Modelización en diseño de fármacos: Exploración conformacional de ligandos y diseño de inhibidores multipotentes

[spa] La presente Tesis Doctoral, presentada como compendio de publicaciones, tiene como objetivo general la aplicación de diversas técnicas computacionales al estudio de los determinantes moleculares que subyacen en la interacción ligando-receptor. Para ello, se han definido dos líneas de investigación, una de desarrollo metodológico y otra de aplicación. En el capítulo metodológico se incluye un único manuscrito (Juárez-Jiménez et. al., J. Phys. Chem. B, 2014) en el que extiende la metodología Multinivel desarrollada por Forti et. al. (J. Chem. Theory Comput. 2012) al uso de campos de fuerza de mecánica clásica. Dicho estudio es relevante de cara a optimizar la exploración de las preferencias conformacionales de fármacos y otras pequeñas moléculas orgánicas. En concreto, se evalúa el uso de campos de fuerza clásicos para llevar a cabo un muestreo a bajo nivel de teoría de las preferencias conformacionales de moléculas tipo feniletilamina y se comprueba que los resultados obtenidos son consistentes con los datos experimentales descritos en la bibliografia y con los resultados obtenidos con la implementación original del método, donde se empleaba el hamiltoniano semiempírico RM1. En segundo lugar, la nueva implementación del método se utiliza para predecir las preferencias conformacionales del antibiótico estreptomicina, obteniéndose resultados similares a los obtenidos experimentalmente por resonancia magnética nuclear, si bien sugerentes de posibles mejoras en la metodología. El capítulo de aplicación incluye tres publicaciones (Bolea et. al. J. Med. Chem. 2011; Juárez Jiménez et. al. BBA Proteins and Proteomics, 2014 y Viayna et. al. J. Med. Chem. 2014), obtenidas a partir de una colaboración multidisciplinar entre diversos grupos de diseño, síntesis y evaluación farmacológica de compuestos de potencial interés en la enfermedad de Alzheimer. Concretamente, se describe el desarrollo de dos familias de fármacos multipotentes: compuestos iMAO-iAChE y compuestos iBACE-1-iAChE, centrándose la discusión en la elucidación de los modos de unión y las relaciones estructura-actividad de dos series de compuestos multipotentes a sus principales dianas terapéuticas mediante la combinación de técnicas de modelización molecular, tales como el docking y la dinámica molecular. En el primer caso, se describen modelos de unión a AChE y MAO de compuestos híbridos donepezilo-PFN9601, abordándose en detalle los determinantes moleculares de las relaciones estructura-actividad y proponiéndose las causas estructurales del diferente comportamiento cinético observado para el mismo inhibidor en MAO A y en MAO B. Con el fin de mejorar el perfil farmacológico de los inhibidores, también se describe, mediante el empleo de compuestos selectivos MAO A, los determinantes moleculares de la selectividad entre las isoformas A y B que muestran algunos inhibidores de MAO. En el segundo caso, la modelización molecular ha permitido proponer un modo de unión a BACE-1 de compuestos híbridos huprina Y-rheína. Dicho modo de unión, sitúa la unidad de huprina Y en el sitio catalítico, mientras que sugiere que la unidad de rheína quedaría acomodada en un sitio de unión diferente al sitio catalítico, que hasta la fecha no ha sido explorado mediante técnicas de diseño racional de fármacos. Ello abre la puerta al desarrollo de nuevos compuestos que interaccionen con BACE aprovechando dicho centro de unión.[eng] The aim of this doctoral thesis is to use computational techniques to explore the molecular determinants that underlie the ligand-receptor interaction. To this end, it has been elaborated as a compendium of publications following two main research lines. First, the expansion of the Multilevel strategy previously developed in the research group (Forti et. al. J. Chem. Theo. Comput. 2012) to explore the conformational preferences of drug-like molecules. Second, the development of new multitarget directed ligands (MTDL) of potential therapeutic interest against Alzheimer’s disease. The extension of the Multilevel strategy is mainly focused in assessing the suitability of classical force fields as an alternative to the originally implemented RM1 semiempirical Hamiltonian and to investigate the performance of the technique for charged molecules. To this end, the conformational preferences of a set of phenylethyl amines are evaluated with both approximations and the results are compared with experimental data obtained by means of nuclear magnetic resonance techniques, reaching similar conclusions in both cases. Streptomycin is also used as a test case to further test the new implementation of the technique. The results are also consistent with experimental results, but also highlight some aspects that could be improved in future revisions of the methodology. The development of MTDL against Alzheimer’s disease is a multidisciplinary effort in the framework of collaborations with experimental research groups (Bolea et. al. J. Med. Chem. 2011; Juárez Jiménez et. al. BBA Proteins and Proteomics, 2014 y Viayna et. al. J. Med. Chem. 2014). Using several molecular modelling techniques, we disclose putative binding modes to their main therapeutic targets for two families of MTDL: iAChE-iMAO and iAChE-BACE-1. The discussion of structure-activity relationships allows us to rationalize the molecular determinants that justify the isoform selectivity between isoforms A and B of MAO. Furthermore, they also allow us to disclose how compounds bind to BACE-1 taking advantage of an exosite previously unexploited in rational drug design, which pave the way for the development of novel drug candidates

Conformational landscape of small ligands: a multilevel strategy to determine the conformational penalty of bioactive ligands

Determining the conformational penalty required for adopting the bioactive conformation is still a challenging question in drug design, because a small uncertainty in this free energy component can lead to significant errors in the predicted activities. Herein, we use the Multilevel strategy, a methodology recently developed by our group, to explore the conformational preferences of ligands in solution, and to estimate the conformational cost of selecting the bioactive conformation

Lenalidomide Stabilizes Protein-Protein Complexes by Turning Labile Intermolecular H-Bonds into Robust Interactions

Targeted protein degradation is a promising therapeutic strategy, spearheaded by the anti-myeloma drugs lenalidomide and pomalidomide. These drugs stabilize very efficiently the complex between the E3 ligase Cereblon (CRBN) and several non-native client proteins (neosubstrates), including the transcription factors Ikaros and Aiolos and the enzyme Caseine Kinase 1 (CK1,), resulting in their degradation. Although the structures for these complexes have been determined, there are no evident interactions that can account for the high efficiency of formation of the ternary complex. We show that lenalidomide's stabilization of the CRBN-CK1 complex is largely due to hydrophobic shielding of intermolecular hydrogen bonds. We also find a quantitative relationship between hydrogen bond robustness and binding affinities of the ternary complexes. These results pave the way to further understand cooperativity effects in drug-induced protein-protein complexes and could help in the design of improved molecular glues and more efficient protein degraders

Unveiling a Novel Transient Druggable Pocket in BACE-1 through Molecular Simulations: Conformational Analysis and Binding Mode of Multisite Inhibitors

The critical role of BACE-1 in the formation of neurotoxic ß-amyloid peptides in the brain makes it an attractive target for an efficacious treatment of Alzheimer's disease. However, the development of clinically useful BACE-1 inhibitors has proven to be extremely challenging. In this study we examine the binding mode of a novel potent inhibitor (compound 1, with IC50 80 nM) designed by synergistic combination of two fragments - huprine and rhein - that individually are endowed with very low activity against BACE-1. Examination of crystal structures reveals no appropriate binding site large enough to accommodate 1. Therefore we have examined the conformational flexibility of BACE-1 through extended molecular dynamics simulations, paying attention to the highly flexible region shaped by loops 8-14, 154-169 and 307-318. The analysis of the protein dynamics, together with studies of pocket druggability, has allowed us to detect the transient formation of a secondary binding site, which contains Arg307 as a key residue for the interaction with small molecules, at the edge of the catalytic cleft. The formation of this druggable 'floppy' pocket would enable the binding of multisite inhibitors targeting both catalytic and secondary sites. Molecular dynamics simulations of BACE-1 bound to huprine-rhein hybrid compounds support the feasibility of this hypothesis. The results provide a basis to explain the high inhibitory potency of the two enantiomeric forms of 1, together with the large dependence on the length of the oligomethylenic linker. Furthermore, the multisite hypothesis has allowed us to rationalize the inhibitory potency of a series of tacrine-chromene hybrid compounds, specifically regarding the apparent lack of sensitivity of the inhibition constant to the chemical modifications introduced in the chromene unit. Overall, these findings pave the way for the exploration of novel functionalities in the design of optimized BACE-1 multisite inhibitors

New polycyclic dual inhibitors of the wild type and the V27A mutant M2 channel of the influenza A virus with unexpected binding mode

Two new polycyclic scaffolds were synthesized and evaluated as anti-influenza A compounds. The 5-azapentacyclo[6.4.0.02,10.03,7.09,11]dodecane derivatives were only active against the wild-type M2 channel in the low-micromolar range. However, some of the 14-azaheptacyclo[8.6.1.02,5.03,11.04,9.06,17.012,16]heptadecane derivatives were dual inhibitors of the wild-type and the V27A mutant M2 channels. The antiviral activity of these molecules was confirmed by cell culture assays. Their binding mode was analysed through molecular dynamics simulations, which showed the existence of distinct binding modes in the wild type M2 channel and its V27A variant

Tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrids as a new family of anti-Alzheimer agents targeting beta-amyloid, tau, and cholinesterase pathologies

Optimization of an essentially inactive 3,4-dihydro-2H-pyrano[3,2-c]quinoline carboxylic ester derivative as acetylcholinesterase (AChE) peripheral anionic site (PAS)-binding motif by double O → NH bioisosteric replacement, combined with molecular hybridization with the AChE catalytic anionic site (CAS) inhibitor 6-chlorotacrine and molecular dynamics-driven optimization of the length of the linker has resulted in the development of the trimethylene-linked 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine6-chlorotacrine hybrid 5a as a picomolar inhibitor of human AChE (hAChE). The tetra-, penta-, and octamethylene-linked homologues 5bd have been also synthesized for comparison purposes, and found to retain the nanomolar hAChE inhibitory potency of the parent 6-chlorotacrine. Further biological profiling of hybrids 5ad has shown that they are also potent inhibitors of human butyrylcholinesterase and moderately potent Aβ42 and tau anti-aggregating agents, with IC50 values in the submicromolar and low micromolar range, respectively. Also, in vitro studies using an artificial membrane model have predicted a good brain permeability for hybrids 5ad, and hence, their ability to reach their targets in the central nervous system. The multitarget profile of the novel hybrids makes them promising leads for developing anti-Alzheimer drug candidates with more balanced biological activities

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

Design, synthesis and biological evaluation of N-methyl-N-[(1,2,3-triazol-4-yl)alkyl]propargylamines as novel monoamine oxidase B inhibitors

Different azides and alkynes have been coupled via Cu-catalyzed 1,3-dipolar Huisgen cycloaddition to afford a novel family of N1- and C5-substituted 1,2,3-triazole derivatives that feature the propargylamine group typical of irreversible MAO-B inhibitors at the C4-side chain of the triazole ring. All the synthesized compounds were evaluated against human MAO-A and MAO-B. Structure-activity relationships and molecular modeling were utilized to gain insight into the structural and chemical features that enhance the binding affinity and selectivity between the two enzyme isoforms. Several lead compounds, in terms of potency (submicromolar to low micromolar range), MAO-B selective recognition, and brain permeability, were identified. One of these leads (MAO-B IC50 of 3.54 μM, selectivity MAO-A/MAO-B index of 27.7) was further subjected to reversibility and time-dependence inhibition studies, which disclosed a slow and irreversible inhibition of human MAO-B. Overall, the results support the suitability of the 4-triazolylalkyl propargylamine scaffold for exploring the design of multipotent anti-Alzheimer compounds endowed with irreversible MAO-B inhibitory activity