Addressing Latent Tuberculosis: New Advances in Mimicking the Disease, Discovering Key Targets, and Designing Hit Compounds

Despite being discovered and isolated more than one hundred years ago, tuberculosis (TB) remains a global public health concern arch. Our inability to eradicate this bacillus is strongly related with the growing resistance, low compliance to current drugs, and the capacity of the bacteria to coexist in a state of asymptomatic latency. This last state can be sustained for years or even decades, waiting for a breach in the immune system to become active again. Furthermore, most current therapies are not efficacious against this state, failing to completely clear the infection. Over the years, a series of experimental methods have been developed to mimic the latent state, currently used in drug discovery, both in vitro and in vivo. Most of these methods focus in one specific latency inducing factor, with only a few taking into consideration the complexity of the granuloma and the genomic and proteomic consequences of each physiological factor. A series of targets specifically involved in latency have been studied over the years with promising scaffolds being discovered and explored. Taking in account that solving the latency problem is one of the keys to eradicate the disease, herein we compile current therapies and diagnosis techniques, methods to mimic latency and new targets and compounds in the pipeline of drug discovery

Targeting bacterial glyoxalase I for the development of novel antibacterials

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2016A via metabólica da glioxalase é essencial na metabolização do metilglioxal, um metabolito tóxico para a célula, recorrendo à glutationa como co-factor. Um aumento nos níveis de metilglioxal pode conduzir a glicação de proteínas e DNA, destabilizando e prejudicando a função celular. Os derivados da glutationa são inibidores já conhecidos da glioxalase. Ao longo dos anos, esta classe de compostos tem sido estudada para diversas doenças como cancro, diabetes e doenças neurodegenerativas. É agora proposto o uso desta via de destoxificação como o novo alvo na descoberta de novos compostos com potencial antibiótico. Como antibacteriano e sendo a glioxalase essencial na sobrevivência celular, espera-se que ao inibir a glioxalase bacteriana, se dê um aumento do metilglioxal para níveis tóxicos, conduzindo a morte celular. Se for obtido um composto com elevada especificidade para a glioxalase bacteriana, esse pode ser um importante passo na descoberta de uma nova classe de antibióticos. Partindo deste objetivo, foram sintetizados vários derivados da glutationa. Estes foram testados contra a glioxalase I usando differential scanning fluorimetry throughout. Vários dos compostos testados mostraram uma afinidade para a enzima superior à da glutationa, demostrando assim o seu potencial como inibidores competitivos. Foram ainda desenvolvidos dois fragmentos da glutationa com o objetivo de determinar qual a parte do péptido que é mais determinante na ligação à enzima. O fragmento composto por glutamato e cisteína mostrou uma afinidade superior ao fragmento de glicina e cisteína e à glutationa em si, podendo ter-se aberto o caminho para uma nova linha de investigação e desenvolvimento de inibidores da glioxalase.The Glyoxalase system is essential in the metabolization of methylglyoxal, a toxic cellular metabolite, using glutathione as a co-factor. The increase in methylglyoxal levels within the cell leads to protein and DNA glycation, destabilizing and prejudicing the cellular function. Glutathione S-conjugates are well known Glyoxalase inhibitors. Over the years, this class of compounds has been studied for several diseases such as cancer, diabetes and neurodegenerative diseases. It is now proposed the use of the glyoxalase system as a novel target for antimicrobial drug discovery. Being the glyoxalase essential to cellular survival, by inhibiting the bacterial glyoxalase, the methylglyoxal may increase to toxic levels, leading to cellular death. If a hit compound with high specificity for the bacterial glyoxalase can be achieved, that could be the first step to create a whole new class of antibiotics. With this goal in mind, several Glutathione S-conjugates were synthesized and tested against the Glyoxalase I using differential scanning fluorimetry throughout. A number of the tested compounds showed a higher affinity for the enzyme than glutathione itself, proving their potential as competitive inhibitors. Two fragments based on the glutathione scaffold were also developed in order to determine which part of the peptide is more important for the enzyme binding. The Glutamate-Cysteine fragment showed a significantly higher affinity for the enzyme than the Glycine-Cysteine fragment or the Glutathione itself

Development of new drug leads for tuberculosis

Tuberculosis (TB) is a deadly disease caused by a single infectious agent, Mycobacterium tuberculosis (M. tuberculosis). The complexity and duration of the treatment lead to misuse and low compliance by patients, increasing disease burden and the appearance of multidrug-resistant strains of M. tuberculosis. Thus, new antibiotics active against drug-resistant M. tuberculosis and useful for short period therapeutic regimens at lower required doses are urgently needed. A family of azaaurone-based derivatives, from a chemical library developed in iMed.ULisboa, revealed to be active against M. tuberculosis, including multidrug- and extensively drug-resistant clinical isolates, at a submicromolar level. [3] Despite the promising activities, this new scaffold displayed poor ADME properties. In this thesis, we report the complete SAR exploration and ADME profiling of newly synthesized derivatives. All chemical sites of the azaaurone scaffold were extensively explored. More metabolically stable moieties were introduced, generating compounds with improved in vitro half-lives. More hydrophilic groups were inserted and nonaromatic heterocycles were introduced to disrupt the scaffold planarity, two strategies to improve overall solubility. The double bond within the scaffold was also reduced to a single bond, generating a new family of saturated analogues of azaaurones. The new synthesized derivatives displayed improved potency against M. tuberculosis and retained its activity on drug-resistant isolates, remaining non-cytotoxic and selective for the bacteria. Identification of the target was attempted through isolation of resistant mutants, but it was not successful as no azaaurone-resistant isolates were found. Final azaaurones are typically obtained as mixtures of E/Z isomers. The isomers were isolated and differentially studied. Both display similar antitubercular activity. Furthermore, azaaurone isomers were found to isomerize in the presence of mouse liver microsomes (MLM) and under the influence of light. Concerning photoisomerization, it was concluded that this family of azaaurones does not display the optimal characteristics to be used as a photoswitch system

Addressing Latent Tuberculosis: New Advances in Mimicking the Disease, Discovering Key Targets, and Designing Hit Compounds

Despite being discovered and isolated more than one hundred years ago, tuberculosis (TB) remains a global public health concern arch. Our inability to eradicate this bacillus is strongly related with the growing resistance, low compliance to current drugs, and the capacity of the bacteria to coexist in a state of asymptomatic latency. This last state can be sustained for years or even decades, waiting for a breach in the immune system to become active again. Furthermore, most current therapies are not efficacious against this state, failing to completely clear the infection. Over the years, a series of experimental methods have been developed to mimic the latent state, currently used in drug discovery, both in vitro and in vivo. Most of these methods focus in one specific latency inducing factor, with only a few taking into consideration the complexity of the granuloma and the genomic and proteomic consequences of each physiological factor. A series of targets specifically involved in latency have been studied over the years with promising scaffolds being discovered and explored. Taking in account that solving the latency problem is one of the keys to eradicate the disease, herein we compile current therapies and diagnosis techniques, methods to mimic latency and new targets and compounds in the pipeline of drug discovery