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Small molecules for modulating biological targets

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Universitat de Barcelona. Facultat de Química

# metadata.dc.description.abstract[].value
[cat] CAPÍTOL 1: Les malalties respiratòries infeccioses com per exemple la pneumònia, la bronquitis, la fibrosis quística i la malaltia pulmonar obstructiva crònica són les principals causes de morbidesa i mortalitat a tot el món. Aquestes infeccions són causades per un nombre creixent de bacteris multi resistents als antibiòtics que existeixen actualment. Uns enzims indispensables pels bacteris són els enzims ribonucleòtids reductases (RNR), els quals produeixen els desoxiribonucleòtids per la síntesi d’ADN. Una molècula capaç d’inhibir l’enzim RNR pararia el creixement del bacteri, esdevenint una estratègia terapèutica en el tractament de les malalties respiratòries infeccioses. La hidroxiurea i els derivats de la N-hidroxilamina són inhibidors de l’enzim RNR. Per tant, vam decidir sintetitzar una llibreria d’hidroxilamines, a partir d’aldehids per aminació reductiva. Els compostos van ser avaluats en quatre línies diferents de bacteris: Pseudomonas aeruginosa, Burkholderia cepacia, Staphylococcus aureus i Bacillus anthracis, que causen infeccions oportunistes en pacients que pateixen malalties respiratòries. CAPÍTOL 2: La mitofusina 2 és una proteïna mitocondrial que participa en la fusió mitocondrial i en regula el metabolisme. A més, Mfn2 està disminuïda en el múscul de pacients que pateixen obesitat o diabetis de tipus 2. Per tant, activadors de l’expressió de Mfn2 es podrien utilitzar com a tractament terapèutic. Per trobar activadors de Mfn2 es va portar a terme un cribratge d’alt rendiment amb la llibreria de compostos Prestwick utilitzant les cèl•lules HeLa que expressen de forma estable la proteïna luciferasa sota el promotor humà de Mfn2. El compost LM1, que va ser identificat, augmenta l’expressió de les proteïnes de fusió Mfn2, Mfn1 i OPA1, i disminueix la proteïna de fissió Drp1 en cèl•lules, produint una elongació de la xarxa mitocondrial. El compost LM1 i el LM2, que inhibeixen l’enzim DHODH directa o indirectament, causen la disminució de la síntesi de pirimidines que produeix un estrès cel•lular i l’activació i acumulació de p53. p53 produeix una parada de cicle cel•lular i modula les proteïnes involucrades en dinàmica mitocondrial promovent l’elongació mitocondrial. L’allargament de la xarxa mitocondrial proporciona més resistència a les cèl•lules que pateixen un estrès cel•lular.

# metadata.dc.description.abstract[].value
[eng] CHAPTER 1: Respiratory Infectious Diseases (RID) are diseases affecting the air passages, the bronchi and the lungs. They range from acute infections, such as pneumonia and bronchitis, to chronic conditions such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Respiratory lung infections are leading causes of morbidity and mortality worldwide with a considerable human, social and financial burden. RID can be caused by an increasing number of multi resistant bacteria to the existing antibiotics. Bacteria, which normally inhabit the mucus, grow out of control and consequently colonize and infect the lungs. The altered mucus leads to formation of bacterial microenvironments known as biofilms, which are difficult for antibiotics and inmunitary cells to penetrate. A potential key enzyme of bacteria, associated with chronic lung infections, to be targeted by antiproliferative drugs are ribonucleotide reductase enzymes (RNR). RNR enzyme provides deoxyribonucleotides for DNA synthesis which are needed for growth and spore germination of the pathogen. Inhibition of RNR enzymes could be a valuable therapeutic strategy for the treatment of RID. In the literature, it has been suggested that hydroxyurea (HU) and N-hydroxylamine derivatives can be used as antiproliferative drugs against bacterial lung infections. Therefore, a library of these compounds was synthesized and evaluated against four different bacteria lines: Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cenocepacia and Bacillus anthracis; all of them are opportunistic infectious pathogens affecting respiratory system. CHAPTER 2: Mitofusin-2 (Mfn2) participates in mitochondrial fusion and moreover, regulates mitochondrial metabolism. We have previously reported that Mfn2 is down regulated in muscle from obese or type 2 diabetic patients, and recently we have demonstrated that Mfn2 deficiency in liver or muscle leads to glucose intolerance and insulin resistance in mice. Therefore, activators of Mfn2 expression could be used as a valuable potential therapeutic strategy for the treatment of type 2 diabetes and obesity. For this purpose, we decided to search activators of mitofusin-2 expression by High Throughput Screening using a FDA-approved library. HeLa cells stably expressing luciferase under the control of 2 kb of human Mfn2 promoter were incubated with the library and compound LM1 was identified as a potent activator of Mfn2 transcriptional activity. Importantly, LM1 increased Mfn2 expression in HeLa and C2C12 cells. Furthermore, LM1 also induced the expression of the mitochondrial fusion protein Mfn1 and repressed the mitochondrial fission protein Drp1 in both cell lines. LM1 modulates the mitochondrial proteins involved in mitochondrial dynamics producing an elongation of the mitochondrial network. In addition, LM1 was able to increase Mfn2, Mfn1 and OPA1 protein levels and promoted mitochondrial elongation in MEFwt, MEF Mfn2-/- and MEF Mfn1-/- cells. However, mitochondrial elongation is more dependent of Mfn1 than Mfn2 due to the higher mitochondrial elongation of MEF Mfn2-/- compared to MEF Mfn1-/- cells. LM1 decreases the synthesis of pyrimidines by inhibiting dihydroorotate dehydrogenase (DHODH), which causes cell stress and p53 activation by phosphorylation at Ser15. p53 triggers Mfns up-regulation promoting mitochondrial elongation. The addition of external uridine, which reverses the deficiency in pyrimidines synthesis, prevents p53 and Mfns increase in C2C12 cells. Moreover, LM2, an inhibitor of complex III that indirectly inhibit DHODH, and consequently pyrimidine synthesis, also up-regulates p53 producing an increase in Mfns protein expression. LM2 also promotes mitochondrial elongation in HeLa, C2C12 and MEFwt cells. We can conclude that the depletion of pyrimidine pools, by complex III or DHODH inhibitors, cause cell stress, and trigger p53 accumulation and activation by p53 phosphorylation at Ser15, which causes cell cycle arrest. p53 up-regulates Mfns and down-regulates Drp1 promoting mitochondrial elongation that confer stress resistance on cells. Therefore, mitochondrial elongation represents an adaptive response against cell stress caused by LM1 and LM2.

# metadata.dc.title[].value
Small molecules for modulating biological targets