Linking pathway gene expressions to the growth inhibition response from the National Cancer Institute's anticancer screen and drug mechanism of action.

Novel strategies are proposed to quantitatively analyze and relate biological pathways to drug responses using gene expression and small-molecule growth inhibition data (GI(50)) derived from the National Cancer Institute's 60 cancer cells (NCI(60)). We have annotated groups of drug GI(50) responses with pathways defined by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioCarta, and functional categories defined by Gene Ontology (GO), through correlations between pathway gene expression patterns and drug GI(50) profiles. Drug-gene-pathway relationships may then be utilized to find drug targets or target-specific drugs. Significantly correlated pathways and the gene products involved represent interesting targets for further exploration, whereas drugs that are significantly correlated with only certain pathways are more likely to be target specific. Separate pathway clustering finds that pathways engaged in the same biological process tend to have similar drug correlation patterns. The biological and statistical significances of our method are established by comparison to known small-molecule inhibitor-gene target relationships reported in the literature and by standard randomization procedures. The results of our pathway, gene expression and drug-induced growth inhibition associations, can serve as a basis for proposing testable hypotheses about potential anticancer drugs, their targets, and mechanisms of action.

Molecular motions and conformational changes of HPPK.

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) belongs to a class of catalytic enzymes involved in phosphoryl transfer and is a new target for the development of novel antimicrobial agents. In the present study, the fundamental consideration is to view the overall structure of HPPK as a network of interacting residues and to extract the most cooperative collective motions that define its global dynamics. A coarse-grained model, harmonically constrained according to HPPK's crystal structure is used. Four crystal structures of HPPK (one apo and three holo forms with different nucleotide and pterin analogs) are studied with the goal of providing insights about the function-dynamic correlation and ligand induced conformational changes. The dynamic differences are examined between HPPK's apo- and holo-forms, because they are involved in the catalytic reaction steps. Our results indicate that the palm-like structure of HPPK is nearly rigid, whereas the two flexible loops: L2 (residues 43-53) and L3 (residues 82-92) exhibit the most concerted motions for ligand recognition and presumably, catalysis. These two flexible loops are involved in the recognition of HPPKs nucleotide and pterin ligands, whereas the rigid palm region is associated with binding of these cognate ligands. Six domains of collective motions are identified, comprised of structurally close but not necessarily sequential residues. Two of these domains correspond to the flexible loops (L2 and L3), whereas the remaining domains correspond to the rigid part of the molecule.

Molecular mechanisms of chaperonin GroEL-GroES function.

The dynamics of the GroEL-GroES complex is investigated with a coarse-grained model. This model is one in which single-residue points are connected to other such points, which are nearby, by identical springs, forming a network of interactions. The nature of the most important (slowest) normal modes reveals a wide variety of motions uniquely dependent upon the central cavity of the structure, including opposed torsional rotation of the two GroEL rings accompanied by the alternating compression and expansion of the GroES cap binding region, bending, shear, opposed radial breathing of the cis and trans rings, and stretching and contraction along the protein assembly's long axis. The intermediate domains of the subunits are bifunctional due to the presence of two hinges, which are alternatively activated or frozen by an ATP-dependent mechanism. ATP binding stabilizes a relatively open conformation (with respect to the central cavity) and hinders the motion of the hinge site connecting the intermediate and equatorial domains, while enhancing the flexibility of the second hinge that sets in motion the apical domains. The relative flexibilities of the hinges are reversed in the nucleotide-free form. Cooperative cross-correlations between subunits provide information about the mechanism of action of the protein. The mechanical motions driven by the different modes provide variable binding surfaces and variable sized cavities in the interior to enable accommodation of a broad range of protein substrates. These modes of motion could be used to manipulate the substrate's conformations.

Reactivity of zinc finger cores: analysis of protein packing and electrostatic screening.

The chemical stability of 207 zinc fingers, derived from 92 experimental protein structures, is evaluated according to the protein packing and electrostatic screening of their zinc cores. These properties are used as measures of the protein protection of zinc cores, to predictively rank relative zinc finger reactivities and assess differences in function. On average, there is a substantial and concomitant increase in the screening of increasingly anionic core motifs, suggesting zinc fingers have evolved in a manner that promotes shielding of their potentially reactive core thiolates. In contrast, enzymatic zinc cores are functionally differentiated by negative electrostatic screening. Zinc finger cores are predominantly screened by networks of backbone:core NH-S hydrogen bonds that electronically stabilize core thiolates and enhance backbone packing. Stabilizing protein:core interactions can be mapped to conserved residues, including [Arg,Lys]:core salt-bridges in some protein families. Labile zinc fingers are identified by poorly screened cores, possibly indicating redox or metallothionein (MT) regulated function. Consistent with experiment, the cores of the C-terminal finger of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein p7 (NCp7) and Escherichia coli Ada protein (Ada) "finger" are identified as reactive. The C-terminal zinc fingers of nuclear receptors are predicted to be the most labile in this study, particularly the human estrogen receptor (hER), which contains a triad of reactive thiolates. We propose that hER DNA binding is redox and MT regulated through the C-terminal finger and that weak electrophilic agents may inhibit hER-mediated transcription, implicated in breast cancer progression.

Screening the molecular surface of human anticoagulant protein C: a search for interaction sites.

Protein C (PC), a 62 kDa multi-modular zymogen, is activated to an anticoagulant serine protease (activated PC or APC) by thrombin bound to thrombomodulin on the surface of endothelial cells. PC/APC interacts with many proteins and the characterisation of these interactions is not trivial. However, molecular modelling methods help to study these complex biological processes and provide basis for rational experimental design and interpretation of the results. PC/APC consists of a Gla domain followed by two EGF modules and a serine protease domain. In this report, we present two structural models for full-length APC and two equivalent models for full-length PC, based on the X-ray structures of Gla-domainless APC and of known serine protease zymogens. The overall elongated shape of the models is further cross-validated using size exclusion chromatography which allows evaluation of the Stokes radius (rs for PC = 33.15 A; rs for APC = 34.19 A), frictional ratio and axial ratio. We then propose potential binding sites at the surface of PC/APC using surface hydrophobicity as a determinant of the preferred sites of intermolecular recognition. Most of the predicted binding sites are consistent with previously reported experimental data, while some clusters highlight new regions that should be involved in protein-protein interactions.

Mapping the binding site of colchicinoids on beta -tubulin. 2-Chloroacetyl-2-demethylthiocolchicine covalently reacts predominantly with cysteine 239 and secondarily with cysteine 354.

2-Chloroacetyl-2-demethylthiocolchicine (2CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3CTC) resemble colchicine in binding to tubulin and react covalently with beta-tubulin, forming adducts with cysteine residues 239 and 354. The adducts at Cys-239 are less stable than those at Cys-354 during formic acid digestion. Extrapolating to zero time, the Cys-239 to Cys-354 adduct ratio is 77:23 for 2CTC and 27:73 for 3CTC. Using energy minimization modeling to dock colchicinoids into the electron crystallographic model of beta-tubulin in protofilaments (Nogales, E. , Wolf, S. G., and Downing, K. H. (1998) Nature 391, 199-203), we found two potential binding sites. At one, entirely encompassed within beta-tubulin, the C2- and C3-oxygen atoms of 2CTC and 3CTC overlapped poorly with those of colchicine and thiocolchicine, but distances from the reactive carbon atoms of the analogs to the sulfur atoms of the cysteine residues were qualitatively consistent with reactivity. The other potential binding site was located at the alpha/beta interface. Here, the oxygen atoms of the analogs overlapped well with those of colchicine, but relative distances of the reactive carbons to the cysteine sulfur atoms did not correlate with the observed reactivity. A significant conformational change must occur in the colchicine binding site of tubulin in the transition from the unpolymerized to the polymerized state.

Characterization of anticancer agents by their growth inhibitory activity and relationships to mechanism of action and structure.

An analysis of the growth inhibitory potency of 122 anticancer agents available from the National Cancer Institute anticancer drug screen is presented. Methods of singular value decomposition (SVD) were applied to determine the matrix of distances between all compounds. These SVD-derived dissimilarity distances were used to cluster compounds that exhibit similar tumor growth inhibitory activity patterns against 60 human cancer cell lines. Cluster analysis divides the 122 standard agents into 25 statistically distinct groups. The first eight groups include structurally diverse compounds with reactive functionalities that act as DNA-damaging agents while the remaining 17 groups include compounds that inhibit nucleic acid biosynthesis and mitosis. Examination of the average activity patterns across the 60 tumor cell lines reveals unique 'fingerprints' associated with each group. A diverse set of structural features are observed for compounds within these groups, with frequent occurrences of strong within-group structural similarities. Clustering of cell types by their response to the 122 anticancer agents divides the 60 cell types into 21 groups. The strongest within-panel groupings were found for the renal, leukemia and ovarian cell panels. These results contribute to the basis for comparisons between log(GI(50)) screening patterns of the 122 anticancer agents and additional tested compounds.

Relating the Structure of HIV-1 Reverse Transcriptase to Its Processing Step.

Abstract By treating an enzyme as a coarse-grained uniform block of material, utilizing only the α-Carbon positions, the normal modes of motion can be obtained. For reverse transcriptase the slower of these motions are suggestive of being involved in the processing step, where the RNA or DNA strand is copied onto a new DNA strand at a polymerase site, and the RNA strand is subsequently cut up at the distant Ribonuclease H site. The slowest mode of motion involves hinge bending about a site midway between the polymerase and Ribonuclease H sites, suggesting that it can push or pull the RNA strand between these two sites. Pulling the nucleic acid strand would require tight binding to the RNase H site. The next slowest mode involves a hinge that opens and closes the protein like a clamp, which could facilitate the release of the nucleic acids for their step-wise progression. The third mode could rotate the substrate. An overall description of the step-wise processing step would involve close coordination among these steps. Results suggest that the smaller p51 subunit serves only as ballast to support the various modes of motion involving the different parts of the p66 subunit.

Cooperative folding units of escherichia coli tryptophan repressor.

A previously published computational procedure was used to identify cooperative folding units within tryptophan repressor. The theoretical results predict the existence of distinct stable substructures in the protein chain for the monomer and the dimer. The predictions were compared with experimental data on structure and folding of the repressor and its proteolytic fragments and show excellent agreement for the dimeric form of the protein. The results suggest that the monomer, the structure of which is currently unknown, is likely to have a structure different from the one it has within the context of the highly intertwined dimer. Application of this method to the repressor monomer represents an extension of the computations into the realm of evaluating hypothetical structures such as those produced by threading.

Collective motions in HIV-1 reverse transcriptase: examination of flexibility and enzyme function.

In order to study the inferences of structure for mechanism, the collective motions of the retroviral reverse transcriptase HIV-1 RT (RT) are examined using the Gaussian network model (GNM) of proteins. This model is particularly suitable for elucidating the global dynamic characteristics of large proteins such as the presently investigated heterodimeric RT comprising a total of 982 residues. Local packing density and coordination order of amino acid residues is inspected by the GNM to determine the type and range of motions, both at the residue level and on a global scale, such as the correlated movements of entire subdomains. Of the two subunits, p66 and p51, forming the RT, only p66 has a DNA-binding cleft and a functional polymerase active site. This difference in the structure of the two subunits is shown here to be reflected in their dynamic characteristics: only p66 has the potential to undergo large-scale cooperative motions in the heterodimer, while p51 is essentially rigid. Taken together, the global motion of the RT heterodimer is comprised of movements of the p66 thumb subdomain perpendicular to those of the p66 fingers, accompanied by anticorrelated fluctuations of the RNase H domain and p51 thumb, thus providing information about the details of one processivity mechanism. A few clusters of residues, generally distant in sequence but close in space, are identified in the p66 palm and connection subdomains, which form the hinge-bending regions that control the highly concerted motion of the subdomains. These regions include the catalytically active site and the non-nucleoside inhibitor binding pocket of p66 polymerase, as well as sites whose mutations have been shown to impair enzyme activity. It is easily conceivable that this hinge region, indicated by GNM analysis to play a critical role in modulating the global motion, is locked into an inactive conformation upon binding of an inhibitor. Comparative analysis of the dynamic characteristics of the unliganded and liganded dimers indicates severe repression of the mobility of the p66 thumb in RT's global mode, upon binding of non-nucleoside inhibitors.

Synthesis and biological properties of novel pyridinioalkanoyl thiolesters (PATE) as anti-HIV-1 agents that target the viral nucleocapsid protein zinc fingers.

Nucleocapsid p7 protein (NCp7) zinc finger domains of the human immunodeficiency virus type 1 (HIV-1) are being developed as antiviral targets due to their key roles in viral replication and their mutationally nonpermissive nature. On the basis of our experience with symmetrical disulfide benzamides (DIBAs; Rice et al. Science 1995, 270, 1194-1197), we synthesized and evaluated variants of these dimers, including sets of 4,4'- and 3,3'-disubstituted diphenyl sulfones and their monomeric benzisothiazolone derivatives (BITA). BITAs generally exhibited diminished antiviral potency when compared to their disulfide precursors. Novel, monomeric structures were created by linking haloalkanoyl groups to the benzamide ring through -NH-C(=O)- (amide) or -S-C(=O)- (thiolester) bridges. Amide-linked compounds generally lacked antiviral activity, while haloalkanoyl thiolesters and non-halogen-bearing analogues frequently exhibited acceptable antiviral potency, thus establishing thiolester benzamides per se as a new anti-HIV chemotype. Pyridinioalkanoyl thiolesters (PATEs) exhibited superior anti-HIV-1 activity with minimal cellular toxicity and appreciable water solubility. PATEs were shown to preferentially target the NCp7 Zn finger when tested against other molecular targets, thus identifying thiolester benzamides, and PATEs in particular, as novel NCp7 Zn finger inhibitors for in vivo studies.

The C4b-binding protein-protein S interaction is hydrophobic in nature.

C4b-binding protein (C4BP) is a major regulatory molecule of the complement system. By forming a non covalent complex with the anticoagulant cofactor protein S (PS), it also plays an important role in blood coagulation. C4BP is composed of one beta-chain and seven alpha-chains that are essentially built from complement control protein (CCP)-modules. Our group has previously reported that the first (N-terminal) CCP module of the beta-chain (betaCCP1) contains the entire binding site for protein S. We now investigate further the binding of protein S to C4BP and show that the complex formation is essentially dependent on hydrophobic forces with minor contribution from electrostatic interactions. This result is in agreement with homology modeling experiments carried out in conjunction with inter-species sequence comparison and theoretical enumeration of potential binding sites. These methods pinpoint a solvent exposed hydrophobic cluster at the surface of the betaCCP1 module that is of crucial importance for the binding process.

Reactivity of the HIV-1 nucleocapsid protein p7 zinc finger domains from the perspective of density-functional theory.

The reaction of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein p7 (NCp7) with a variety of electrophilic agents was investigated by experimental measurements of Trp37 fluorescence decay and compared with theoretical measures of reactivity based on density-functional theory in the context of the hard and soft acids and bases principle. Statistically significant correlations were found between rates of reaction and the ability of these agents to function as soft electrophiles. Notably, the molecular property that correlated strongest was the ratio of electronegativity to hardness, chi2/eta, a quantity related to the capacity of an electrophile to promote a soft (covalent) reaction. Electronic and steric determinants of the reaction were also probed by Fukui function and frontier-orbital overlap analysis in combination with protein-ligand docking methods. This analysis identified selective ligand docking regions within the conserved zinc finger domains that promoted reaction. The Cys49 thiolate was found overall to be the NCp7 site most susceptible to electrophilic attack.

Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41.

The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e-gp41), consisting of residues 27-149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e-gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N-terminal (residues 30-80) and C-terminal (residues 107-147) helices connected by a 26 residue loop (residues 81-106). The N-terminal helices of each subunit form a parallel coiled-coil structure in the interior of the complex which is surrounded by the C-terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non-sequential intramolecular contacts. The helical core of SIV e-gp41 is similar to recent X-ray structures of truncated constructs of the helical core of HIV-1 e-gp41. The present structure establishes unambiguously the connectivity of the N- and C-terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e-gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e-gp41 represents one of the largest protein structures determined by NMR to date.

Solution structure of cyanovirin-N, a potent HIV-inactivating protein.

The solution structure of cyanovirin-N, a potent 11,000 Mr HIV-inactivating protein that binds with high affinity and specificity to the HIV surface envelope protein gp120, has been solved by nuclear magnetic resonance spectroscopy, including extensive use of dipolar couplings which provide a priori long range structural information. Cyanovirin-N is an elongated, largely beta-sheet protein that displays internal two-fold pseudosymmetry. The two sequence repeats (residues 1-50 and 51-101) share 32% sequence identity and superimpose with a backbone atomic root-mean-square difference of 1.3 A. The two repeats, however, do not form separate domains since the overall fold is dependent on numerous contacts between them. Rather, two symmetrically related domains are formed by strand exchange between the two repeats. Analysis of surface hydrophobic clusters suggests the location of potential binding sites for protein-protein interactions.

Structural investigation of C4b-binding protein by molecular modeling: localization of putative binding sites.

C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one beta-chain and seven alpha-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its alpha-chains and with protein S through its beta-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP alpha-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the alpha-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein-protein interactions.

Anti-HIV agents that selectively target retroviral nucleocapsid protein zinc fingers without affecting cellular zinc finger proteins.

Agents that target the two highly conserved Zn fingers of the human immunodeficiency virus (HIV) nucleocapsid p7 (NCp7) protein are under development as antivirals. These agents covalently modify Zn-coordinating cysteine thiolates of the fingers, causing Zn ejection, loss of native protein structure and nucleic acid binding capacity, and disruption of virus replication. Concentrations of three antiviral agents that promoted in vitro Zn ejection from NCp7 and inhibited HIV replication did not impact the functions of cellular Zn finger proteins, including poly(ADP-ribose) polymerase and the Sp1 and GATA-1 transcription factors, nor did the compounds inhibit HeLa nuclear extract mediated transcription. Selectivity of interactions of these agents with NCp7 was supported by molecular modeling analysis which (1) identified a common saddle-shaped nucleophilic region on the surfaces of both NCp7 Zn fingers, (2) indicated a strong correspondence between computationally docked positions for the agents tested and overlap of frontier orbitals within the nucleophilic loci of the NCp7 Zn fingers, and (3) revealed selective steric exclusion of the agents from the core of the GATA-1 Zn finger. Further modeling analysis suggests that the thiolate of Cys49 in the carboxy-terminal finger is the site most susceptible to electrophilic attack. These data provide the first experimental evidence and rationale for antiviral agents that selectively target retroviral nucleocapsid protein Zn fingers.

Correlation between native-state hydrogen exchange and cooperative residue fluctuations from a simple model.

Recently, we developed a simple analytical model based on local residue packing densities and the distribution of tertiary contacts for describing the conformational fluctuations of proteins in their folded state. This so-called Gaussian network model (GNM) is applied here to the interpretation of experimental hydrogen exchange (HX) behavior of proteins in their native state or under weakly denaturing conditions. Calculations are performed for five proteins: bovine pancreatic trypsin inhibitor, cytochrome c, plastocyanin, staphylococcal nuclease, and ribonuclease H. The results are significant in two respects. First, a good agreement is reached between calculated fluctuations and experimental measurements of HX despite the simplicity of the model and within computational times 2 or 3 orders of magnitude faster than earlier, more complex simulations. Second, the success of a theory, based on the coupled conformational fluctuations of residues near the native state, to satisfactorily describe the native-state HX behavior indicates the significant contribution of local, but cooperative, fluctuations to protein conformational dynamics. The correlation between the HX data and the unfolding kinetics of individual residues further suggests that local conformational susceptibilities as revealed by the GNM approach may have implications relevant to the global dynamics of proteins.

A cooperative folding unit in HIV-1 protease. Implications for protein stability and occurrence of drug-induced mutations.

We investigated the HIV-1 protease molecule for the occurrence of cooperative folding units, i.e. structural units that exhibit a relatively stronger protection against unfolding than do other parts of the molecule. Calculated unfolding penalties are used to delineate folding units. This procedure identifies a folding core in HIV-1 protease, based on an ensemble of denatured states derived from native structures, comprising a spatially close unit of residues 84-91, 74-78 and 22-32, the last of which contains the active site residues D25, T26 and G27. Observed enzyme mutations of HIV-1 protease, either naturally occurring or induced by drug therapy, are found in regions that are not structurally designed to withstand unfolding. These mutations are especially likely to occur in the flap region, a part of the protein which is not essential for the stability of the protein, but does contribute significantly to the stability of protease-drug complexes. A similar avoidance of structurally protected regions in the reverse transcriptase enzyme is also observed.

Identification of cooperative folding units in a set of native proteins.

Cooperative unfolding penalties are calculated by statistically evaluating an ensemble of denatured states derived from native structures. The ensemble of denatured states is determined by dividing the native protein into short contiguous segments and defining all possible combinations of native, i.e., interacting, and non-native, i.e., non-interacting, segments. We use a novel knowledge-based scoring function, derived from a set of non-homologous proteins in the Protein Data Bank, to describe the interactions among residues. This procedure is used for the structural identification of cooperative folding cores for four globular proteins: bovine pancreatic trypsin inhibitor, horse heart cytochrome c, French bean plastocyanin, and staphylococcal nuclease. The theoretical folding units are shown to correspond to regions that exhibit enhanced stability against denaturation as determined from experimental hydrogen exchange protection factors. Using a sequence similarity score for related sequences, we show that, in addition to residues necessary for enzymatic function, those amino acids comprising structurally important folding cores are also preferentially conserved during evolution. This implies that the identified folding cores may be part of an array of fundamental structural folding units.