil hormonal, a concentração de interleucina 6 e a coexistência de fibromialgia. Quase no 50% dos pacientes os valores de IL 6 foram seis vezes superiores ao valor considerado normal. O hábito de fumar, a idade e o tipo de tratamento não influíram nos valores de IL 6. No subgrupo de pacientes com fibromialgia observa-se tanto altos níveis de IL 6 como baixos níveis de DHEA.

Estudio psico-inmunológico y hormonal en pacientes con enfermedades reumáticas (autoinmunes?) / Psycho-immunological and hormonal study in patients with rheumatic diseases (autoimmune?)

Se realizó un estudio clínico y psicológico a pacientes con enfermedades autoinmunes. Se seleccionaron a portadores de lupus, artritis reumatoidea, fibromialgia y esclerodermia con distintos estadios de enfermedad. Se efectuaron mediciones bioquímicas para evaluar el eje endocrino (tiroideo, adrenal y gonadal) y el sistema inmune en su expresión humoral mediada por anticuerpos en el sector celular y en las secreciones, representadas por las citoquinas. Se correlacionaron los hallazgos en cada enfermedad con la situación psicológica evaluada a través de cuestionarios

We developed a clinical and psychological study of patients with autoimmune diseases, selecting those patients who had different stages of systemic lupus erythematous, rheumatoid arthritis, fibromyalgia and scleroderma. We made biochemical tests to examine the endocrine axis (thyroid, adrenal and reproductive), humoral and cellular expressions of the immunesystem through the concentrations of antibodies and cytokines. We correlated observed values with the psychological situation of patients as measured by means of specific questionnaires