The Diverse Nature of the Molecular Interactions That Govern the COV-2 Variants' Cell Receptor Affinity Ranking and Its Experimental Variability.

A critical determinant of infectivity and virulence of the most infectious and or lethal variants of concern (VOCs): Wild Type, Delta and Omicron is related to the binding interactions between the receptor-binding domain of the spike and its host receptor, the initial step in cell infection. It is of the utmost importance to understand how mutations of a viral strain, especially those that are in the viral spike, affect the resulting infectivity of the emerging VOC, knowledge that could help us understand the variant virulence and inform the therapies applied or the vaccines developed. For this sake, we have applied a battery of computational protocols of increasing complexity to the calculation of the spike binding affinity for three variants of concern to the ACE2 cell receptor. The results clearly illustrate that the attachment of the spikes of the Delta and Omicron variants to the receptor originates through different molecular interaction mechanisms. All our protocols unanimously predict that the Delta variant has the highest receptor-binding affinity, while the Omicron variant displays a substantial variability in the binding affinity of the spike that relates to the structural plasticity of the Omicron spike-receptor complex. We suggest that the latter result could explain (at least in part) the variability of the in vitro binding results for this VOC and has led us to suggest a reason for the lower virulence of the Omicron variant as compared to earlier strains. Several hypotheses have been developed around this subject.

Chain-Branched Polyhydroxylated Octahydro-1H-Indoles as Potential Leads against Lysosomal Storage Diseases.

Here, the synthesis and glycosidase inhibition properties of the two first known 3-ethyloctahydro-1H-indole-4,5,6-triols are reported. This study shows the transformation of d-glucose into polyhydroxylated 1-(2-nitrocyclohexane) acetaldehydes, followed by a protocol involving the formation of the azacyclopentane ring. Results of inhibitory potency assays and docking calculations show that at least one of them could be a lead for optimization in the search for compounds that behave like folding chaperones in lysosomal storage diseases.

Environmental Effects Determine the Structure of Potential ß-Amino Acid Based Foldamers.

This work shows that hybrid peptides formed by alternating trans-2-aminocyclopentanecarboxylic acid (trans-ACPC) and trans-2-aminocyclohexanecarboxylic acid (trans-ACHC) do not fold in the solvents typically used in the study of their homo-oligomers. Only when the peptides are assayed in SDS micelles are the predicted helical structures obtained. This indicates that the environment could play an equally important role (as the backbone stereochemistry) in determining their fold, possibly by providing a sequestered environment.

Computer-aided structure-based design of multitarget leads for Alzheimer's disease.

Alzheimer's 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.

Effect of pH and ligand charge state on BACE-1 fragment docking performance.

In this work we propose a protocol for estimating the effect of pH on the docking performance to BACE-1, which affords the charge state of the inhibitor as well as the protonation state of all ionisable residues in the protein at a given pH value. To the best of our knowledge, this is the first report of a protocol predicting the BACE-1 ligand docking poses not only at the neutral pH at which most crystallographic structures were obtained, but also at the optimal pH of the enzyme (in the acidic range), at which most of the BACE-1 binding affinity assays are performed. We have applied this protocol to a set of 23 fragment-like BACE-1 ligands that span four orders of magnitude in their binding affinities. The pK a values of the BACE-1 acidic residues deviate substantially from the estimates for model compounds in solution and display a ligand dependent variability, especially in the case of the catalytic Asp dyad residues. This outcome should have a strong bearing on the design of protocols for docking based BACE-1 screening campaigns. Finally, we were able to find an explanation for the poor docking success rate of some fragments based on the availability of anchoring points, a rationale that could help to improve hit rates in BACE-1 screening campaigns.

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 AP's 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 (LBHB's) in the enzymatic path. Catalytic rate enhancement in AP's 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 LBHB's. 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.

Experimental and 'in silico' analysis of the effect of pH on HIV-1 protease inhibitor affinity: implications for the charge state of the protein ionogenic groups.

The pH dependence of the HIV-1 protease inhibitor affinity was studied by determining the interaction kinetics of a series of inhibitors at three pH values by surface plasmon resonance (SPR) biosensor analysis. The results were rationalized by molecular mechanics based protocols that have as a starting point the structures of the HIV-1 protease inhibitor complexes differing in the protonation states as predicted by our calculations. The SPR experiments indicate a variety of binding affinity pH dependencies which are rather well reproduced by our simulations. Moreover, our calculations are able to pinpoint the possible changes in the charged state of the protein binding site and of the inhibitor that underlie the observed effects of the pH on binding affinity. The combination of SPR and molecular mechanics calculations has afforded novel insights into the pH dependence of inhibitor interactions with their target. This work raises the possibility of designing inhibitors with different pH binding affinity profiles to the ones described here.

Design, synthesis, evaluation, and structure of vitamin D analogues with furan side chains.

Based on the crystal structures of human vitamin D receptor (hVDR) bound to 1α,25-dihydroxy-vitamin D(3) (1,25 D) and superagonist ligands, we previously designed new superagonist ligands with a tetrahydrofuran ring at the side chain that optimize the aliphatic side-chain conformation through an entropy benefit. Following a similar strategy, four novel vitamin D analogues with aromatic furan side chains (3a, 3b, 4a, 4b) have now been developed. The triene system has been constructed by an efficient stereoselective intramolecular cyclization of an enol triflate (A-ring precursor) followed by a Suzuki-Miyaura coupling of the resulting intermediate with an alkenyl boronic ester (CD-side chain, upper fragment). The furan side chains have been constructed by gold chemistry. These analogues exhibit significant pro-differentiation effects and transactivation potency. The crystal structure of 3a in a complex with the ligand-binding domain of hVDR revealed that the side-chain furanic ring adopts two conformations.

Synthesis and biological evaluation of 1α,25-dihydroxyvitamin D(3) analogues hydroxymethylated at C-26.

We designed by docking and synthesized two novel analogues of 1α,25-dihydroxyvitamin D(3) hydroxymethylated at C-26 (2 and 3). The syntheses were carried out by the convergent Wittig-Horner approach via epoxide 12a as a common key intermediate. The antiproliferative and transactivation potency of the compounds was evaluated in colon and breast cancer cell lines. The analogues showed a similar but reduced activity compared to 1,25(OH)(2)D(3). Analogue 3 was more potent than analogue 2, and in some assays it exhibited potency similar to that of the natural ligand.

On a possible neutral charge state for the catalytic dyad in ß-secretase when bound to hydroxyethylene transition state analogue inhibitors.

ß-Secretase is one of the aspartic proteases involved in the formation of amyloid plaques in Alzheimer's disease patients. Our previous results using a combination of surface plasmon resonance experiments with molecular modeling calculations suggested that the Asp dyad in ß-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.

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 Alzheimer's 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.

Searching the conformational space of cyclic beta-amino acid peptides.

There is an increasing interest in the secondary structure of beta-amino-acid-containing peptides, since these compounds exhibit an intrinsic propensity to form stable folds even for short peptides, a feature that is rarely observed in alpha-amino-acid-containing peptides. In this work, we use a multiple trajectory molecular dynamics approach to study a panel of cyclic beta-amino-acid-containing peptides with a variety of motifs that differ in the ring size, ring substituents, and terminal protecting groups. We find a reasonable agreement between the predicted and the experimentally observed structures, in spite of the simple solvent representation used, indicating that in most cases the folding proceeds energetically downhill and it is driven to a great extent by structural preferences coded in the internal degrees of freedom, a result supported by our energy partition analysis. Our results also show that when the N-terminal end is unprotected, it is likely to be charged in a protic polar solvent. In that case, we find that only a molecular dynamics simulation with an "all atom" solvent representation is capable of reproducing the experimentally observed secondary structure of the peptide. Finally, the time evolution analysis of the hydrogen-bond-induced turns as well as of the root-mean-square deviation from the observed structure indicates that some peptides could have a higher intrinsic flexibility than others, within a given fold, a result that correlates to some degree with our molecular mechanics energy analysis.

Prediction and analysis of binding affinities for chemically diverse HIV-1 PR inhibitors by the modified SAFE_p approach.

One of the biggest challenges in the "in silico" screening of enzyme ligands is to have a protocol that could predict the ligand binding free energies. In our group we have developed a very simple screening function (referred to as solvent accessibility free energy of binding predictor, SAFE_p) which we have applied previously to the study of peptidic HIV-1 protease (HIV-1 PR) inhibitors and later to cyclic urea type HIV-1 PR inhibitors. In this work, we have extended the SAFE_p protocol to a chemically diverse set of HIV-1 PR inhibitors with binding constants that differ by several orders of magnitude. The resulting function is able to reproduce the ranking and in many cases the value of the inhibitor binding affinities for the HIV-1 PR, with accuracy comparable with that of costlier protocols. We also demonstrate that the binding pocket SAFE_p analysis can contribute to the understanding of the physical forces that participate in ligand binding. The analysis tools afforded by our protocol have allowed us to identify an induced fit phenomena mediated by the inhibitor and have demonstrated that larger fragments do not necessarily contribute the most to the binding free energy, an outcome partially brought about by the substantial role the desolvation penalty plays in the energetics of binding. Finally, we have revisited the effect of the Asp dyad protonation state on the predicted binding affinities.

Design, synthesis, evaluation, and crystallographic-based structural studies of HIV-1 protease inhibitors with reduced response to the V82A mutation.

In our quest for HIV-1 protease inhibitors that are not affected by the V82A resistance mutation, we have synthesized and tested a second generation set of C2-symmetric HIV-1 protease inhibitors that contain a cyclohexane group at P1 and/or P1'. The binding affinity results indicate that these compounds have an improved response to the appearance of the V82A mutation than the parent compound. The X-ray structure of one of these compounds with the V82A HIV-1 PR variant provides the structural rationale for the better resistance profile of these compounds. Moreover, scrutiny of the X-ray structure suggests that the ring of the Cha side chain might be in a boat rather than in the chair conformation, a result supported by molecular dynamics simulations.

The search for drug leads targeted to the beta-secretase: an example of the roles of computer assisted approaches in drug discovery.

The inhibition of beta-secretase has become a very promising approach to control the onset and progression of Alzheimer's disease due to its involvement in the generation of amyloid plaques. The main goal of the many drug discovery projects targeting this enzyme is the identification of highly specific, non-peptidic compounds with low molecular weight, characteristics that are desirable for drug leads with low toxicity that have to transverse the blood brain barrier. We describe the main approaches used in the discovery of novel inhibitors, including substrate specificity, target structure based design, and high throughput screening (HTS), both in vitro and in silico. We place special emphasis in the receptor based design and in silico HTS, two strategies that make wide use of computer assisted tools. To exemplify the usefulness and versatility of computer tools in this endeavor we use the computer generated 'enzyme's binding site cast' to rationalize qualitatively some salient structural features of known beta-secretase second generation inhibitors, and for guiding the review of many of the ligands whose complexes with the enzyme have been studied by X-ray crystallography. We discuss the use made by other authors of molecular modelling for the understanding of the very special characteristics of ligand binding to beta-secretase and for the design of new inhibitors. Finally, we review the quest for non-peptidic inhibitors that has led to the use of HTS in vitro and in silico. The screening of extensive libraries resulted in a few low affinity compounds that do not fit into the key S1/S1' pockets, indicating that this is not an easy target to block.

Inhibitor docking screened by the modified SAFE_p scoring function: application to cyclic urea HIV-1 PR inhibitors.

Our laboratory has in the past developed a method for the prediction of ligand binding free energies to proteins, referred to as SAFE_p (Solvent free energy predictor). Previously, we have applied this protocol for the prediction of the binding free energy of peptidic and cyclic urea HIV-1 PR inhibitors, whose X-ray structures bound to enzyme are known. In this work, we present the first account of a docking simulation, where the ligand conformations were screened and inhibitor ranking was predicted on the basis of a modified SAFE_p approach, for a set of cyclic urea-HIV-1 PR complexes whose structures are not known. We show that the optimal dielectric constant for docking is rather high, in line with the values needed to reproduce some protein residue properties, like pKa's. Our protocol is able to reproduce most of the observed binding ranking, even in the case that the components of the equation are not fitted to experimental data. Partition of the binding free energy into pocket and residue contributions sheds light into the importance of the inhibitor's fragments and on the prediction of "hot spots" for resistance mutations.

Ligand recognition by RAR and RXR receptors: binding and selectivity.

Fundamental biological functions, most notably embriogenesis, cell growth, cell differentiation, and cell apoptosis, are in part regulated by a complex genomic network that starts with the binding (and activation) of retinoids to their cognate receptors, members of the superfamily of nuclear receptors. We have studied ligand recognition of retinoic receptors (RXRalpha and RARgamma) using a molecular-mechanics-based docking method. The protocol used in this work is able to rank the affinity of pairs of ligands for a single retinoid receptor, the highest values corresponding to those that adapt better to the shape of the binding site and generate the optimal set of electrostatic and apolar interactions with the receptor. Moreover, our studies shed light onto some of the energetic contributions to retinoid receptor ligand selectivity. In this regard we show that there is a difference in polarity between the binding site regions that anchor the carboxylate in RAR and RXR, which translates itself into large differences in the energy of interaction of both receptors with the same ligand. We observe that the latter energy change is canceled off by the solvation energy penalty upon binding. This energy compensation is borne out as well by experiments that address the effect of site-directed mutagenesis on ligand binding to RARgamma. The hypothesis that the difference in binding site polarity might be exploited to build RXR-selective ligands is tested with some compounds having a thiazolidinedione anchoring group.

Predicting the cell differentiation activity of 1 alpha,25-dihydroxyvitamin D3 side chain analogues from docking simulations.

We present a receptor-based protocol for the prediction of the cell differentiation activities of a series of side chain analogues of 1 alpha,25-dihydroxyvitamin D(3), a compound that exhibits a very large variety of biological functions. Our protocol is able to reproduce the activity of the compounds studied here. It also sheds light on the relative importance of binding site residues in the biological activity and on the mechanism behind it.

(9Z)- and (11Z)-8-methylretinals for artificial visual pigment studies: stereoselective synthesis, structure, and binding models.

Artificial visual pigment formation was studied by using 8-methyl-substituted retinals in an effort to understand the effect that alkyl substitution of the chromophore side chain has on the visual cycle. The stereoselective synthesis of the 9-cis and 11-cis isomers of 8-methylretinal, as well as the 5-demethylated analogues is also described. The key bond formations consist of a thallium-accelerated Suzuki cross-coupling reaction between cyclohexenylboronic acids and dienyliodides (C6-C7), and a highly stereocontrolled Horner-Wadsworth-Emmons or Wittig condensation (C11-C12). The cyclohexenylboronic acid was prepared by trapping the precursor cyclohexenyllithium species with B(OiPr)(3) or B(OMe)(3). The cyclohexenyllithium species is itself obtained by nBuLi-induced elimination of a trisylhydrazone (Shapiro reaction), or depending upon the steric hindrance of the ring, by iodine-metal exchange. In binding experiments with the apoprotein opsin, only 9-cis-5-demethyl-8-methylretinal yielded an artificial pigment; 9-cis-8-methylretinal simply provided residual binding, while evidence of artificial pigment formation was not found for the 11-cis analogues. Molecular-mechanics-based docking simulations with the crystal structure of rhodopsin have allowed us to rationalize the lack of binding displayed by the 11-cis analogues. Our results indicate that these isomers are highly strained, especially when bound, due to steric clashes with the receptor, and that these interactions are undoubtedly alleviated when 9-cis-5-demethyl-8-methylretinal binds opsin.

Modified solvent accessibility free energy prediction analysis of cyclic urea inhibitors binding to the HIV-1 protease.

One of the most successful drug targets against AIDS in the last decade has been the HIV-1 protease (HIV-1 PR), an enzyme that processes the polyprotein gene products into active replicative viral proteins. In our quest for a wide-ranging, binding free energy function we have extended the solvent accessibility free energy predictor (SAFE_p) method, recently developed for peptidic HIV-1 PR inhibitors, to the study of the binding of cyclic urea (CU) HIV-1 PR inhibitors. Our results show that there is a need for a specific term depicting polar contacts to be added to the original SAFE_p analytical expression, an outcome not seen in our studies of HIV-1 PR peptidic inhibitors. Nevertheless, despite the higher profile of the electrostatic interactions in the binding of the CU inhibitors, our analysis indicates that CU inhibitor binding is still driven by the hydrophobic entropic contribution, as much as for the peptidic inhibitors.