The Influence of Hydrogen Atoms on the Performance of Radial Distribution Function-Based Descriptors in the Chemoinformatic Studies of HIV-1 Protease Complexes with Inhibitors.

AIMS: The aim of this letter is to explore the influence of adding hydrogen atoms to the crystallographic structures of HIV-1 protease complexes with a series of inhibitors on the performance of radial distribution function based descriptors recently introduced in chemoinformatic studies. BACKGROUND: Quite recently the successful application of molecular descriptors based on a radial distribution function to correlate it with biologically interesting properties of a ligand - enzyme complex was demonstrated. Except its predictive power, the analysis of atoms with dominant contributions to the RDFs can be used to identify relevant atoms and interactions. Since original paper was published on dataset consisting of the X-ray structures of complexes without hydrogen atoms, we wonder weather addition of light atoms can provide us new piece of information. OBJECTIVE: The primarily objective is to create the model correlating the RDF based descriptors and physicochemical properties of the HIV-1 protease complexes with inhibitors with hydrogen atoms. Then, we will compare the performance of new model with previous one, where the hydrogen atoms were discarded. Information about interactions between the enzyme and the inhibitors will be extracted from the analysis of the RDF. METHODS: The radial distribution function descriptor weighted by the number of valence shell electrons has proven to be sensitive to the changes in the structure of the enzyme and enzyme-ligand complexes. For each structure in our data set, RDF will be calculated and using multiple linear regression method the mathematical model will be designed correlating RDF based descriptors and the physicochemical properties. Statistical analysis of the atom's contribution to the total RDF will reveal relevant interactions. RESULTS: The applicability of RDF based descriptor for the correlation with pKi and EC50 values is demonstrated, while simple models containing only two or three parameters are able to explain 78 and 86 % of the variance, respectively. The models with explicitly included hydrogens are of comparable quality with the previous models without hydrogens. The analysis of the atom's dominant contributions highlighted the importance of the hydroxyl groups of the inhibitor near the Asp25 and Asp25' residues when it is bounded to the protease. CONCLUSION: Models based on the RDF weighted by the number of valence shell electrons for correlating small number of molecular descriptors and physicocehmical properties for structures with and without hydrogens are of comparable quality and both can be used for identification of relevant functional groups and interactions. Other: Our approach can be integrated to the next generation virtual screening methods, because is fast, reliable with high predictability potential.

Comparative molecular dynamics study of dimeric and monomeric forms of HIV-1 protease in ligand bound and unbound state.

Human immunodeficiency virus type 1 protease is a viral-encoded enzyme and it is essential for replication and assembly of the virus. Inactivation of HIV-1 protease causes production of immature, noninfectious viral particles and thus HIV-1 protease is an attractive target in anti-AIDS drug design. In our current work, we performed molecular dynamics (MD) calculations (500 ns) for two different ligands (COM5 - designed in our previous study, and Darunavir) and made effort to understand dynamics behaviour of our designed compound COM5. An apo form of HIV-1 protease as monomer and dimer form was also studied in order to analyze response of protein to the ligand. MD results suggest that presence of ligand in hinders the stability of HIV-1 protease and one monomer from dimer systems is dominant on other monomer in terms of interaction made with ligands. We were able to trace functional residues as well as continuous motion of opening and closing (clapping) of flap region in HIV-1 protease (apo form) during entire 1000 ns of MD simulation. COM5 showed almost similar behaviour towards HIV-1 protease enzyme as Darunavir and propose as promising lead compound for the development of new inhibitor for HIV-1 protease.

Exploring the antiviral activity of juglone by computational method.

Nature has been the best source of medicines for a long time. Many plant extracts have been used as drugs. Juglone occurs in all parts of the Juglandaceae family and is found extensively in the black walnut plants. It possesses antifungal, antimalarial, antibacterial and antiviral properties besides exhibiting cytotoxic effects. Juglone has gained interest by the researchers for its anticancer properties. This article elucidates the antiviral activity of the Juglone by the computational method.

Molecular modeling studies and comparative analysis on structurally similar HTLV and HIV protease using HIV-PR inhibitors.

Retroviruses are most perilous viral family, which cause much damage to the Homo sapiens. HTLV-1 mechanism found to more similar with HIV-1 and both retroviruses are causative agents of severe and fatal diseases including adult T-cell leukemia (ATL) and the acquired immune deficiency syndrome (AIDS). Both viruses code for a protease (PR) that is essential for replication and therefore represents a key target for drugs interfering with viral infection. In this work, the comparative study of HIV-1 and HTLV-1 PR enzymes through sequence and structural analysis is reported along with approved drugs of HIV-PR. Conformation of each HIV PR drugs have been examined with different parameters of interactions and energy scorings parameters. MD simulations with respect to timescale event of 20 ns favors that, few HIV-PR inhibitors can be more active inside the HTLV-1 PR binding pocket. Overall results suggest that, some of HIV inhibitors like Tipranavir, Indinavir, Darunavir and Amprenavir are having good energy levels with HTLV-1. Due to absence of interactions with MET37, here we report that derivatives of these compounds can be much better inhibitors for targeting HTLV-1 proteolytic activity.

Atomistic simulations of the HIV-1 protease folding inhibition.

Biochemical experiments have recently revealed that the p-S8 peptide, with an amino-acid sequence identical to the conserved fragment 83-93 (S8) of the HIV-1 protease, can inhibit catalytic activity of the enzyme by interfering with protease folding and dimerization. In this study, we introduce a hierarchical modeling approach for understanding the molecular basis of the HIV-1 protease folding inhibition. Coarse-grained molecular docking simulations of the flexible p-S8 peptide with the ensembles of HIV-1 protease monomers have revealed structurally different complexes of the p-S8 peptide, which can be formed by targeting the conserved segment 24-34 (S2) of the folding nucleus (folding inhibition) and by interacting with the antiparallel termini beta-sheet region (dimerization inhibition). All-atom molecular dynamics simulations of the inhibitor complexes with the HIV-1 PR monomer have been independently carried out for the predicted folding and dimerization binding modes of the p-S8 peptide, confirming the thermodynamic stability of these complexes. Binding free-energy calculations of the p-S8 peptide and its active analogs are then performed using molecular dynamics trajectories of the peptide complexes with the HIV-1 PR monomers. The results of this study have provided a plausible molecular model for the inhibitor intervention with the HIV-1 PR folding and dimerization and have accurately reproduced the experimental inhibition profiles of the active folding inhibitors.

Insight into the folding inhibition of the HIV-1 protease by a small peptide.

It has recently been shown that the highly protected segments 24-34 (S2) and 83-93 (S8) of each of the two 99-mers of human immunodeficiency virus type 1 protease play an essential role in the folding of the monomers, giving rise to the so-called (postcritical) folding nucleus ((FN) minimum condensation unit ensuring folding) when they dock. This scenario received further support from model calculations that demonstrated that the peptide p-S8, displaying an amino acid sequence identical to the corresponding (83-93) segment of the monomer, can be used to interfere with the formation of the FN and eventually to inhibit folding by docking the fragment 24-34. Experiments in vitro and in cells infected with ex vivo wild-type and multiresistant HIV isolates confirm that the inhibition power of p-S8 is robust. On the other hand, there is no direct evidence demonstrating the validity of the proposed mechanism of inhibition associated with p-S8. To shed light on this question and to provide the basis for the design of a molecule mimetic to p-S8, to be used as lead of an eventual drug against AIDS, we study, in this paper, with the help of all-atom simulations in explicit solvent and the novel method of metadynamics combined with parallel tempering: a), the free energy and the equilibrium structure of each of the peptides p-S2 and p-S8; b), the details of the docking mechanism of the two peptides and the free energy associated with this process. Whereas p-S8 is found to be well structured, p-S2 is rather flexible, wrapping itself around p-S8 to give rise to the FN, which is stabilized by three particular hydrogen bonds.

HIV-1 protease substrate binding and product release pathways explored with coarse-grained molecular dynamics.

We analyze the encounter of a peptide substrate with the native HIV-1 protease, the mechanism of substrate incorporation in the binding cleft, and the dissociation of products after substrate hydrolysis. To account for the substrate, we extend a coarse-grained model force field, which we previously developed to study the flap opening dynamics of HIV-1 protease on a microsecond timescale. Molecular and Langevin dynamics simulations show that the flaps need to open for the peptide to bind and that the protease interaction with the substrate influences the flap opening frequency and interval. On the other hand, release of the products does not require flap opening because they can slide out from the binding cleft to the sides of the enzyme. Our data show that in the protease-substrate complex the highest fluctuations correspond to the 17- and 39-turns and the substrate motion is anticorrelated with the 39-turn. Moreover, the active site residues and the flap tips move in phase with the peptide. We suggest some mechanistic principles for how the flexibility of the protein may be involved in ligand binding and release.

Insights into saquinavir resistance in the G48V HIV-1 protease: quantum calculations and molecular dynamic simulations.

The spread of acquired immune deficiency syndrome has increasingly become a great concern owing largely to the failure of chemotherapies. The G48V is considered the key signature residue mutation of HIV-1 protease developing with saquinavir therapy. Molecular dynamics simulations of the wild-type and the G48V HIV-1 protease complexed with saquinavir were carried out to explore structure and interactions of the drug resistance. The molecular dynamics results combined with the quantum-based and molecular mechanics Poisson-Boltzmann surface area calculations indicated a monoprotonation took place on D25, one of the triad active site residues. The inhibitor binding of the triad residues and its interaction energy in the mutant were similar to those in the wild-type. The overall structure of both complexes is almost identical. However, the steric conflict of the substituted valine results in the conformational change of the P2 subsite and the disruption of hydrogen bonding between the -NH of the P2 subsite and the backbone -CO of the mutated residue. The magnitude of interaction energy changes was comparable to the experimental K(i) data. The designing for a new drug should consider a reduction of steric repulsion on P2 to enhance the activity toward this mutant strain.

Solvation studies of DMP323 and A76928 bound to HIV protease: analysis of water sites using grand canonical Monte Carlo simulations.

We examine the water solvation of the complex of the inhibitors DMP323 and A76928 bound to HIV-1 protease through grand canonical Monte Carlo simulations, and demonstrate the ability of this method to reproduce crystal waters and effectively predict water positions not seen in the DMP323 or A76928 structures. The simulation method is useful for identifying structurally important waters that may not be resolved in the crystal structures. It can also be used to identify water positions around a putative drug candidate docked into a binding pocket. Knowledge of these water positions may be useful in designing drugs to utilize them as bridging groups or displace them in the binding pocket. In addition, the method should be useful in finding water sites in homology models of enzymes for which crystal structures are unavailable.

Comparison of protein surfaces using a genetic algorithm.

A genetic algorithm (GA) is described which is used to compare the solvent-accessible surfaces of two proteins or fragments of proteins, represented by a dot surface calculated using the Connolly algorithm. The GA is used to move one surface relative to the other to locate the most similar surface region between the two. The matching process is enhanced by the use of the surface normals and shape terms provided by the Connolly program and also by a simple hydrogen-bonding descriptor and an additional shape descriptor. The algorithm has been tested in applications ranging from the comparison of small surface patches to the comparison of whole protein surfaces, and it has performed correctly in all cases. Examples of the matches are given and a quantitative analysis of the quality of the matches is performed. A number of possible future enhancements to the program are described which would allow the GA to be used for more complex surface comparisons.

Effect of point mutations on the kinetics and the inhibition of human immunodeficiency virus type 1 protease: relationship to drug resistance.

Mutations of human immunodeficiency virus type 1 (HIV-1) protease at four positions, Val82, Asp30, Gly48, and Lys45 were analyzed for the resulting effects on kinetics and inhibition. In these mutants, Val82 was substituted separately by Asn, Glu, Ala, Ser, Asp, and Gln; Asp30 was individually substituted by Phe or Trp; Gly48 by His, Asp, and Tyr, respectively; and Lys45 by Glu. By examination of the inhibition of a single inhibitor, the differences in Ki values between the native and mutant enzymes can range from very large to insignificant even for the mutants with substitutions at the same position. By examination of a single mutant enzyme, the same broad range of Ki changes was observed for a group of inhibitors: Thus, how much the inhibition changes from the wild-type enzyme to a mutant is dependent on both the mutation and the inhibitor. The examination of Ki changes of inhibitors with closely related structures binding to Val82 mutants also reveals that the change of inhibition involves subsites in which Val82 is not in direct contact, indicating a considerable flexibility of the conformation of HIV protease. For the catalytic activities of the mutants, the kcat and Km values of many Val82 mutants and a Lys45 mutant are comparable to the native enzyme. Surprisingly, Gly48 mutations produce enzymes with catalytic efficiency superior to that of the wild-type enzyme by as much as 10-fold. Modeling of the structure of the mutants suggests that the high catalytic efficiency of some substrates is related to an increase of rigidity of the flap region of the mutants. The examination of the relative changes of inhibition and catalysis of mutants suggests that some of the Val82 and Gly48 mutants are potential resistance mutants. However, the resistance is specific with respect to individual inhibitors.

A priori prediction of activity for HIV-1 protease inhibitors employing energy minimization in the active site.

We have observed a high correlation between the intermolecular interaction energy (Einter) calculated for HIV-1 protease inhibitor complexes and the observed in vitro enzyme inhibition. A training set of 33 inhibitors containing modifications in the P1' and P2' positions was used to develop a regression equation which relates Einter and pIC50. This correlation was subsequently employed to successfully predict the activity of proposed HIV-1 protease inhibitors in advance of synthesis in a structure-based design program. This included a precursor, 47, to the current phase II clinical candidate, L-735,524 (51). The development of the correlation, its applications, and its limitations are discussed, and the force field (MM2X) and host molecular mechanics program (OPTIMOL) used in this work are described.

Reactivity of cysteine residues in the protease from human immunodeficiency virus: identification of a surface-exposed region which affects enzyme function.

The protease encoded by the human immunodeficiency virus (HIV) is essential for the processing of viral polyproteins encoded by the gag and pol genes into mature viral proteins. The 99-residue protease from HIV-1 contains two cysteine residues (Cys-67 and Cys-95), both of which are usually conserved in viruses isolated from patients. Despite this conservation, neither residue is required for enzymatic activity. Certain site-specific cysteine mutants of HIV-1 protease are catalytically active, and the protease from HIV-2 lacks both cysteines. Copper is a potent inhibitor of HIV-1 protease, but not of mutants lacking cysteine (A. R. Karlström and R. L. Levine, 1991, Proc. Natl. Acad. Sci. USA 88, 5552-5556). The addition of copper to the protease at pH 5.5 induced aggregation of the protein, providing a possible basis for the inhibitory action of copper. However, addition of both copper and dithiothreitol still led to inhibition of activity but did not cause aggregation. These findings led to a study of the reactivity of the cysteine residues to 5,5'-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent), a sulfhydryl compound which reacts with the ionized form of cysteine residues. At pH 6.2 in 6 M guanidine, no derivatization of cysteine residues occurred, consistent with the typical pKa of cysteine expected for the denatured protein. However, in the same buffer without guanidine, the native protease reacted rapidly with concomitant loss of proteolytic activity. Peptic mapping demonstrated that both Cys-67 and Cys-95 were derivatized. A catalytically active fusion protein of protease with protein A domains was then studied with the expectation that access to Cys-95 would be hindered. This was confirmed, with only Cys-67 reacting rapidly with Ellman's reagent. Enzymatic activity was again lost, indicating that derivatization of the surface-accessible Cys-67 was sufficient to inactivate the enzyme. The reactivity and accessibility of these residues suggest an interesting approach for the development of protease inhibitors which are not directed to the substrate-binding site.

Entrapment and inhibition of human immunodeficiency virus proteinase by alpha 2-macroglobulin and structural changes in the inhibitor.

The inhibitory effect of alpha 2-macroglobulin (alpha 2M), a major plasma proteinase inhibitor, on human immunodeficiency virus (HIV) proteinase was investigated. The activity of HIV proteinase toward the Moloney murine sarcoma virus-derived gag protein (a high-molecular-mass substrate) was found to be inhibited by alpha 2M at pH 5.5-7.4. On the other hand, the activity toward the B chain of oxidized insulin (a low-molecular-mass substrate) was scarcely inhibited. The complex of alpha 2M and HIV proteinase was isolated by gel filtration and the enzyme was shown to be significantly protected by the complex formation from autoinactivation under nonreducing conditions. The stoichiometry of the complex formation was found to be 2:1 (enzyme: alpha 2M, mol/mol). These results demonstrate the entrapment and concomitant inhibition of HIV proteinase by alpha 2M.

Maturation of human immunodeficiency virus particles assembled from the gag precursor protein requires in situ processing by gag-pol protease.

The vaccinia virus expression system was used to determine the role of human immunodeficiency virus type 1 (HIV-1) protease in viral morphogenesis and maturation. The unprocessed p55 gag precursor polyprotein alone was assembled to form HIV-1 particles which budded from cells. The particles were spherical and immature, containing an electron-dense shell in the particle submembrane; there was no evidence of core formation. Expression of both gag and pol proteins from a recombinant containing the complete gag-pol coding sequences resulted in intracellular processing of gag-pol proteins and the production of mature particles with electron-dense cores characteristic of wild-type HIV virions. To ascertain the role of protein processing in particle maturation, the pol ORF in the gag-pol recombinant was truncated to limit expression of the pol gene to the protease domain. With this recombinant expressing p55 gag and protease, intracellular processing was observed. Some of the resultant particles were partially mature and contained processed gag protein subunits. In contrast, particle maturation was not observed when the HIV-1 protease and p55 gag were coexpressed from separate recombinants, despite evidence of intracellular gag processing. These findings suggest that HIV-1 protease must be an integral component of the full-length gag-pol precursor for optimal processing and virion maturation.

Human immunodeficiency virus proteinase dimer as component of the viral polyprotein prevents particle assembly and viral infectivity.

Enzymatically active retroviral proteinases are dimers of identical polypeptide chains with a fold similar to that of other aspartic proteinases. Each polypeptide chain, encoded on one of the viral polyproteins, is less than half the size of cellular aspartic proteinases and contains only one of the two active-site aspartate residues. A plasmid was constructed to generate a genetically linked dimer of the proteinase (PR) of human immunodeficiency virus (HIV) type 1, composed of two copies of the PR sequence linked by a structurally flexible hinge region. The expression product was stable and active against HIV polyprotein substrates. Mutational analysis revealed that the linked dimer, and not multimers thereof, contained the proteolytic activity. Expression of the linked dimer as a component of a HIV polyprotein by in vitro translation gave rapid autocatalytic processing, whereas the wild-type polyprotein was stable on prolonged incubation. Transfection of HIV subviral or proviral constructs, containing the linked dimer of HIV PR, gave premature processing of the viral polyproteins, thus preventing particle formation and infectivity. Premature processing also led to increased cell toxicity.

Domain communication in the dynamical structure of human immunodeficiency virus 1 protease.

A dynamical model for the structure of the human immunodeficiency virus 1 (HIV-1) protease dimer in aqueous solution has been developed on the basis of molecular dynamics simulation. The model provides an accurate account of the crystal geometry and also a prediction of the structural reorganization expected to occur in the protein in aqueous solution compared to the crystalline environment. Analysis of the results by means of dynamical cross-correlation coefficients for atomic displacements indicates that domain-domain communication is present in the protein in the form of a molecular "cantilever" and is likely to be involved in enzyme function at the molecular level. The dynamical structure also suggests information that may ultimately be useful in understanding and further development of specific inhibitors of HIV-1 protease.