Amedeo Caflisch - Academia.edu (original) (raw)

Papers by Amedeo Caflisch

Die folgende Dissertation befasst sich mit der Entwicklung und Anwendung dreier neur Ansatze fur ... more Die folgende Dissertation befasst sich mit der Entwicklung und Anwendung dreier neur Ansatze fur die Erzeugung und Untersuchung von Energy Landscapes mit dem Ziel, die Proteinfaltung zu charakterisieren und besser zu verstehen. Das Paradigma der Energy Landscapes hat sich in den letzten zehn Jahren bei der Untersuchung des Faltungsverhaltens von Proteinen bewahrt. Dieses Paradigma fuhrt dazu, dass die Energy Landscape eines Proteins vereinfacht als Trichter dargestellt werden kann. Projektionen dieser Energy Landscape auf Ordnungsparameter, wie zum Beispiel die Projektion auf die freie Energie zur Untersuchung von Stabilitat, sollten eine vereinfachte Sicht der Zustande und Energiebarrieren wiedergeben, wobei oftmals versteckte Annahmen getroffen werden, die nicht erfullt sind. Basierend auf der Theorie komplexer Netzwerke wird als erstes ein neur Ansatz zur Untersuchung der Proteinzustande vorgestellt. Dabei sind die Konformationen, welche im Verlauf einer molecular dynamics (MD) Simulation angenommen werden, die Knoten und die zeitlichen Uebergange die Verbindungen. Dieses Netzwerk reprasentiert die vollstandige multi-dimensionale Energy Landscape ohne Projektion auf willkurlich gewahlte Ordnungsparameter. Die Anwendung von Netzwerken enthullt ein komplexes Zusammenspiel von Minima, Basins und Super-Basins, die in der Energy Landscape die Rolle von Attraktoren spielen. Als erstes wird die MD-Simulation eines strukturierten Peptides (Beta3s) untersucht. Dabei werden Basins mit kleiner Entropie/ kleiner Enthalpie, sowie Basins mit grosser Entropie/grosser Enthalpie gefunden. Hierbei ist interessant, dass Beta3s im gefalteten Zustand (im Unterschied zu einer kinetischen Trap) nicht nur enthalpisch, sondern auch entropisch stabilisiert wird. Free Energy Basins entsprechen Subgraphen (Communities) des gesamten Netzwerks, wobei die Knoten in einen Basin dicht miteinander verbunden sind. Das Finden dieser Communities und die Validierung einer Unterteilung des gesamten Netzwerkes in solche ist trotz der Fulle von existierenden Algorithmen noch nicht zufriedenstllend gelost. In dieser Dissertation wird dazu ein Mass, die Goodness Deviation, vorgeschlagen. Zweitens ist die Kinetik der Proteinfaltung ebenfalls eng mit der Topographie von Energy Landscapes verknupft. Die schwer fassbare Natur des Transition State Ensemble (TSE) erlaubt kein einfaches Bild fur Faltunsubergange. Zur Charakterisierung des TSE wird oft die Faltungswahrscheinlichkeit pfold einer Proteinstruktur benutzt. Obwohl g fur die Analyse von Faltungsubergangen sehr nutzlich ist, erfordert dessen Berechnung einen grossen rechnerischen Aufwand, was die praktische Anwendungen limitiert. Die kinetische Homogenitat von strukturell ahnlichen Snapshots erlaubt jedoch einen statistischen Zugang fur die Analyse einzelner Konformationen. Die Idee des sogenannten Cluster-pfold ermoglicht es in dieser Dissertation, g fur Strukturen einer MD-simulation approximativ zu berechnen. Die Anwendung dieser Methode auf Beta3s zeigt ein breites, heterogenes TSE, sowie zwei Haupt-Pathways. Diese Ergebnisse decken sich interessanterweise mit der Netzwerk-Analyse aus vorhergehenden Untersuchungen. Der marginale Rechenaufwand fur den Cluster-pfold ermoglichte die Analyse des TSE von vielen Beta3s-Mutanten und die Berechnung von Phi-Werten. Mit herkommlichen Methoden ware dies fur unsere riesige Menge Simulationsdaten (0.65ms) unmoglich gewesen. Drittens sind in dieser Dissertation alle Simulationen mit konstanter Temperatur bei einer Temperatur T gemacht worden, welche hoher ist als die physiologische Temperatur. Es sei erwahnt, dass die Hohe der Energie-Barrieren proportional zu exp(-(DeltaE/kT) ansteigt, wobei k die Boltzmann-Konstante ist. Daher bleiben MD-Simulationen bei physiologischer Temperatur leicht in lokalen Minima stecken und ergeben somit kein korrektes Sampling der Energy Landscape innert nutzlicher Rechenzeit. In dieser Dissertation wird die Replica-exchange Methode (REM) benutzt, um die fruhen Stadium der Peptid-Aggregation eines amyloidogenetischen Peptides des Prionen-Proteins Sup35

PLoS ONE, 2010

The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previou... more The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimer's disease-associated b-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Ab oligomerization and fibrillization, as well as the cytotoxic effect of Ab oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimer's disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Ab while immuno-staining of the 3 rd instar larval brains showed a significant reduction in Ab accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Ab. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimer's disease.

The Journal of Physical Chemistry B, 2000

The folding of an R-helix and a-hairpin was studied by 862 molecular dynamics simulations with an... more The folding of an R-helix and a-hairpin was studied by 862 molecular dynamics simulations with an implicit solvation model that allowed sampling of a total of 4 µs. The average effective energy is rather flat for conformations having less than about 50% of the folded state contacts formed, except for the R-helix at very high temperatures. For both peptides there is a smooth decrease of the effective energy close to the folded state. The free energy landscape shows that the helix-coil transition is not first order, while the-hairpin has one or two minima, depending on the temperature. At low temperature (T < 1.1T m) there is an increase in the folding rate with increasing temperature as expected from an activation energy limited process. At higher temperatures the rate decreases for both peptides which is consistent with an activation entropy dominated process. The unfolding rate, by contrast, shows an Arrhenius-like behavior; i.e., it increases monotonously with temperature. The-hairpin peptide folds about 30 times slower than the R-helix peptide at 300 K. Multiple folding pathways are present for the R-helix, whereas the-hairpin initiates folding mainly at the-turn.

The Journal of Physical Chemistry B, 2000

The folding of the helical peptide Y(MEARA) 6 was studied by a series of molecular dynamics simul... more The folding of the helical peptide Y(MEARA) 6 was studied by a series of molecular dynamics simulations with an implicit solvation model that allowed sampling of a total of more than 4 µs. In the 44 runs at 360 K started from all-coil conformations the peptide assumed an R-helical structure within the first 30 ns, with an average folding time of 10 ns. The free energy surface shows that the coil to helix transition has a small barrier at the helix nucleation step which consists of two to three i, i + 4 hydrogen bonds and does not show a strong preference along the sequence. On the helix side of the barrier, there is a very broad basin corresponding to conformations having more than one helical turn. Although the R-helical content is predominant, there is a nonnegligible percentage of conformations with one or more π-helical turns stabilized in part by interactions between Met side chains. Control simulations with two different helical sequences, a 31-residue polyalanine and A 5 (AAARA) 3 A, did not reveal a significant π-helix population, which indicates that the π-helical content of Y(MEARA) 6 is not an artifact of the force field and solvation model. The folding mechanism and free energy surface presented here are in agreement with previous theoretical models and experimental data on different helical sequences, which suggest that they may be valid for the folding of helical peptides, in general.

The Journal of Physical Chemistry B, 1998

The potential of mean force in aqueous solution for rotation around the two backbone dihedrals φ ... more The potential of mean force in aqueous solution for rotation around the two backbone dihedrals φ and ψ of the alanine dipeptide is computed in explicit water and in the continuum approximation by numerical integration of the self-energies and the generalized Born (GB) equation. The two models show good agreement. The experimentally observed increase in the gauche/trans population ratio for dichloroethane in going from the gas phase to the pure liquid is reproduced by the GB solvation model with a solvent dielectric constant of 10.5. This test case shows that the GB model gives accurate predictions also for solvents with much lower polarizability than water. For both test systems additional calculations with a finite difference Poisson equation solver yield somewhat more accurate results at a much higher computational cost than the GB solvation model.

The FASEB Journal, 2007

The self-perpetuating conversion of cellular prion proteins (PrP C) into an aggregated ␤-sheet ri... more The self-perpetuating conversion of cellular prion proteins (PrP C) into an aggregated ␤-sheet rich conformation is associated with transmissible spongiform encephalopathies (TSE). The loop 166-175 (L1) in PrP C , which displays sequence and structural variation among species, has been suggested to play a role in species barrier, in particular against transmission of TSE from cervids to domestic and laboratory animals. L1 is ordered in elk PrP, as well as in a mouse/elk hybrid (in which L1 of mouse is replaced by elk) but not in other species such as mice, humans, and bovine. To investigate the source and significance of L1 dynamics, we carried out explicit solvent molecular dynamics simulations (Ϸ0.5 s in total) of the mouse prion protein, the mouse/elk hybrid, and control simulations, in which the mouse sequence is reintroduced into the structure of the mouse/elk hybrid. We found that the flexibility of L1 correlates with the backbone dynamics of Ser170. Furthermore, L1 mobility promotes a substantial displacement of Tyr169, rupture of the Asp178-Tyr128 and Asp178-Tyr169 side chain hydrogen bonds, as well as disruption of Tyr169-Phe175-stacking interaction. The simulation results go beyond the available experimental data because they highlight the dependence of this network of interactions on residue 170 and L1 plasticity.-Gorfe A. A., Caflisch A. Ser170 controls the conformational multiplicity of the loop 166-175 in prion proteins: implication for conversion and species barrier.

Proteins: Structure, Function, and Genetics, 2001

A solvation term based on the solvent accessible surface area (SASA) is combined with the CHARMM ... more A solvation term based on the solvent accessible surface area (SASA) is combined with the CHARMM polar hydrogen force field for the efficient simulation of peptides and small proteins in aqueous solution. Only two atomic solvation parameters are used: one is negative for favoring the direct solvation of polar groups and the other positive for taking into account the hydrophobic effect on apolar groups. To approximate the water screening effects on the intrasolute electrostatic interactions, a distance-dependent dielectric function is used and ionic side chains are neutralized. The use of an analytical approximation of the SASA renders the model extremely efficient (i.e., only about 50% slower than in vacuo simulations). The limitations and range of applicability of the SASA model are assessed by simulations of proteins and structured peptides. For the latter, the present study and results reported elsewhere show that with the SASA model it is possible to sample a significant amount of folding/unfolding transitions, which permit the study of the thermodynamics and kinetics of folding at an atomic level of detail. Proteins 2002;46:24-33.

Proteins: Structure, Function, and Genetics, 1999

A new method is presented for docking molecular fragments to a rigid protein with evaluation of t... more A new method is presented for docking molecular fragments to a rigid protein with evaluation of the binding energy. Polar fragments are docked with at least one hydrogen bond with the protein while apolar fragments are positioned in the hydrophobic pockets. The electrostatic contribution to the binding energy, which consists of screened intermolecular energy and protein and fragment desolvation terms, is evaluated efficiently by a numerical approach based on the continuum dielectric approximation. The latter is also used to predetermine the hydrophobic pockets of the protein by rolling a low dielectric sphere over the protein surface and calculating the electrostatic desolvation of the protein and van der Waals interaction energy. The method was implemented in the program SEED (solvation energy for exhaustive docking). The SEED continuum electrostatic approach has been successfully validated by a comparison with finite difference solutions of the Poisson equation for more than 2,500 complexes of small molecules with thrombin and the monomer of HIV-1 aspartic proteinase. The fragments docked by SEED in the active site of thrombin reproduce the structural features of the interaction patterns between known inhibitors and thrombin. Moreover, the combinatorial connection of these fragments yields a number of compounds that are very similar to potent inhibitors of thrombin.

Protein Science, 2005

The mechanisms by which peptides and proteins form ordered aggregates are not well understood. He... more The mechanisms by which peptides and proteins form ordered aggregates are not well understood. Here we focus on the physicochemical properties of amino acids that favor ordered aggregation and suggest a parameter-free model that is able to predict the change of aggregation rates over a large set of natural sequences. Furthermore, the results of the parameter-free model correlate well with the aggregation propensities of a set of peptides designed by computer simulations.

Protein Science, 2009

Amyloid aggregation is linked to a number of neurodegenerative syndromes, the most prevalent one ... more Amyloid aggregation is linked to a number of neurodegenerative syndromes, the most prevalent one being Alzheimer's disease. In this pathology, the b-amyloid peptides (Ab) aggregate into oligomers, protofibrils, and fibrils and eventually into plaques, which constitute the characteristic hallmark of Alzheimer's disease. Several low-molecular-weight compounds able to impair the Ab aggregation process have been recently discovered; yet, a detailed description of their interactions with oligomers and fibrils is hitherto missing. Here, molecular dynamics simulations are used to investigate the influence of two relatively similar tricyclic, planar compounds, that is, 9, 10-anthraquinone (AQ) and anthracene (AC), on the early phase of the aggregation of the Ab heptapeptide segment H 14 QKLVFF 20 , the hydrophobic stretch that promotes the Ab self-assembly. The simulations show that AQ interferes with b-sheet formation more than AC. In particular, AQ intercalates into the b-sheet because polar interactions between the compound and the peptide backbone destabilize the interstrand hydrogen bonds, thereby favoring disorder. The thioflavin T-binding assay indicates that AQ, but not AC, sensibly reduces the amount of aggregated Ab 1-40 peptide. Taken together, the in silico and in vitro results provide evidence that structural perturbations by AQ can remarkably affect ordered oligomerization. Moreover, the simulations shed light at the atomic level on the interactions between AQ and Ab oligomers, providing useful insights for the design of small-molecule inhibitors of aggregation with therapeutic potential in Alzheimer's disease.

Proceedings of the National Academy of Sciences, 2003

Understanding the early steps of aggregation at atomic detail might be crucial for the rational d... more Understanding the early steps of aggregation at atomic detail might be crucial for the rational design of therapeutics preventing diseases associated with amyloid deposits. In this paper, aggregation of the heptapeptide GNNQQNY, from the N-terminal prion-determining domain of the yeast protein Sup35, was studied by 20 molecular dynamics runs for a total simulation time of 20 μs. The simulations generate in-register parallel packing of GNNQQNY β-strands that is consistent with x-ray diffraction and Fourier transform infrared data. The statistically preferred aggregation pathway does not correspond to a purely downhill profile of the energy surface because of the presence of enthalpic barriers that originate from out-of-register interactions. The parallel β-sheet arrangement is favored over the antiparallel because of side-chain contacts; in particular, stacking interactions of the tyrosine rings and hydrogen bonds between amide groups. No ordered aggregation was found in control simu...

Proceedings of the National Academy of Sciences, 2000

Protein folding is a grand challenge of the postgenomic era. In this paper, 58 folding events sam... more Protein folding is a grand challenge of the postgenomic era. In this paper, 58 folding events sampled during 47 molecular dynamics trajectories for a total simulation time of more than 4 μs provide an atomic detail picture of the folding of a 20-residue synthetic peptide with a stable three-stranded antiparallel β-sheet fold. The simulations successfully reproduce the NMR solution conformation, irrespective of the starting structure. The sampling of the conformational space is sufficient to determine the free energy surface and localize the minima and transition states. The statistically predominant folding pathway involves the formation of contacts between strands 2 and 3, starting with the side chains close to the turn, followed by association of the N-terminal strand onto the preformed 2–3 β-hairpin. The folding mechanism presented here, formation of a β-hairpin followed by consolidation, is in agreement with a computational study of the free energy surface of another synthetic t...

PLoS Neglected Tropical Diseases, 2009

Background: The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefor... more Background: The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus (WNV) and dengue infections. Methodology: In this work, a small-molecule inhibitor of the WNV NS3pro has been identified by automatic fragment-based docking of about 12000 compounds and testing by nuclear magnetic resonance (NMR) spectroscopy of only 22 molecules. Specific binding of the inhibitor into the active site of NS3pro and its binding mode are confirmed by 15 N-HSQC NMR spectra. The inhibitory activity is further validated by an enzymatic assay and a tryptophan fluorescence quenching assay. Conclusion: The inhibitor [4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl]-methylsulfanylmethanimidamide has a good ratio of binding affinity versus molecular weight (ligand efficiency of 0.33 kcal/mol per non-hydrogen atom), and thus has good potential as lead compound for further development to combat West Nile virus infections.

Journal of Molecular Graphics and Modelling, 2001

Farnesyltransferase (FTase) catalyzes the attachment of a 15-carbon isoprenoid moiety, farnesyl, ... more Farnesyltransferase (FTase) catalyzes the attachment of a 15-carbon isoprenoid moiety, farnesyl, through a thioether linkage to a cysteine near the C-terminus of oncogenic Ras proteins. These transform animal cells to a malignant phenotype when farnesylated. Hence, FTase is an interesting target for the development of antitumor agents. In this work we first investigate the active site of FTase by mapping its hydrophobic patches. Then the program SEED is used to dock functional groups into the active site by an exhaustive search and efficient evaluation of the binding energy with solvation. The electrostatic energy in SEED is based on the continuum dielectric approximation and consists of screened intermolecular energy and protein and fragment desolvation terms. The results are found to be consistent with the sequence variability of the tetrapeptide substrate. The distribution of functional groups (functionality maps) on the substrate binding site allows for identification of modifications of the tetrapeptide sequence that are consistent with potent peptidic inhibitors. Furthermore, the best minima of benzene match corresponding moieties of an inhibitor in clinical trials. The functionality maps are also used to design a library of disubstituted indoles that might prevent the binding of the protein substrates.

Journal of Molecular Graphics and Modelling, 1999

Phakellistatin 8 is a cyclic decapeptide that inhibits cancer cell growth and has sequence and st... more Phakellistatin 8 is a cyclic decapeptide that inhibits cancer cell growth and has sequence and structure similar to antamanide. In molecular dynamics simulations of phakellistatin 8 in water, the decapeptide ring undergoes a conformational change from the saddle-like crystal structure to a more elongated conformation by a transition of the Tyr9 main chain from the ␣ L to an extended structure. This is coupled to the loss of the NH9-O6 ␤-turn hydrogen bond and the transient dissociation of the Pro7-Tyr9 side-chain packing. Furthermore, the water molecule acting as a transannular bridge forms an additional hydrogen bond with phakellistatin 8, namely with the NH group of Val5 besides those already present in the crystal structure, i.e., with the NH of Ile10 and the CO of Leu6. The ␣-turn hydrogen bond between the Phe4 amide hydrogen and the Ile10 carbonyl oxygen is always present. The solution conformations of the two cyclic decapeptides are similar, in particular in the region involving the NH4-O10 ␣ turn of phakellistatin 8 and the NH5-O1 ␣ turn of antamanide. The simulation results suggest that in aqueous solution the conformation of phakellistatin 8 is more extended than in the crystalline state, and on a nanosecond time scale phakellistatin 8 is more flexible than antamanide.

Journal of Molecular Biology, 2001

The unbinding of¯uorescein from the single-chain Fv fragment of the 4D5Flu antibody is investigat... more The unbinding of¯uorescein from the single-chain Fv fragment of the 4D5Flu antibody is investigated by biased molecular dynamics with an implicit solvation model. To obtain statistically meaningful results, a large number of unbinding trajectories are calculated; they involve a total simulation time of more than 200 ns. Simulations are carried out with a time-dependent perturbation and in the presence of a constant force. The two techniques, which provide complementary information, induce unbinding by favoring an increase in the distance between the ligand and the antibody. This distance is an appropriate progress variable for the dissociation reaction and permits direct comparison of the unbinding forces in the simulations with data from atomic force microscopy (AFM). The time-dependent perturbation generates unfolding pathways that are close to equilibrium and can be used to reconstruct the mean force; i.e. the derivative of the potential of mean force, along the reaction coordinate. This is supported by an analysis of the overall unbinding pro®le and the magnitude of the mean force, which are similar to those of the unbinding force (i.e. the external force due to the time-dependent perturbation) averaged over several unbinding events. The multiple simulations show that unbinding proceeds along a rather well-de®ned pathway for a broad range of effective pulling speeds. Initially, there is a distortion of the protein localized in the C-terminal region followed by the¯uorescein exit from the binding site. This occurs in steps that involve breaking of speci®c electrostatic and van der Waals interactions. It appears that the simulations do not explore the same barriers as those measured in the AFM experiments because of the much higher unfolding speed in the former. The dependence of the force on the logarithm of the loading rate is linear and the slope is higher than in the AFM, in agreement with experiment in other systems, where different slopes were observed for different regimes. Based on the unbinding events, mutations in the 4D5Flu antigen binding site are predicted to result in signi®cant changes in the unbinding force.

Journal of Molecular Biology, 2001

Fifty-®ve molecular dynamics runs of two three-stranded antiparallel b-sheet peptides were perfor... more Fifty-®ve molecular dynamics runs of two three-stranded antiparallel b-sheet peptides were performed to investigate the relative importance of amino acid sequence and native topology. The two peptides consist of 20 residues each and have a sequence identity of 15 %. One peptide has Gly-Ser (GS) at both turns, while the other has D-Pro-Gly (D PG). The simulations successfully reproduce the NMR solution conformations, irrespective of the starting structure. The large number of folding events sampled along the trajectories at 360 K (total simulation time of about 5 ms) yield a projection of the free-energy landscape onto two signi®cant progress variables. The two peptides have compact denatured states, similar free-energy surfaces, and folding pathways that involve the formation of a b-hairpin followed by consolidation of the unstructured strand. For the GS peptide, there are 33 folding events that start by the formation of the 2-3 b-hairpin and 17 with ®rst the 1-2 b-hairpin. For the D PG peptide, the statistical predominance is opposite, 16 and 47 folding events start from the 2-3 b-hairpin and the 1-2 b-hairpin, respectively. These simulation results indicate that the overall shape of the free-energy surface is de®ned primarily by the native-state topology, in agreement with an ever-increasing amount of experimental and theoretical evidence, while the amino acid sequence determines the statistically predominant order of the events.

Journal of Medicinal Chemistry, 2004

The linear interaction energy (LIE) method is combined with energy minimization and finitediffere... more The linear interaction energy (LIE) method is combined with energy minimization and finitedifference Poisson calculation of electrostatic solvation for the estimation of the absolute free energy of binding. A predictive accuracy of about 1.0 kcal/mol is obtained for 13 and 29 inhibitors of-secretase (BACE) and HIV-1 protease (HIV-1 PR), respectively. The multiplicative coefficients for the van der Waals and electrostatic terms are not transferable between BACE and HIV-1 PR although they are both aspartic proteases. The present approach is about 2 orders of magnitude faster than previous LIE methods and can be used for ranking large libraries of structurally diverse compounds docked by automatic computational tools.

Journal of Medicinal Chemistry, 2008

To take into account polarization effects, the linear interaction energy model with continuum ele... more To take into account polarization effects, the linear interaction energy model with continuum electrostatic solvation (LIECE) is supplemented by the linear-scaling semiempirical quantum mechanical calculation of the intermolecular electrostatic energy (QMLIECE). QMLIECE and LIECE are compared on three enzymes belonging to different classes: the West Nile virus NS3 serine protease (WNV PR), the aspartic protease of the human immunodeficiency virus (HIV-1 PR), and the human cyclin-dependent kinase 2 (CDK2). QMLIECE is superior for 44 peptidic inhibitors of WNV PR because of the different amount of polarization due to the broad range of formal charges of the inhibitors (from 0 to 3). On the other hand, QMLIECE and LIECE show similar accuracy for 24 peptidic inhibitors of HIV-1 PR (20 neutral and 4 with one formal charge) and for 73 CDK2 inhibitors (all neutral). These results indicate that quantum mechanics is essential when the inhibitor/protein complexes have highly variable charge-charge interactions.

Journal of Medicinal Chemistry, 1993

Rational ligand design is a complex problem that can be divided into three parts: the search for ... more Rational ligand design is a complex problem that can be divided into three parts: the search for optimal positions and orientations of functional groups in the binding site, the connection of such positions to form candidate ligands, and the estimation of their binding constants. Approaches for addressing the first two parts of the problem are described in the present work. They are applied to the construction of peptide ligands in the binding site of the human immunodeficiency virus 1 (HIV-1) proteinase. The primary objective is to test the method by comparison of the results with the MVT-101 complex structure for which coordinates are available; the results obtained with the liganded and unliganded proteinase structure are used to examine the utility of the latter for binding studies. A secondary objective is to show how to find new inhibitor candidates. The multiple copy simultaneous search (MCSS) method is utilized to search for optimal positions and orientations of a set of functional groups. For peptide ligands, functional groups corresponding to the protein main chain (N-methylacetamide) and to protein side chains (e.g., methanol, ethyl guanidinium) are used. The resulting N-methylacetamide minima are connected to form hexapeptide main chains with a simple pseudoenergy function that permits a complete search of all possible ways of connecting the minima. Side chains are added to the main-chain candidates by application of the same pseudoenergy function to the appropriate functional group minima. A set of 15 hexapeptides with the sequence of MVT-101 is then minimized by a Monte Carlo scheme, which allows for escape from local minima. Comparison of the MCSS results with the structure of MVT-101 in the HIV-1 binding site showed that all of its functional group positions correspond (within 2.4 A) to some (usually more than one) MCSS minima. There were also many other low-energy MCSS minima which do not appear in any known inhibitors, e.g., methyl ammonium minima in the neighborhood of the catalytic aspartates. Among the 15 lowest minima are seven hexapeptides with the same main-chain orientation as the one found by X-ray crystallography for the inhibitor MVT-101 in the binding site and eight with the main chain oriented in the opposite direction; the latter tend to be more stable. [Addendum: These results are in agreement with recent high-resolution crystallographic data provided after the study was completed. They show that the MVT-101 binds in two orientations and that the published orientation represents the minor conformer. (M. Miller et al. Private communication.)l A set of terminal blocked dipeptides were constructed from low-energy MCSS minima at one open end of the HIV-1 aspartic proteinase binding site and their interactions with the protein were analyzed. It was shown that some of the dipeptides can be connected to known hexapeptide ligands. The paper demonstrates that the combination of a method for an exhaustive search of the binding site for functional group minima (MCSS) with a highly efficient method for constructing molecules from them provides a novel and effective approach to the theoretical design and docking of candidate peptide ligands. The results of the present analysis suggest several modifications of MVT-101 that may have increased affinity and/or specificity for the HIV-1 aspartic proteinase binding site.

Die folgende Dissertation befasst sich mit der Entwicklung und Anwendung dreier neur Ansatze fur ... more Die folgende Dissertation befasst sich mit der Entwicklung und Anwendung dreier neur Ansatze fur die Erzeugung und Untersuchung von Energy Landscapes mit dem Ziel, die Proteinfaltung zu charakterisieren und besser zu verstehen. Das Paradigma der Energy Landscapes hat sich in den letzten zehn Jahren bei der Untersuchung des Faltungsverhaltens von Proteinen bewahrt. Dieses Paradigma fuhrt dazu, dass die Energy Landscape eines Proteins vereinfacht als Trichter dargestellt werden kann. Projektionen dieser Energy Landscape auf Ordnungsparameter, wie zum Beispiel die Projektion auf die freie Energie zur Untersuchung von Stabilitat, sollten eine vereinfachte Sicht der Zustande und Energiebarrieren wiedergeben, wobei oftmals versteckte Annahmen getroffen werden, die nicht erfullt sind. Basierend auf der Theorie komplexer Netzwerke wird als erstes ein neur Ansatz zur Untersuchung der Proteinzustande vorgestellt. Dabei sind die Konformationen, welche im Verlauf einer molecular dynamics (MD) Simulation angenommen werden, die Knoten und die zeitlichen Uebergange die Verbindungen. Dieses Netzwerk reprasentiert die vollstandige multi-dimensionale Energy Landscape ohne Projektion auf willkurlich gewahlte Ordnungsparameter. Die Anwendung von Netzwerken enthullt ein komplexes Zusammenspiel von Minima, Basins und Super-Basins, die in der Energy Landscape die Rolle von Attraktoren spielen. Als erstes wird die MD-Simulation eines strukturierten Peptides (Beta3s) untersucht. Dabei werden Basins mit kleiner Entropie/ kleiner Enthalpie, sowie Basins mit grosser Entropie/grosser Enthalpie gefunden. Hierbei ist interessant, dass Beta3s im gefalteten Zustand (im Unterschied zu einer kinetischen Trap) nicht nur enthalpisch, sondern auch entropisch stabilisiert wird. Free Energy Basins entsprechen Subgraphen (Communities) des gesamten Netzwerks, wobei die Knoten in einen Basin dicht miteinander verbunden sind. Das Finden dieser Communities und die Validierung einer Unterteilung des gesamten Netzwerkes in solche ist trotz der Fulle von existierenden Algorithmen noch nicht zufriedenstllend gelost. In dieser Dissertation wird dazu ein Mass, die Goodness Deviation, vorgeschlagen. Zweitens ist die Kinetik der Proteinfaltung ebenfalls eng mit der Topographie von Energy Landscapes verknupft. Die schwer fassbare Natur des Transition State Ensemble (TSE) erlaubt kein einfaches Bild fur Faltunsubergange. Zur Charakterisierung des TSE wird oft die Faltungswahrscheinlichkeit pfold einer Proteinstruktur benutzt. Obwohl g fur die Analyse von Faltungsubergangen sehr nutzlich ist, erfordert dessen Berechnung einen grossen rechnerischen Aufwand, was die praktische Anwendungen limitiert. Die kinetische Homogenitat von strukturell ahnlichen Snapshots erlaubt jedoch einen statistischen Zugang fur die Analyse einzelner Konformationen. Die Idee des sogenannten Cluster-pfold ermoglicht es in dieser Dissertation, g fur Strukturen einer MD-simulation approximativ zu berechnen. Die Anwendung dieser Methode auf Beta3s zeigt ein breites, heterogenes TSE, sowie zwei Haupt-Pathways. Diese Ergebnisse decken sich interessanterweise mit der Netzwerk-Analyse aus vorhergehenden Untersuchungen. Der marginale Rechenaufwand fur den Cluster-pfold ermoglichte die Analyse des TSE von vielen Beta3s-Mutanten und die Berechnung von Phi-Werten. Mit herkommlichen Methoden ware dies fur unsere riesige Menge Simulationsdaten (0.65ms) unmoglich gewesen. Drittens sind in dieser Dissertation alle Simulationen mit konstanter Temperatur bei einer Temperatur T gemacht worden, welche hoher ist als die physiologische Temperatur. Es sei erwahnt, dass die Hohe der Energie-Barrieren proportional zu exp(-(DeltaE/kT) ansteigt, wobei k die Boltzmann-Konstante ist. Daher bleiben MD-Simulationen bei physiologischer Temperatur leicht in lokalen Minima stecken und ergeben somit kein korrektes Sampling der Energy Landscape innert nutzlicher Rechenzeit. In dieser Dissertation wird die Replica-exchange Methode (REM) benutzt, um die fruhen Stadium der Peptid-Aggregation eines amyloidogenetischen Peptides des Prionen-Proteins Sup35

PLoS ONE, 2010

The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previou... more The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimer's disease-associated b-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Ab oligomerization and fibrillization, as well as the cytotoxic effect of Ab oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimer's disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Ab while immuno-staining of the 3 rd instar larval brains showed a significant reduction in Ab accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Ab. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimer's disease.

The Journal of Physical Chemistry B, 2000

The folding of an R-helix and a-hairpin was studied by 862 molecular dynamics simulations with an... more The folding of an R-helix and a-hairpin was studied by 862 molecular dynamics simulations with an implicit solvation model that allowed sampling of a total of 4 µs. The average effective energy is rather flat for conformations having less than about 50% of the folded state contacts formed, except for the R-helix at very high temperatures. For both peptides there is a smooth decrease of the effective energy close to the folded state. The free energy landscape shows that the helix-coil transition is not first order, while the-hairpin has one or two minima, depending on the temperature. At low temperature (T < 1.1T m) there is an increase in the folding rate with increasing temperature as expected from an activation energy limited process. At higher temperatures the rate decreases for both peptides which is consistent with an activation entropy dominated process. The unfolding rate, by contrast, shows an Arrhenius-like behavior; i.e., it increases monotonously with temperature. The-hairpin peptide folds about 30 times slower than the R-helix peptide at 300 K. Multiple folding pathways are present for the R-helix, whereas the-hairpin initiates folding mainly at the-turn.

The Journal of Physical Chemistry B, 2000

The folding of the helical peptide Y(MEARA) 6 was studied by a series of molecular dynamics simul... more The folding of the helical peptide Y(MEARA) 6 was studied by a series of molecular dynamics simulations with an implicit solvation model that allowed sampling of a total of more than 4 µs. In the 44 runs at 360 K started from all-coil conformations the peptide assumed an R-helical structure within the first 30 ns, with an average folding time of 10 ns. The free energy surface shows that the coil to helix transition has a small barrier at the helix nucleation step which consists of two to three i, i + 4 hydrogen bonds and does not show a strong preference along the sequence. On the helix side of the barrier, there is a very broad basin corresponding to conformations having more than one helical turn. Although the R-helical content is predominant, there is a nonnegligible percentage of conformations with one or more π-helical turns stabilized in part by interactions between Met side chains. Control simulations with two different helical sequences, a 31-residue polyalanine and A 5 (AAARA) 3 A, did not reveal a significant π-helix population, which indicates that the π-helical content of Y(MEARA) 6 is not an artifact of the force field and solvation model. The folding mechanism and free energy surface presented here are in agreement with previous theoretical models and experimental data on different helical sequences, which suggest that they may be valid for the folding of helical peptides, in general.

The Journal of Physical Chemistry B, 1998

The potential of mean force in aqueous solution for rotation around the two backbone dihedrals φ ... more The potential of mean force in aqueous solution for rotation around the two backbone dihedrals φ and ψ of the alanine dipeptide is computed in explicit water and in the continuum approximation by numerical integration of the self-energies and the generalized Born (GB) equation. The two models show good agreement. The experimentally observed increase in the gauche/trans population ratio for dichloroethane in going from the gas phase to the pure liquid is reproduced by the GB solvation model with a solvent dielectric constant of 10.5. This test case shows that the GB model gives accurate predictions also for solvents with much lower polarizability than water. For both test systems additional calculations with a finite difference Poisson equation solver yield somewhat more accurate results at a much higher computational cost than the GB solvation model.

The FASEB Journal, 2007

The self-perpetuating conversion of cellular prion proteins (PrP C) into an aggregated ␤-sheet ri... more The self-perpetuating conversion of cellular prion proteins (PrP C) into an aggregated ␤-sheet rich conformation is associated with transmissible spongiform encephalopathies (TSE). The loop 166-175 (L1) in PrP C , which displays sequence and structural variation among species, has been suggested to play a role in species barrier, in particular against transmission of TSE from cervids to domestic and laboratory animals. L1 is ordered in elk PrP, as well as in a mouse/elk hybrid (in which L1 of mouse is replaced by elk) but not in other species such as mice, humans, and bovine. To investigate the source and significance of L1 dynamics, we carried out explicit solvent molecular dynamics simulations (Ϸ0.5 s in total) of the mouse prion protein, the mouse/elk hybrid, and control simulations, in which the mouse sequence is reintroduced into the structure of the mouse/elk hybrid. We found that the flexibility of L1 correlates with the backbone dynamics of Ser170. Furthermore, L1 mobility promotes a substantial displacement of Tyr169, rupture of the Asp178-Tyr128 and Asp178-Tyr169 side chain hydrogen bonds, as well as disruption of Tyr169-Phe175-stacking interaction. The simulation results go beyond the available experimental data because they highlight the dependence of this network of interactions on residue 170 and L1 plasticity.-Gorfe A. A., Caflisch A. Ser170 controls the conformational multiplicity of the loop 166-175 in prion proteins: implication for conversion and species barrier.

Proteins: Structure, Function, and Genetics, 2001

A solvation term based on the solvent accessible surface area (SASA) is combined with the CHARMM ... more A solvation term based on the solvent accessible surface area (SASA) is combined with the CHARMM polar hydrogen force field for the efficient simulation of peptides and small proteins in aqueous solution. Only two atomic solvation parameters are used: one is negative for favoring the direct solvation of polar groups and the other positive for taking into account the hydrophobic effect on apolar groups. To approximate the water screening effects on the intrasolute electrostatic interactions, a distance-dependent dielectric function is used and ionic side chains are neutralized. The use of an analytical approximation of the SASA renders the model extremely efficient (i.e., only about 50% slower than in vacuo simulations). The limitations and range of applicability of the SASA model are assessed by simulations of proteins and structured peptides. For the latter, the present study and results reported elsewhere show that with the SASA model it is possible to sample a significant amount of folding/unfolding transitions, which permit the study of the thermodynamics and kinetics of folding at an atomic level of detail. Proteins 2002;46:24-33.

Proteins: Structure, Function, and Genetics, 1999

A new method is presented for docking molecular fragments to a rigid protein with evaluation of t... more A new method is presented for docking molecular fragments to a rigid protein with evaluation of the binding energy. Polar fragments are docked with at least one hydrogen bond with the protein while apolar fragments are positioned in the hydrophobic pockets. The electrostatic contribution to the binding energy, which consists of screened intermolecular energy and protein and fragment desolvation terms, is evaluated efficiently by a numerical approach based on the continuum dielectric approximation. The latter is also used to predetermine the hydrophobic pockets of the protein by rolling a low dielectric sphere over the protein surface and calculating the electrostatic desolvation of the protein and van der Waals interaction energy. The method was implemented in the program SEED (solvation energy for exhaustive docking). The SEED continuum electrostatic approach has been successfully validated by a comparison with finite difference solutions of the Poisson equation for more than 2,500 complexes of small molecules with thrombin and the monomer of HIV-1 aspartic proteinase. The fragments docked by SEED in the active site of thrombin reproduce the structural features of the interaction patterns between known inhibitors and thrombin. Moreover, the combinatorial connection of these fragments yields a number of compounds that are very similar to potent inhibitors of thrombin.

Protein Science, 2005

The mechanisms by which peptides and proteins form ordered aggregates are not well understood. He... more The mechanisms by which peptides and proteins form ordered aggregates are not well understood. Here we focus on the physicochemical properties of amino acids that favor ordered aggregation and suggest a parameter-free model that is able to predict the change of aggregation rates over a large set of natural sequences. Furthermore, the results of the parameter-free model correlate well with the aggregation propensities of a set of peptides designed by computer simulations.

Protein Science, 2009

Amyloid aggregation is linked to a number of neurodegenerative syndromes, the most prevalent one ... more Amyloid aggregation is linked to a number of neurodegenerative syndromes, the most prevalent one being Alzheimer's disease. In this pathology, the b-amyloid peptides (Ab) aggregate into oligomers, protofibrils, and fibrils and eventually into plaques, which constitute the characteristic hallmark of Alzheimer's disease. Several low-molecular-weight compounds able to impair the Ab aggregation process have been recently discovered; yet, a detailed description of their interactions with oligomers and fibrils is hitherto missing. Here, molecular dynamics simulations are used to investigate the influence of two relatively similar tricyclic, planar compounds, that is, 9, 10-anthraquinone (AQ) and anthracene (AC), on the early phase of the aggregation of the Ab heptapeptide segment H 14 QKLVFF 20 , the hydrophobic stretch that promotes the Ab self-assembly. The simulations show that AQ interferes with b-sheet formation more than AC. In particular, AQ intercalates into the b-sheet because polar interactions between the compound and the peptide backbone destabilize the interstrand hydrogen bonds, thereby favoring disorder. The thioflavin T-binding assay indicates that AQ, but not AC, sensibly reduces the amount of aggregated Ab 1-40 peptide. Taken together, the in silico and in vitro results provide evidence that structural perturbations by AQ can remarkably affect ordered oligomerization. Moreover, the simulations shed light at the atomic level on the interactions between AQ and Ab oligomers, providing useful insights for the design of small-molecule inhibitors of aggregation with therapeutic potential in Alzheimer's disease.

Proceedings of the National Academy of Sciences, 2003

Understanding the early steps of aggregation at atomic detail might be crucial for the rational d... more Understanding the early steps of aggregation at atomic detail might be crucial for the rational design of therapeutics preventing diseases associated with amyloid deposits. In this paper, aggregation of the heptapeptide GNNQQNY, from the N-terminal prion-determining domain of the yeast protein Sup35, was studied by 20 molecular dynamics runs for a total simulation time of 20 μs. The simulations generate in-register parallel packing of GNNQQNY β-strands that is consistent with x-ray diffraction and Fourier transform infrared data. The statistically preferred aggregation pathway does not correspond to a purely downhill profile of the energy surface because of the presence of enthalpic barriers that originate from out-of-register interactions. The parallel β-sheet arrangement is favored over the antiparallel because of side-chain contacts; in particular, stacking interactions of the tyrosine rings and hydrogen bonds between amide groups. No ordered aggregation was found in control simu...

Proceedings of the National Academy of Sciences, 2000

Protein folding is a grand challenge of the postgenomic era. In this paper, 58 folding events sam... more Protein folding is a grand challenge of the postgenomic era. In this paper, 58 folding events sampled during 47 molecular dynamics trajectories for a total simulation time of more than 4 μs provide an atomic detail picture of the folding of a 20-residue synthetic peptide with a stable three-stranded antiparallel β-sheet fold. The simulations successfully reproduce the NMR solution conformation, irrespective of the starting structure. The sampling of the conformational space is sufficient to determine the free energy surface and localize the minima and transition states. The statistically predominant folding pathway involves the formation of contacts between strands 2 and 3, starting with the side chains close to the turn, followed by association of the N-terminal strand onto the preformed 2–3 β-hairpin. The folding mechanism presented here, formation of a β-hairpin followed by consolidation, is in agreement with a computational study of the free energy surface of another synthetic t...

PLoS Neglected Tropical Diseases, 2009

Background: The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefor... more Background: The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus (WNV) and dengue infections. Methodology: In this work, a small-molecule inhibitor of the WNV NS3pro has been identified by automatic fragment-based docking of about 12000 compounds and testing by nuclear magnetic resonance (NMR) spectroscopy of only 22 molecules. Specific binding of the inhibitor into the active site of NS3pro and its binding mode are confirmed by 15 N-HSQC NMR spectra. The inhibitory activity is further validated by an enzymatic assay and a tryptophan fluorescence quenching assay. Conclusion: The inhibitor [4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl]-methylsulfanylmethanimidamide has a good ratio of binding affinity versus molecular weight (ligand efficiency of 0.33 kcal/mol per non-hydrogen atom), and thus has good potential as lead compound for further development to combat West Nile virus infections.

Journal of Molecular Graphics and Modelling, 2001

Farnesyltransferase (FTase) catalyzes the attachment of a 15-carbon isoprenoid moiety, farnesyl, ... more Farnesyltransferase (FTase) catalyzes the attachment of a 15-carbon isoprenoid moiety, farnesyl, through a thioether linkage to a cysteine near the C-terminus of oncogenic Ras proteins. These transform animal cells to a malignant phenotype when farnesylated. Hence, FTase is an interesting target for the development of antitumor agents. In this work we first investigate the active site of FTase by mapping its hydrophobic patches. Then the program SEED is used to dock functional groups into the active site by an exhaustive search and efficient evaluation of the binding energy with solvation. The electrostatic energy in SEED is based on the continuum dielectric approximation and consists of screened intermolecular energy and protein and fragment desolvation terms. The results are found to be consistent with the sequence variability of the tetrapeptide substrate. The distribution of functional groups (functionality maps) on the substrate binding site allows for identification of modifications of the tetrapeptide sequence that are consistent with potent peptidic inhibitors. Furthermore, the best minima of benzene match corresponding moieties of an inhibitor in clinical trials. The functionality maps are also used to design a library of disubstituted indoles that might prevent the binding of the protein substrates.

Journal of Molecular Graphics and Modelling, 1999

Phakellistatin 8 is a cyclic decapeptide that inhibits cancer cell growth and has sequence and st... more Phakellistatin 8 is a cyclic decapeptide that inhibits cancer cell growth and has sequence and structure similar to antamanide. In molecular dynamics simulations of phakellistatin 8 in water, the decapeptide ring undergoes a conformational change from the saddle-like crystal structure to a more elongated conformation by a transition of the Tyr9 main chain from the ␣ L to an extended structure. This is coupled to the loss of the NH9-O6 ␤-turn hydrogen bond and the transient dissociation of the Pro7-Tyr9 side-chain packing. Furthermore, the water molecule acting as a transannular bridge forms an additional hydrogen bond with phakellistatin 8, namely with the NH group of Val5 besides those already present in the crystal structure, i.e., with the NH of Ile10 and the CO of Leu6. The ␣-turn hydrogen bond between the Phe4 amide hydrogen and the Ile10 carbonyl oxygen is always present. The solution conformations of the two cyclic decapeptides are similar, in particular in the region involving the NH4-O10 ␣ turn of phakellistatin 8 and the NH5-O1 ␣ turn of antamanide. The simulation results suggest that in aqueous solution the conformation of phakellistatin 8 is more extended than in the crystalline state, and on a nanosecond time scale phakellistatin 8 is more flexible than antamanide.

Journal of Molecular Biology, 2001

The unbinding of¯uorescein from the single-chain Fv fragment of the 4D5Flu antibody is investigat... more The unbinding of¯uorescein from the single-chain Fv fragment of the 4D5Flu antibody is investigated by biased molecular dynamics with an implicit solvation model. To obtain statistically meaningful results, a large number of unbinding trajectories are calculated; they involve a total simulation time of more than 200 ns. Simulations are carried out with a time-dependent perturbation and in the presence of a constant force. The two techniques, which provide complementary information, induce unbinding by favoring an increase in the distance between the ligand and the antibody. This distance is an appropriate progress variable for the dissociation reaction and permits direct comparison of the unbinding forces in the simulations with data from atomic force microscopy (AFM). The time-dependent perturbation generates unfolding pathways that are close to equilibrium and can be used to reconstruct the mean force; i.e. the derivative of the potential of mean force, along the reaction coordinate. This is supported by an analysis of the overall unbinding pro®le and the magnitude of the mean force, which are similar to those of the unbinding force (i.e. the external force due to the time-dependent perturbation) averaged over several unbinding events. The multiple simulations show that unbinding proceeds along a rather well-de®ned pathway for a broad range of effective pulling speeds. Initially, there is a distortion of the protein localized in the C-terminal region followed by the¯uorescein exit from the binding site. This occurs in steps that involve breaking of speci®c electrostatic and van der Waals interactions. It appears that the simulations do not explore the same barriers as those measured in the AFM experiments because of the much higher unfolding speed in the former. The dependence of the force on the logarithm of the loading rate is linear and the slope is higher than in the AFM, in agreement with experiment in other systems, where different slopes were observed for different regimes. Based on the unbinding events, mutations in the 4D5Flu antigen binding site are predicted to result in signi®cant changes in the unbinding force.

Journal of Molecular Biology, 2001

Fifty-®ve molecular dynamics runs of two three-stranded antiparallel b-sheet peptides were perfor... more Fifty-®ve molecular dynamics runs of two three-stranded antiparallel b-sheet peptides were performed to investigate the relative importance of amino acid sequence and native topology. The two peptides consist of 20 residues each and have a sequence identity of 15 %. One peptide has Gly-Ser (GS) at both turns, while the other has D-Pro-Gly (D PG). The simulations successfully reproduce the NMR solution conformations, irrespective of the starting structure. The large number of folding events sampled along the trajectories at 360 K (total simulation time of about 5 ms) yield a projection of the free-energy landscape onto two signi®cant progress variables. The two peptides have compact denatured states, similar free-energy surfaces, and folding pathways that involve the formation of a b-hairpin followed by consolidation of the unstructured strand. For the GS peptide, there are 33 folding events that start by the formation of the 2-3 b-hairpin and 17 with ®rst the 1-2 b-hairpin. For the D PG peptide, the statistical predominance is opposite, 16 and 47 folding events start from the 2-3 b-hairpin and the 1-2 b-hairpin, respectively. These simulation results indicate that the overall shape of the free-energy surface is de®ned primarily by the native-state topology, in agreement with an ever-increasing amount of experimental and theoretical evidence, while the amino acid sequence determines the statistically predominant order of the events.

Journal of Medicinal Chemistry, 2004

The linear interaction energy (LIE) method is combined with energy minimization and finitediffere... more The linear interaction energy (LIE) method is combined with energy minimization and finitedifference Poisson calculation of electrostatic solvation for the estimation of the absolute free energy of binding. A predictive accuracy of about 1.0 kcal/mol is obtained for 13 and 29 inhibitors of-secretase (BACE) and HIV-1 protease (HIV-1 PR), respectively. The multiplicative coefficients for the van der Waals and electrostatic terms are not transferable between BACE and HIV-1 PR although they are both aspartic proteases. The present approach is about 2 orders of magnitude faster than previous LIE methods and can be used for ranking large libraries of structurally diverse compounds docked by automatic computational tools.

Journal of Medicinal Chemistry, 2008

To take into account polarization effects, the linear interaction energy model with continuum ele... more To take into account polarization effects, the linear interaction energy model with continuum electrostatic solvation (LIECE) is supplemented by the linear-scaling semiempirical quantum mechanical calculation of the intermolecular electrostatic energy (QMLIECE). QMLIECE and LIECE are compared on three enzymes belonging to different classes: the West Nile virus NS3 serine protease (WNV PR), the aspartic protease of the human immunodeficiency virus (HIV-1 PR), and the human cyclin-dependent kinase 2 (CDK2). QMLIECE is superior for 44 peptidic inhibitors of WNV PR because of the different amount of polarization due to the broad range of formal charges of the inhibitors (from 0 to 3). On the other hand, QMLIECE and LIECE show similar accuracy for 24 peptidic inhibitors of HIV-1 PR (20 neutral and 4 with one formal charge) and for 73 CDK2 inhibitors (all neutral). These results indicate that quantum mechanics is essential when the inhibitor/protein complexes have highly variable charge-charge interactions.

Journal of Medicinal Chemistry, 1993

Rational ligand design is a complex problem that can be divided into three parts: the search for ... more Rational ligand design is a complex problem that can be divided into three parts: the search for optimal positions and orientations of functional groups in the binding site, the connection of such positions to form candidate ligands, and the estimation of their binding constants. Approaches for addressing the first two parts of the problem are described in the present work. They are applied to the construction of peptide ligands in the binding site of the human immunodeficiency virus 1 (HIV-1) proteinase. The primary objective is to test the method by comparison of the results with the MVT-101 complex structure for which coordinates are available; the results obtained with the liganded and unliganded proteinase structure are used to examine the utility of the latter for binding studies. A secondary objective is to show how to find new inhibitor candidates. The multiple copy simultaneous search (MCSS) method is utilized to search for optimal positions and orientations of a set of functional groups. For peptide ligands, functional groups corresponding to the protein main chain (N-methylacetamide) and to protein side chains (e.g., methanol, ethyl guanidinium) are used. The resulting N-methylacetamide minima are connected to form hexapeptide main chains with a simple pseudoenergy function that permits a complete search of all possible ways of connecting the minima. Side chains are added to the main-chain candidates by application of the same pseudoenergy function to the appropriate functional group minima. A set of 15 hexapeptides with the sequence of MVT-101 is then minimized by a Monte Carlo scheme, which allows for escape from local minima. Comparison of the MCSS results with the structure of MVT-101 in the HIV-1 binding site showed that all of its functional group positions correspond (within 2.4 A) to some (usually more than one) MCSS minima. There were also many other low-energy MCSS minima which do not appear in any known inhibitors, e.g., methyl ammonium minima in the neighborhood of the catalytic aspartates. Among the 15 lowest minima are seven hexapeptides with the same main-chain orientation as the one found by X-ray crystallography for the inhibitor MVT-101 in the binding site and eight with the main chain oriented in the opposite direction; the latter tend to be more stable. [Addendum: These results are in agreement with recent high-resolution crystallographic data provided after the study was completed. They show that the MVT-101 binds in two orientations and that the published orientation represents the minor conformer. (M. Miller et al. Private communication.)l A set of terminal blocked dipeptides were constructed from low-energy MCSS minima at one open end of the HIV-1 aspartic proteinase binding site and their interactions with the protein were analyzed. It was shown that some of the dipeptides can be connected to known hexapeptide ligands. The paper demonstrates that the combination of a method for an exhaustive search of the binding site for functional group minima (MCSS) with a highly efficient method for constructing molecules from them provides a novel and effective approach to the theoretical design and docking of candidate peptide ligands. The results of the present analysis suggest several modifications of MVT-101 that may have increased affinity and/or specificity for the HIV-1 aspartic proteinase binding site.