Design and synthesis of selective MMP-9 inhibitors for the treatment of epilepsy

Abstract

Matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases involved in the degradation of the extracellular matrix. They make a major contribution to the progression of a vast number of diseases, particularly cancer and some central nervous system (CNS) disorders, such as epilepsy and Alzheimer’s disease. Although a number of MMP inhibitors (MMPi) have been developed for the treatment of cancer, they have all failed in clinical trials due to lack of efficacy and/or, most importantly, severe side effects. The latter can be explained by their lack of selectivity. Elevated levels of gelatinases (MMP-2 and MMP-9) have been described in various animal models and in clinical studies in humans with epilepsy after seizures, being MMP-9 the most overexpressed in all these cases. Thus, a selective inhibitor for MMP- 9, and to some extent MMP-2, might be beneficial for the treatment of this disease. In this thesis, computer-aided drug design was used to design novel MMP-9 inhibitors which were synthesised and evaluated using in vitro enzyme assays. Following this approach, we obtained several molecules that show selectivity for gelatinases over other MMP family members (MMP-1, MMP-3 and MMP-7). These compounds may be of particular interest for the treatment of epileptogenesis, the process by which a normal brain is functionally altered and leads to the development of epilepsy, because of the prominent role of MMP-9 in this process. The permeability across the Blood-brain barrier (BBB) is the main obstacle and a prerequisite for drugs targeting the central nervous system. In this thesis, I worked in the refinement of the structure of the molecules to improve the BBB permeability of the compounds under development. One of the developed compounds, was selected for further studies because its enzymatic and selectivity for Gelatinases and high proteolytic stability. Once the lead candidate was selected, this compound was studied in vivo. First, the pharmacokinetics of the compound were studied in mice and rats, indicating that the molecule was absorbed in the body and distributed through the BBB. Later, I evaluated the effects of the lead compound on the progression of epilepsy in three animal models

(the pentylenetetrazole (PTZ) acute injection mouse model, the intrahippocampal kainic acid (KA) mouse model and the rapid kindling rat model) of epilepsy. Results from these experiments indicate that the selected compound has the capacity to inhibit MMP-9 in vivo, has anticonvulsive effects and is able to attenuate the progression of epilepsy in the tested epilepsy models, without any side effects. Moreover, the effects of increased MMP-9 activity in memory and learning were also reduced by using the tested compound. The last part of this thesis was focused on the lead compound optimization addressing metabolic deficiencies through structural modification. Based on this approach, two optimized lead candidates were obtained showing higher potency and selectivity for gelatinases and improved drug-like properties (in vitro BBB permeability, cytotoxicity and stability in serum, intestinal fluids and liver microsomes). As a conclusion, this thesis followed the general structure of the drug discovery phase in the drug research process. MMP-9 was identified and selected as a potential therapeutic target for the treatment of epileptogenesis. The hit identification and hit- to-lead process were achieved using in silico calculations in combination with in vitro tools to design MMPi that selectively target gelatinases and have the ability to cross the BBB. The pharmacokinetic study, the efficacy and toxicity of the lead compounds were demonstrated in different models of epilepsy. Finally, the lead compound was optimized for oral administration.

Les metal·loproteinases de matriu (MMPs) constitueixen una família de proteases dependents del zinc i involucrades en la degradació de la matriu extracel·lular. Participen en la progressió d’un gran nombre de malalties, principalment en càncer i en malalties del sistema nerviós central, com ara l’Alzheimer i l’epilèpsia. En les últimes dècades, s’han desenvolupat una serie d’inhibidors de MMPs (MMPi) pel tractament del càncer, però aquests han fallat en estudis clínics degut a la seva ineficàcia o efectes secundaris adversos, principalment deguts a la falta de selectivitat del inhibidors. En la literatura s’ha descrit un increment en els nivells d’expressió i activitat de gelatinases (MMP-2 i MMP-9) en diferents models animals d’epilèpsia, així com també en humans amb aquesta condició, sent la MMP-9 les més sobreexpressada. Per això, un inhibidor selectiu de la MMP-9, i en part també de la MMP-2, podria ser beneficial pel tractament d’aquesta malaltia. En aquesta tesis, el disseny de fàrmacs assistit per ordinador s’ha utilitzat per a dissenyar inhibidors de la MMP-9 que han estat sintetitzats i avaluats in vitro mitjançant assajos enzimàtics. Els compostos més potents i selectius obtinguts per la inhibició de gelatinases s’han seleccionat i optimitzat per creuar la barrera hematoencefàlica. Per la molècula més prometedora, s’han dut a terme en ratolins i rates. A més, la seva eficàcia i potencial toxicitat s’ha estudiat en 3 models animals d’epilèpsia, demostrant que aquest candidat a fàrmac inhibeix la MMP-9 in vivo i pot atenuar els efectes convulsius i epileptogènics presents en els models animals, a més a més no s’han observat símptomes clínics adversos durant els experiments amb animals. Finalment, aquest compost s’ha optimitzat amb l’objectiu de millorar la seva estabilitat metabòlica i poder ser administrat per via oral. Després d’aquest procés d’optimització s’han obtingut dos compostos potents i selectius amb bones propietats ADME .