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The family of concentrative Na+/nucleoside cotransporters in humans is constituted by three subtypes, namely, hCNT1, hCNT2, and hCNT3. Besides their different nucleoside selectivity, hCNT1 and hCNT2 have a Na+/nucleoside stoichiometry of 1:1, while for hCNT3 it is 2:1. This distinct stoichiometry of subtype 3 might hint the existence of a secondary sodium-binding site that is not present in the other two subtypes, but to date their three-dimensional structures remain unknown and the residues implicated in Na+binding are unclear. In this work, we have identified and characterized the Na+binding sites of hCNT3 by combining molecular modeling and mutagenesis studies. A model of the transporter was obtained by homology modeling, and key residues of two sodium-binding sites were identified and verified with a mutagenesis strategy. The structural model explains the altered sodium-binding properties of the hCNT3C602R polymorphic variant and supports previously generated data identifying the determinant residues of nucleoside selectivity, paving the way to understand how drugs can target this plasma membrane transporter.

One of the current challenges in docking studies is the inclusion of receptor flexibility. This is crucial because the binding sites of many therapeutic targets sample a wide range of conformational states, which has major consequences on molecular recognition. In this paper, we make use of very large sets of X-ray structures of cyclin dependent kinase 2 (CDK2) and heat shock protein 90 (HSP90) to assess the performance of flexible receptor docking in binding-mode prediction and virtual screening experiments. Flexible receptor docking performs much better than rigid receptor docking in the former application. Regarding the latter, we observe a significant improvement in the prediction of binding affinities, but owing to an increase in the number of false positives, this is not translated into better hit rates. A simple scoring scheme to correct this limitation is presented. More importantly, pitfalls inherent to flexible receptor docking have been identified and guidelines are presented to avoid them.

The crystal structure of a previously reported screening hit 1 (CCT018159) bound to the N terminal domain of molecular chaperone Hsp90 has been used to design 5-amide analogues. These exhibit enhanced potency against the target in binding and functional assays with accompanying appropriate cellular pharmacodynamic changes. Compound 11 (VER-49009) compares favorably with the clinically evaluated 17-AAG.

Recent advances in structure determination and computational methods have encouraged the development of structure-based virtual screening. Here we survey progress in the field and review the most recent methods, validation experiments and real applications, including an in-house example of hit identification for the oncology target Hsp90. These results provide a basis for discussing the current state of structure-based virtual screening and to outline the developments that are expected to have a major impact in the near future.

This study examines the transferability of fragmental contributions to the octanol/water partition coefficient. As a previous step, we report the parameterization of the AM1 and PM3 versions of the MST model for n‐octanol. The final AM1 and PM3 MST models reproduce the experimental free energy of solvation and the octanol/water partition coefficient (log Pow) with a root‐mean‐square deviation of around 0.7 kcal/mol and 0.5 (in units of log P), respectively. Based on this parameterization, an NNDO‐based procedure is presented to dissect the free energy of transfer between octanol and water in contributions directly associated with specific atoms or functional groups. The application of this procedure to a set of representative molecular systems illustrates the dependence of the log Powfragmental contribution due to electronic, hydrogen bonding, and steric effects, which cannot be easily accounted for in simple additive‐based empirical schemes. The results point out the potential use of theoretical methods to refine the fragmental contributions in empirical methods. © 2002 Wiley Periodicals, Inc. J Comput Chem 24: 32–45, 2003

A similarity index based on the hydrophilic/hydrophobic properties of molecules is presented. Such an index is defined based on the fractional partition of the free energy of solvation developed within the framework of the self‐consistent reaction field MST model, which divides the free energy of solvation or the free energy of transfer into contributions assigned to the surface elements defining the solute/solvent interface. These surface contributions can be integrated to derive atomic or group contributions. The suitability of the index to compute the molecular similarity based on hydrophobic/hydrophilic properties is examined by considering their application in a variety of test systems, including structure‐activity relationships, absorption properties, and molecular recognition. The similarity index is expected to be a very powerful tool in molecular similarity studies for compounds of chemical, biochemical, and pharmaceutical interest. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 554–563, 2002; DOI 10.1002/jcc.10055

Two 12-amino-6,7,8,11-tetrahydro-7,11-methanocycloocta[b]quinoline derivatives [9-Me(Et)] (syn-huprines) have been obtained by condensation of known 7-alkylbicyclo[3.3.1]non-6-en-3-ones with 2-(trifluoromethyl)aniline, followed by basic cyclization of the resulting imine, and chromatographic separation of the regioisomeric mixture of products, thus obtained. The new (±)-syn-huprines were shown to be slightly less active bovine or human acetylcholinesterase inhibitors than the corresponding anti-derivatives. Molecular modeling simulations allow us to explain the differences in inhibitory activity of these compounds on the basis of an inverse solvation effect.

During the last years, solving the X-ray crystallographic structure of both the unliganded acetylcholinesterase (AChE) and AChE complexes with various inhibitors has provided valuable knowledge of the interactions that mediate inhibitor binding. This structural information allows us to rationalize differences in binding affinities for related analogues, and more importantly opens new strategies to design compounds with improved pharmacological properties. This is illustrated in the case of the recently reported huprines, which are a new class of very potent and selective acetylcholinesterase inhibitors.

During the last years, solving the X-ray crystallographic structure of both the unliganded acetylcholinesterase (AChE) and AChE complexes with various inhibitors has provided valuable knowledge of the interactions that mediate inhibitor binding. This structural information allows us to rationalize differences in binding affinities for related analogues, and more importantly opens new strategies to design compounds with improved pharmacological properties. This is illustrated in the case of the recently reported huprines, which are a new class of very potent and selective acetylcholinesterase inhibitors.

Abstract.: This study examines the accuracy of molecular dynamics-linear response (MD/LR) and Poisson–Boltzmann/solvent accessible surface (PB/SAS) calculations to predict relative binding affinities. A series of acetylcholinesterase (AChE) huprine inhibitors has been chosen as a test system owing to the availability of free-energy (thermodynamic integration) calculations. The results obtained with the MD/LR approach point out a clear relationship between the experimental affinity and the electrostatic interaction energy alone for a subset of huprines, but the suitability of the MD/LR approach to predict the binding affinity of the whole series of compounds is limited. On the other hand, PB/SAS calculations show a marked dependence on both the computational protocol and the nature of the inhibitor–enzyme complex.

Several new 12-amino-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinoline derivatives (tacrine−huperzine A hybrids, huprines) have been synthesized and tested as acetylcholinesterase (AChE) inhibitors. All of the new compounds contain either a methyl or ethyl group at position 9 and one or two (chloro, fluoro, or methyl) substituents at positions 1, 2, or 3. Among the monosubstituted derivatives, the more active are those substituted at position 3, their activity following the order 3-chloro > 3-fluoro > 3-methyl > 3-hydrogen. For the 1,3-difluoro and 1,3-dimethyl derivatives, the effect of the substituents is roughly additive. No significant differences were observed for the inhibitory activity of 9-methyl vs 9-ethyl derivatives mono- or disubstituted at positions 1 and/or 3. The levorotatory enantiomers of these hybrid compounds are much more active (eutomers) than the dextrorotatory forms (distomers) as AChE inhibitors. Compounds rac-20, (−)-20, rac-26, (−)-26, rac-30, (−)-30, and rac-31 showed human AChE inhibitory activities up to 28.5-fold higher than for the corresponding bovine enzyme. Also, rac-19, (−)-20, (−)-30, and rac-31 were very selective for human AChE vs butyrylcholinesterase (BChE), the AChE inhibitory activities being 438−871-fold higher than for BChE. Several hybrid compounds, specially (−)-20 and (−)-30, exhibited tight-binding character, showing higher activity after incubation of the enzyme with the inhibitor than without incubation, though the reversible nature of the enzyme−inhibitor interaction was demonstrated by dialysis. The results of the ex vivo experiments also supported the tight-binding character of compounds (−)-20 and (−)-30 and showed their ability to cross the blood−brain barrier. Molecular modeling simulations of the AChE−inhibitor complex provided a basis to explain the differences in inhibitory activity of these compounds.

The binding of the 9-methyl derivative of tacrine−huperzine A hybrid to Torpedo californica acetylcholinesterase (AChE) has been studied by computational methods. Molecular dynamics simulations have been performed for the AChE−drug complex considering two different ionization states of the protein and two different orientations of the drug in the binding pocket, which were chosen from a previous screening procedure. Analysis of structural fluctuations and of the pattern of interactions between drug and enzyme clearly favor one binding mode for the tacrine−huperzine A hydrid, which mixes effectively some of the binding features of tacrine and huperzine A. The differences in inhibitory activity for a series of related derivatives have been successfully predicted by free energy calculations, which reinforces the confidence in the binding mode and its usefulness for molecular modeling studies. The same techniques have been used to make de novo predictions for a new 3-fluoro-9-ethyl derivative, which can be used to verify a posteriori the goodness of the binding mode. Finally, we have also investigated the effect of replacing Phe300 in the Torpedo californica enzyme by Tyr, which is present in the human AChE. The results indicate that the Phe330→Tyr mutation is expected to have little effect on the binding affinities. Overall, the whole of results supports the validity of the putative binding model to explain the binding of tacrine−huperzine A hybrids to AChE.

Eleven new 12-amino-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinoline derivatives [tacrine (THA)−huperzine A hybrids, rac-21−31] have been synthesized as racemic mixtures and tested as acetylcholinesterase (AChE) inhibitors. For derivatives unsubstituted at the benzene ring, the highest activity was obtained for the 9-ethyl derivative rac-20, previously prepared by our group. More bulky substituents at position 9 led to less active compounds, although some of them [9-isopropyl (rac-22), 9-allyl (rac-23), and 9-phenyl (rac-26)] show activities similar to that of THA. Substitution at position 1 or 3 with methyl or fluorine atoms always led to more active compounds. Among them, the highest activity was observed for the 3-fluoro-9-methyl derivative rac-28 [about 15-fold more active than THA and about 9-fold more active than (−)-huperzine A]. The activity of some THA−huperzine A hybrids (rac-19, rac-20, rac-28, and rac-30), which were separated into their enantiomers by chiral medium-pressure liquid chromatography (chiral MPLC), using microcrystalline cellulose triacetate as the chiral stationary phase, showed the eutomer to be always the levorotatory enantiomer, their activity being roughly double that of the corresponding racemic mixture, the distomer being much less active. Also, the activity of some of these compounds inhibiting butyrylcholinesterase (BChE) was tested. Most of them [rac-27−31, (−)-28, and (−)-30], which are more active than (−)-huperzine A as AChE inhibitors, turned out to be quite selective for AChE, although not so selective as (−)-huperzine A. Most of the tested compounds 19−31 proved to be much more active than THA in reversing the neuromuscular blockade induced by d-tubocurarine. Molecular modeling of the interaction of these compounds with AChE from Torpedo californica showed them to interact as truly THA−huperzine A hybrids:  the 4-aminoquinoline subunit of (−)-19 occupies the same position of the corresponding subunit in THA, while its bicyclo[3.3.1]nonadiene substructure roughly occupies the same position of the corresponding substructure in (−)-huperzine A, in agreement with the absolute configurations of (−)-19 and (−)-huperzine A.

A theoretical study on the stability of the salt bridges in the gas phase, in solution, and in the interior of proteins is presented. The study is mainly focused on the interaction between acetate and methylguanidinium ions, which were used as model compounds for the salt bridge between Asp (Glu) and Arg. Two different solvents (water and chloroform) were used to analyze the effect of varying the dielectric constant of the surrounding media on the salt bridge interaction. Calculations in protein environments were performed by using a set of selected protein crystal structures. In all cases attention was paid to the difference in stability between the ion pair and neutral hydrogen‐bonded forms. Comparison of the results determined in the gas phase and in solution allows us to stress the large influence of the environment on the binding process, as well as on the relative stability between the ionic and neutral complexes. The high anisotropy of proteins and the local microenvironment in the interior of proteins make a decisive contribution in modulating the energetics of the salt bridge. In general, the formation of salt bridges in proteins is not particularly favored, with the ion pair structure being preferred over the interaction between neutral species. Proteins 32:67–79, 1998. © 1998 Wiley‐Liss, Inc.

A theoretical study on the stability of the salt bridges in the gas phase, in solution, and in the interior of proteins is presented. The study is mainly focused on the interaction between acetate and methylguanidinium ions, which were used as model compounds for the salt bridge between Asp (Glu) and Arg. Two different solvents (water and chloroform) were used to analyze the effect of varying the dielectric constant of the surrounding media on the salt bridge interaction. Calculations in protein environments were performed by using a set of selected protein crystal structures. In all cases attention was paid to the difference in stability between the ion pair and neutral hydrogen-bonded forms. Comparison of the results determined in the gas phase and in solution allows us to stress the large influence of the environment on the binding process, as well as on the relative stability between the ionic and neutral complexes. The high anisotropy of proteins and the local microenvironment in the interior of proteins make a decisive contribution in modulating the energetics of the salt bridge. In general, the formation of salt bridges in proteins is not particularly favored, with the ion pair structure being preferred over the interaction between neutral species. Proteins 32:67–79, 1998. © 1998 Wiley-Liss, Inc.