M. Carmen Villaverde

Effect of the protonation state of the titratable residues on the inhibitor affinity to BACE-1.

BACE-1 is one of the aspartic proteases involved in the cleavage of beta amyloid peptide, an initial step in the formation of amyloid plaques whose toxicity induces neuron death in Alzheimers disease patients. One of the central issues in the search of novel BACE-1 inhibitors is the optimum pH for the binding of inhibitors to the enzyme. It is known that the enzyme has optimal catalytic activity at acidic pH, while cell active inhibitors may bind optimally at higher pH. In this work we determine the effect of the pH on the affinities of a set of inhibitors, with a variety of chemical motifs, for the ectodomain region of BACE-1 by a surface plasmon resonance (SPR) biosensor based assay. In order to understand the molecular interactions that underlie the diverse optimum pH for the binding of the various inhibitors as observed experimentally, we have calculated the titration curves for a set of BACE-1 ligand complexes. The results indicate that the pK(a) values of the titratable residues of the protein depend on the nature of the ligand involved, in disagreement with previous work. The enzyme-inhibitor structures with the resulting protonation states at pH values 4.5 and 7.4 served as the starting point for the prediction of the pH-dependent binding ranking. Our calculations reproduced the entire affinity ranking observed upon pH increase and most of the binding trends among inhibitors, especially at low pH. Finally, our cell-based assays indicate a possible correlation between high inhibitor affinity at both acidic and neutral pH values, with optimal cell response, a result that may open new venues for the search of potent BACE-1 inhibitors that are active at the cellular level.

Computer-Aided Structure-Based Design of Multitarget Leads for Alzheimer’s Disease

Alzheimers disease is a neurodegenerative pathology with unmet clinical needs. A highly desirable approach to this syndrome would be to find a single lead that could bind to some or all of the selected biomolecules that participate in the amyloid cascade, the most accepted route for Alzheimer disease genesis. In order to circumvent the challenge posed by the sizable differences in the binding sites of the molecular targets, we propose a computer-assisted protocol based on a pharmacophore and a set of required interactions with the targets that allows for the automated screening of candidates. We used a combination of docking and molecular dynamics protocols in order to discard nonbinders, optimize the best candidates, and provide a rationale for their potential as inhibitors. To provide a proof of concept, we proceeded to screen the literature and databases, a task that allowed us to identify a set of carbazole-containing compounds that initially showed affinity only for the cholinergic targets in our experimental assays. Two cycles of design based on our protocol led to a new set of analogues that were synthesized and assayed. The assay results revealed that the designed inhibitors had improved affinities for BACE-1 by more than 3 orders of magnitude and also displayed amyloid aggregation inhibition and affinity for AChE and BuChE, a result that led us to a group of multitarget amyloid cascade inhibitors that also could have a positive effect at the cholinergic level.

Radical cyclization to aporphines. A new, efficient total synthesis of the aporphine glaucine and the 4,5-dioxoaporphine pontevedrine, and the first total synthesis of 5-oxoaporphines.

Abstract We describe the radical cyclization of bromobenzylisoquinolines and benzylisoquinolin-3-ones, which afford aporphines or the novel 5-oxoaporphines and 5-oxodehydroaporphines respectively. Oxidation of the latter compounds provides a new route to 4,5-dioxoaporphines.

Tributyltinhydride-induced intramolecular radical cyclization to aporphines and 5-oxoaporphines

Abstract A new synthesis of aporphines by tributyltinhydride-induced intramolecular radical cyclization of bromobenzylisoquinolines is described. This route to aporphines also allowed the first total synthesis of a 5-oxoaporphine.

On the active site protonation state in aspartic proteases: implications for drug design.

Aspartic proteases (AP) are a family of important hydrolytic enzymes in medicinal chemistry, since many of its members have become therapeutical targets for a wide variety of diseases from AIDS to Alzheimer. The enzymatic activity of these proteins is driven by the Asp dyad, a pair of active site Asp residues that participate in the hydrolysis of peptides. Hence, the protonation state of these and other acidic residues present in these enzymes determines the catalytic rate and the affinity for an inhibitor at a given pH. In the present work we have reviewed the effect of the protonation states of the titratable residues in APs both on catalysis and inhibition in this family of enzymes. The first section focuses on the details of the catalytic reaction mechanism picture brought about by a large number of kinetic, crystallographic and computational chemistry analyses. The results indicate that although the mechanism is similar in both retroviral and eukaryotic enzymes, there are some clear differences. For instance, while in the former family branch the binding of the substrate induces a mono-ionic charge state for the Asp dyad, this charge state seems to be already present in the unbound state of the eukaryotic enzymes. In this section we have explored as well the possible existence of low barrier hydrogen bonds (LBHBs) in the enzymatic path. Catalytic rate enhancement in APs could in part be explained by the lowering of the barrier for proton transfer in a hydrogen bond from donor to acceptor, which is a typical feature of LBHBs. Review of the published work indicates that the experimental support for this type of bonds is rather scarce and it may be more probable in the first stages of the hydrolytic mechanism in retroviral proteases. The second section deals with the effect of active site protonation state on inhibitor binding. The design of highly potent AP inhibitors, that could be the basis for drug leads require a deep knowledge of the protonation state of the active site residues induced by their presence. This vital issue has been tackled by experimental techniques like NMR, X-ray crystallography, calorimetric and binding kinetic techniques. Recently, we have developed a protocol that combines monitoring the pH effect on binding affinities by SPR methods and rationalization of the results by molecular mechanics based calculations. We have used this combined method on BACE-1 and HIV-1 PR, two important therapeutic targets. Our calculations are able to reproduce the inhibitor binding trends to either enzyme upon a pH increase. The results indicate that inhibitors that differ in the Asp dyad binding fragments will present different binding affinity trends upon a pH increase. Our calculations have enabled us to predict the protonation states at different pH values that underlie the above mentioned trends. We have found out that these results have many implications not only for in silico hit screening campaigns aimed at finding high affinity binders, but also (in the case of BACE-1) for the discovery of cell active compounds.

Tributyltin(IV) hydride mediated free-radical syntheses of dehydrodibenzochromanones, dehydrodibenzocoumaranones and aristolactams

Abstract We describe free radical cyclization of methyl bromophenylacetylphenylacetates to novel dehydrodibenzo[de,g]chromanones; oxidation of the latter compounds allowed the first total synthesis of dehydrodibenzo[cd,f]coumaranones, which are easily transformed into aristolactams.

On a Possible Neutral Charge State for the Catalytic Dyad in β-Secretase When Bound to Hydroxyethylene Transition State Analogue Inhibitors

β-Secretase is one of the aspartic proteases involved in the formation of amyloid plaques in Alzheimers disease patients. Our previous results using a combination of surface plasmon resonance experiments with molecular modeling calculations suggested that the Asp dyad in β-secretase bound to hydroxylethylene containing inhibitors adopts a neutral charged state. In this work, we show that the Asp dyad diprotonated state reproduced the binding ranking of a set of these inhibitors better than alternative protonation states.

A simple, efficient route to the synthesis of dibenzocoumaranones and aristolactams.

Abstract We report the first synthesis of dibenzo[cd,f]coumaranones that could be readily transformed into their nitrogen analogues, the aristolactams.

ALKALOIDS FROM GUATTERIA GOUDOTIANA

Abstract The leaves and bark of Guatteria goudotiana were investigated for their alkaloid content. From the leaves, 15 alkaloids were isolated: the 1-benzylisoquinolines, (+)-reticuline and (−)-juziphine; the morphinandienone, (−)-pallidine; the noraporphines, (−)-3-hydroxynornuciferine, (+)-laurotetanine and (+)-norisodomesticine; the aporphines, (+)-neolitsine, (+)-isodomesticine, (+)- N -methyllaurotetanine, (+)-isoboldine and (+)-corytuberine; the dehydroaporphines, dehydroneolitsine, dehydronantenine and goudotianine; and the oxoaporphine, liriodenine. From the bark, the following four alkaloids were isolated: the noraporphines, (+)-norisodomesticine and (−)-anolobine; the dehydroaporphine, goudotianine; and the phenanthrene alkaloid, argentinine. This is the first time the dehydroaporphines, dehydroneolitsine and goudotianine, have been isolated from a natural source.

Alkaloids from Platycapnos spicata

Abstract Two new phenanthrene alkaloids, N -methylsecoglaucine and thalicthuberine N -oxide, were isolated from aerial parts of Platycapnos spicata al