Synthesis, in Vitro Profiling, and in Vivo Evaluation of Benzohomoadamantane-Based Ureas for Visceral Pain: A New Indication for Soluble Epoxide Hydrolase Inhibitors

The soluble epoxide hydrolase (sEH) has been suggested as a pharmacological target for the treatment of several diseases, including pain-related disorders. Herein, we report further medicinal chemistry around new benzohomoadamantane-based sEH inhibitors (sEHI) in order to improve the drug metabolism and pharmacokinetics properties of a previous hit. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, two candidates were evaluated in vivo in a murine model of capsaicin-induced allodynia. The two compounds showed an anti-allodynic effect in a dose-dependent manner. Moreover, the most potent compound presented robust analgesic efficacy in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain. Overall, these results suggest painful bladder syndrome as a new possible indication for sEHI, opening a new range of applications for them in the visceral pain field

Discovery and In Vivo Proof of Concept of a Highly Potent Dual Inhibitor of Soluble Epoxide Hydrolase and Acetylcholinesterase for the Treatment of Alzheimer's Disease

With innumerable clinical failures of target-specific drug candidates for multifactorial diseases, such as Alzheimer's disease (AD), which remains inefficiently treated, the advent of multitarget drug discovery has brought a new breath of hope. Here, we disclose a class of 6-chlorotacrine (huprine)‒TPPU hybrids as dual inhibitors of the enzymes soluble epoxide hydrolase (sEH) and acetylcholinesterase (AChE), a multitarget profile to provide cumulative effects against neuroinflammation and memory impairment. Computational studies confirmed the gorge-wide occupancy of both enzymes, from the main site to a secondary site, including a so far non-described AChE cryptic pocket. The lead compound displayed in vitro dual nanomolar potencies, adequate brain permeability, aqueous solubility, and human microsomal stability and lack of neurotoxicity, and rescued memory, synaptic plasticity and neuroinflammation in an AD mouse model, after low dose chronic oral administration

Computational strategies for understanding the molecular basis of biochemical and biocatalytic processes

Enzymes are molecules that play a crucial role in many biological and chemical processes. To understand how they work and how to design enzymes with specific functions, it is important to study their molecular structure and dynamics. However, it can be difficult to capture the transient nature of these processes, so combining experimental techniques with computational methods can provide an atomistic view to explain the molecular basis of biological processes. This thesis focuses on using computational techniques, such as molecular dynamics simulations and quantum mechanics, to explore the molecular basis of biochemical and biocatalytic processes. The goal is to understand enzymatic properties such as allostery, cofactor specificity, and catalytic activity, and use this knowledge to design new enzyme variants. The thesis is divided into three results chapters. In the first chapter (Chapter 4), the focus is on understanding the molecular basis of allosteric regulation in the enzyme Imidazole Glycerol Phosphate Synthase (IGPS). By characterizing the molecular details of the allosteric activation of IGPS in the ternary complex, it was possible to identify the hidden states relevant for IGPS catalytic activity. In the next results chapter (Chapter 5), we designed a computational protocol to unravel the molecular mechanism of the enantioselective N-H insertion in P411 enzyme variants. By exploring the molecular basis of this enzymatic transformation and elucidating the role of key mutations, it was possible to generate a biocatalytic platform for enantiodivergent C-N bond formation. In the last results chapter (Chapter 6), we rationalized the molecular basis of cofactor specificity in engineered formate dehydrogenase variants. By studying the kinetic efficiency with the non-natural NADP+ cofactor, specificity towards the non-natural NADP+ cofactor, and affinity towards the substrate formate, it was possible to understand how to design enzymes with specific cofactor preferences. Overall, this thesis demonstrates the importance of understanding enzyme function at the molecular level in order to design enzyme variants with specific functions. The use of computational techniques allows for a more detailed understanding of enzymatic mechanisms and provides a valuable tool for designing novel enzymes with improved propertiesEls enzims són molècules que tenen un paper crucial en molts processos biològics i químics. Per entendre com funcionen i com es poden dissenyar enzims amb funcions específiques, és important estudiar-ne l'estructura molecular i la seva dinàmica. Tanmateix, pot ser difícil captar la naturalesa transitòria dels processos enzimàtics, de manera que combinar tècniques experimentals amb mètodes computacionals pot proporcionar una visió atomística més detallada que permeti explicar les bases moleculars d’aquests processos biològics. Aquesta tesi es centra en utilitzar tècniques computacionals, com ara simulacions de dinàmica molecular i mecànica quàntica, per explorar les bases moleculars de processos bioquímics i biocatalítics. L'objectiu és comprendre’n propietats enzimàtiques com l'al·losteria, l'especificitat envers cofactors, l'activitat catalítica, i finalment, utilitzar aquest coneixement per a dissenyar noves variants enzimàtiques. La tesi està dividida en tres capítols de resultats. El primer capítol (Capítol 4), es centra en la comprensió de les bases moleculars de la regulació al·lostèrica en l'enzim Imidazol Glicerol Fosfat Sintasa (IGPS). El fet de poder caracteritzar els detalls moleculars de l'activació al·lostèrica de l’enzim IGPS al complex ternari, va permetre identificar els estats ocults rellevants per a l'activitat catalítica d’aquest enzim. En el següent capítol de resultats (Capítol 5), es dissenya un protocol computacional per elucidar el mecanisme molecular de la inserció enantioselectiva de N-H en variants de l'enzim P411. Explorant les bases moleculars d'aquesta transformació enzimàtica i dilucidant el paper de les mutacions clau, ha estat possible generar una plataforma biocatalítica enantiodivergent per a la formació d'enllaços C-N. Al darrer capítol de resultats (Capítol 6), es pretén racionalitzar les bases molecular de l'especificitat envers cofactors en noves variants de format deshidrogenasa (FDH). Mitjançant l'estudi de l'eficiència cinètica i l’especificitat envers el cofactor no-natural NADP+, i l'afinitat envers el substrat (format), s’ha pogut entendre com dissenyar enzims amb preferències específiques envers el cofactor. En conjunt, aquesta tesi demostra la importància d'entendre la funció enzimàtica a nivell molecular per tal de dissenyar variants enzimàtiques amb funcions específiques. L'ús de tècniques computacionals permet una comprensió més detallada dels mecanismes enzimàtics i proporciona una valuosa eina per dissenyar nous enzimsPrograma de Doctorat en Químic

Reversing the enantioselectivity of enzymatic carbene N–H insertion through mechanism-guided protein engineering

In this work, we report a computationally driven approach to access enantiodivergent enzymatic carbene N–H bond insertions catalyzed by P411 enzyme variants. Computational modeling was employed to guide engineering efforts to control the accessible conformations of a key lactone-carbene (LAC) intermediate in the enzyme active site by installing a new H-bond anchoring point. By combining MD simulations and protein engineering, a reversed (R-selective) P411 enzyme variant, L5_FL-B3, was obtained in a single round of semi-rational directed evolution. L5_FL-B3 accepts a broad scope of amine substrates with excellent yields (up to >99%), high efficiency (up to 12,300 TTN) and good enantiocontrol (up to 7:93 er), which complements the previously engineered S-selective P411-L7_LF variant

From the Design to the In Vivo Evaluation of Benzohomoadamantane-Derived Soluble Epoxide Hydrolase Inhibitors for the Treatment of Acute Pancreatitis.

The pharmacological inhibition of soluble epoxide hydrolase (sEH) is efficient for the treatment of inflammatory and pain-related diseases. Numerous potent sEH inhibitors (sEHIs) present adamantyl or phenyl moieties, such as the clinical candidates AR9281 or EC5026. Herein, in a new series of sEHIs, these hydrophobic moieties have been merged in a benzohomoadamantane scaffold. Most of the new sEHIs have excellent inhibitory activities against sEH. Molecular dynamics simulations suggested that the addition of an aromatic ring into the adamantane scaffold produced conformational rearrangements in the enzyme to stabilize the aromatic ring of the benzohomoadamantane core. A screening cascade permitted us to select a candidate for an in vivo efficacy study in a murine model of cerulein-induced acute pancreatitis. The administration of 22 improved the health status of the animals and reduced pancreatic damage, demonstrating that the benzohomoadamantane unit is a promising scaffold for the design of novel sEHIs

Synthesis, In Vitro Profiling, and In Vivo Evaluation of Benzohomoadamantane-Based Ureas for Visceral Pain: A New Indication for Soluble Epoxide Hydrolase Inhibitors

The soluble epoxide hydrolase (sEH) has been suggested as a pharmacological target for the treatment of several diseases, including pain-related disorders. Herein, we report further medicinal chemistry around new benzohomoadamantane-based sEH inhibitors (sEHI) in order to improve the drug metabolism and pharmacokinetics properties of a previous hit. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, two candidates were evaluated in vivo in a murine model of capsaicin-induced allodynia. The two compounds showed an anti-allodynic effect in a dose-dependent manner. Moreover, the most potent compound presented robust analgesic efficacy in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain. Overall, these results suggest painful bladder syndrome as a new possible indication for sEHI, opening a new range of applications for them in the visceral pain field