Structural basis of binding of high-affinity ligands to protein kinase C: prediction of the binding modes through a new molecular dynamics method and evaluation by site-directed mutagenesis

The structural basis of protein kinase C (PKC) binding to several classes of high-affinity ligands has been investigated through complementary computational and experimental methods. Employing a recently developed q-jumping molecular dynamics (MD) simulation method, which allows us to consider the flexibility of both the ligands and the receptor in docking studies, we predicted the binding models of phorbol-13-acetate, phorbol-12,13-dibutyrate (PDBu), indolactam V (ILV), ingenol-3-benzoate, and thymeleatoxin to PKC. The "predicted" binding model for phorbol-13-acetate is virtually identical to the experimentally determined binding model for this ligand. The predicted binding model for PDBU is the same as that for phorbol-13-acetate in terms of the hydrogen-bonding network and hydrophobic contacts. The predicted binding model for ILV is the same as that obtained in a previous docking study using a Monte Carlo method and is consistent with the structure-activity relationships for this class of ligands. Together with the X-ray structure of phorbol-13-acetate in complex with PKCdelta C1b, the predicted binding models of PDBu, ILV, ingenol-3-benzoate, and thymeleatoxin in complex with PKC showed that the binding of these ligands to PKC is governed by a combination of several highly specific and optimal hydrogen bonds and hydrophobic contacts. However, the hydrogen-bonding network for each class of ligand is somewhat different and the number of hydrogen bonds formed between PKC and these ligands has no correlation with their binding affinities. To provide a direct and quantitative assessment of the contributions of several conserved residues around the binding site to PKC-ligand binding, we have made 11 mutations and measured the binding affinities of the high-affinity PKC ligands to these mutants. The results obtained through site-directed mutagenic analysis support our predicted binding models for these ligands and provide new insights into PKC-ligand binding. Although all the ligands have high affinity for the wild-type PKCdelta C1b, our site-directed mutagenic results showed that ILV is the ligand most sensitive to structural perturbations of the binding site while ingenol-3-benzoate is the least sensitive among the four classes of ligands examined here. Finally, we have employed conventional MD simulations to investigate the structural perturbations caused by each mutation to further examine the role played by each individual residue in PKC-ligand binding. MD simulations revealed that several mutations, including Pro11 --> Gly, Leu21 --> Gly, Leu24 --> Gly, and Gln27 --> Gly, cause a rather large conformational alteration to the PKC binding site and, in some cases, to the overall structure of the protein. The complete abolishment or the significant reduction in PKC-ligand binding observed for these mutants thus reflects the loss of certain direct contacts between the side chain of the mutated residue in PKC and ligands as well as the large conformational alteration to the binding site caused by the mutation.     

Structural determinants for high-affinity zolpidem binding to GABA-A receptors

The imidazopyridine zolpidem (Ambien) is one of the most commonly prescribed sleep aids in the United States (Rush, 1998). Similar to classic benzodiazepines (BZDs), zolpidem binds at the extracellular N-terminal alpha/gamma subunit interface of the GABA-A receptor (GABAR). However, zolpidem differs significantly from classic BZDs in chemical structure and neuropharmacological properties. Thus, classic BZDs and zolpidem are likely to have different requirements for high-affinity binding to GABARs. To date, three residues--gamma2Met57, gamma2Phe77, and gamma2Met130--have been identified as necessary for high-affinity zolpidem binding (Proc Natl Acad Sci USA 94:8824-8829, 1997; Mol Pharmacol 52:874-881, 1997). In this study, we used radioligand binding techniques, gamma2/alpha1 chimeric subunits (chi), site-directed mutagenesis, and molecular modeling to identify additional gamma2 subunit residues important for high-affinity zolpidem binding. Whereas alpha1beta2chi receptors containing only the first 161 amino-terminal residues of the gamma2 subunit bind the classic BZD flunitrazepam with wild-type affinity, zolpidem affinity is decreased approximately 8-fold. By incrementally restoring gamma2 subunit sequence, we identified a seven-amino acid stretch in the gamma2 subunit loop F region (amino acids 186-192) that is required to confer high-affinity zolpidem binding to GABARs. When mapped to a homology model, these seven amino acids make up part of loop F located at the alpha/gamma interface. Based on in silico zolpidem docking, three residues within loop F, gamma2Glu189, gamma2Thr193, and gamma2Arg194, emerge as being important for stabilizing zolpidem in the BZD binding pocket and probably interact with other loop F residues to maintain the structural integrity of the BZD binding site.     

Vancomycin binding to low-affinity ligands: delineating a minimum set of interactions necessary for high-affinity binding

Bacterial resistance to vancomycin has been attributed to the loss of an intermolecular hydrogen bond between vancomycin and its peptidoglycan target when cell wall biosynthesis proceeds via depsipeptide intermediates rather than the usual polypeptide intermediates. To investigate the relative importance of this hydrogen bond to vancomycin binding, we have determined crystal structures at 1.0 A resolution for the vancomycin complexes with three ligands that mimic peptides and depsipeptides found in vancomycin-sensitive and vancomycin-resistant bacteria: N-acetylglycine, D-lactic acid, and 2-acetoxy-D-propanoic acid. These, in conjunction with structures that have been reported previously, indicate higher-affinity ligands elicit a structural change in the drug not seen with these low-affinity ligands. They also enable us to define a minimal set of drug-ligand interactions necessary to confer higher-affinity binding on a ligand. Most importantly, these structures point to factors in addition to the loss of an intermolecular hydrogen bond that must be invoked to explain the weaker affinity of vancomycin for depsipeptide ligands. These factors are important considerations for the design of vancomycin analogues to overcome vancomycin resistance.     

Structural basis for computational screening of non-steroidal androgen receptor ligands

Importance of the field: Deep structural and chemical understanding of the protein target and computational methods for detection of receptor-selective ligands are important for the early drug discovery in the steroid receptor field. Areas covered in this review: This review focuses on the use of currently available structural information of the androgen receptor (AR) and known AR ligands to make computational strategies for the discovery of AR ligands in order to offer new chemical platforms for drug development. What the reader will gain: AR is a challenging target for drug discovery and modeling even if there is a wealth of experimental data available. First, only the active structure of AR is currently known, which hampers the design of AR antagonists. Second, the structural similarity between the ligand-binding sites of AR and its mutated forms and closely related steroid receptors (SRs) such as progesterone receptors presents challenges for the development of drugs with receptor-selective function. Take home message: Research indicates that a very small chemical change in the structure of a non-steroidal ligand can cause a complete change in its activity. One source of this effect arises from binding to similar binding sites in related SRs and other proteins in the signaling pathway. Currently, computational methods are not able to predict the subtle differences between AR ligand activities but modeling does offer the possibility of generating new lead structures that might have the desired properties.     

New bivalent PKC ligands linked by a carbon spacer: enhancement in binding affinity

Protein kinase C (PKC) is known to play an important role in many signal transduction pathways involved in hormone release, mitogenesis, and tumor promotion. In continuation of our efforts to find highly potent activators of PKC for possible use as Alzheimer's disease therapeutics, we designed and synthesized molecules containing two binding moieties (amides of benzolactams or esters of naphthylpyrrolidones) connected by a flexible spacer chain, which could theoretically bind to both the C1a and C1b activator binding domains of the catalytic region or to the C1 domains of two adjacent PKC molecules. The dimers 2a-g of benzolactam showed a 200-fold increase in affinity to PKCalpha and -delta as the spacer length increased from 4 to 20 carbon atoms. Replacement of the oligomethylene chain with an oligoethylene glycol unit (compounds 2h, 2i) showed a 4000- to 7000-fold decrease in affinity to PKCalpha. The dimers of naphthylpyrrolidones 4a-g did not show any marked improvement in binding affinities to PKC in comparison to the monomers synthesized earlier. The dimer of benzolactam 2e did not show much selectivity for PKCalpha, -betaIota, -delta, -epsilon, and -gamma. The high binding affinity of compounds 2d-g to PKCs gives us the impetus to design additional molecules that would retain this enhanced activity and would also show selectivity for the PKC isoforms.     

The molecular basis of high-affinity binding of the antiarrhythmic compound vernakalant (RSD1235) to Kv1.5 channels

Vernakalant (RSD1235) is an investigational drug recently shown to convert atrial fibrillation rapidly and safely in patients (J Am Coll Cardiol 44:2355-2361, 2004). Here, the molecular mechanisms of interaction of vernakalant with the inner pore of the Kv1.5 channel are compared with those of the class IC agent flecainide. Initial experiments showed that vernakalant blocks activated channels and vacates the inner vestibule as the channel closes, and thus mutations were made, targeting residues at the base of the selectivity filter and in S6, by drawing on studies of other Kv1.5-selective blocking agents. Block by vernakalant or flecainide of Kv1.5 wild type and mutants was assessed by whole-cell patch-clamp experiments in transiently transfected human embryonic kidney 293 cells. The mutational scan identified several highly conserved amino acids, Thr479, Thr480, Ile502, Val505, and Val508, as important residues for affecting block by both compounds. In general, mutations in S6 increased the IC50 for block by vernakalant; I502A caused an extremely local 25-fold decrease in potency. Specific changes in the voltage-dependence of block with I502A supported the crucial role of this position. A homology model of the pore region of Kv1.5 predicted that, of these residues, only Thr479, Thr480, Val505, and Val508 are potentially accessible for direct interaction, and that mutation at additional sites studied may therefore affect block through allosteric mechanisms. For some of the mutations, the direction of changes in IC50 were opposite for vernakalant and flecainide, highlighting differences in the forces that drive drug-channel interactions.     

Molecular determinants of high-affinity drug binding to HERG channels

Human ether-a-go-go-related gene (HERG) subunits mediate a K+ current that is required for normal repolarization of the cardiac action potential. The unintentional inhibition of HERG currents by numerous medications results in prolongation of the QT interval, as measured on the electrocardiogram, and is associated with increased risk of patients suffering from life-threatening cardiac arrhythmias. QT interval prolongation is considered a major safety concern by worldwide drug regulatory bodies, and early detection of new compounds with this undesirable side effect has become an important objective for pharmaceutical companies. New studies are shedding light on the structural basis of drug binding and the gating-dependent repositioning of key residues in the inner cavity of HERG, which are responsible for the unusual sensitivity of HERG to pharmacological agents. Insights from these studies may help develop novel strategies to reduce the proarrhythmic potential of the next generation of drugs.     

Structural and conformational requirements for high-affinity binding to the SH2 domain of Grb2(1)

Following earlier work on cystine-bridged peptides, cyclic phosphopeptides containing nonreducible mimics of cystine were synthesized that show high affinity and specificity toward the Src homology (SH2) domain of the growth factor receptor-binding protein (Grb2). Replacement of the cystine in the cyclic heptapeptide cyclo(CYVNVPC) by D-alpha-acetylthialysine or D-alpha-lysine gave cyclo(YVNVP(D-alpha-acetyl-thiaK)) (22) and cyclo(YVNVP(D-alpha-acetyl-K)) (30), which showed improved binding 10-fold relative to that of the control peptide KPFYVNVEF (1). NMR spectroscopy and molecular modeling experiments indicate that a beta-turn conformation centered around YVNV is essential for high-affinity binding. X-ray structure analyses show that the linear peptide 1 and the cyclic compound 21 adopt a similar binding mode with a beta-turn conformation. Our data confirm the unique structural requirements of the ligand binding site of the SH2 domain of Grb2. Moreover, the potency of our cyclic lactams can be explained by the stabilization of the beta-turn conformation by three intramolecular hydrogen bonds (one mediated by an H2O molecule). These stable and easily accessible cyclic peptides can serve as templates for the evaluation of phosphotyrosine surrogates and further chemical elaboration.     

Spiropiperidines as high-affinity, selective sigma ligands.

A variety of achiral conformationally restricted spirocyclic piperidines have been prepared in an attempt to investigate the functional role of the central sigma recognition site. All the compounds possessed a lipophilic N-substituent incorporating either a tetralin, indan, or benzocycloheptane skeleton. Their in vitro affinity at the sigma site was assessed in radioligand displacement experiments with guinea pig cerebellum homogenates using the sigma-specific radioligand [3H]-N,N'-di-o-tolylguanidine ([3H]-DTG, [3H]-6). A study of the structure-activity relationships identified the N-butyl and N-dimethylallyl substituents as the optimum groups for high affinity and selectivity at the sigma site (e.g., 3,4-dihydro-1'-(3-methylbut-2-enyl)spiro[1H-indene-1,4'-piperidine ] (48), pIC50 = 8.9 vs [3H]-6 and greater than 10,000-fold selective over the dopamine D2 receptor). Such compounds are amongst the highest affinity sigma ligands reported to date, with excellent selectivity over the dopamine D2 receptor, and may serve as a useful tool for exploring the physiological role of the sigma site.     

Structural basis for the binding of antitumor anthracycline antibiotics to model membranes: circular dichroism studies

Circular dichroism was used to compare the binding of several anthracycline antitumor antibiotics to sonicated phosphatidylcholine vesicles. Daunorubicin analogues, differing from the parent by structural changes in the amino sugar moiety of the molecule, were tested both with vesicles that contained negatively charged phospholipids and with neutral vesicles. The self-association properties of the analogues were also investigated. Binding to negatively charged vesicles was not strictly dependent on electrostatic interactions, since the characteristics of daunorubicin binding were totally different from those of Adriamycin (doxorubicin). Furthermore, the cardiotoxicity of these molecules did not have its origin in their quantitatively preferential electrostatic binding to negatively charged cardiolipin-containing membranes: DR-19, a daunorubicin derivative having lower cardiotoxicity than the parent compound, which bound to negatively charged vesicles in a manner quite similar to that of Adriamycin, whereas DR-10, another daunorubicin derivative with higher cardiotoxicity, bound poorly to negatively charged vesicles.     

Stereoselective high-affinity binding of 3H-alaproclate to membranes from rat cerebral cortex

The binding of 3H-alaproclate, a selective 5-hydroxytryptamine uptake inhibitor, to membranes prepared from the rat cerebral cortex was investigated by a filtration technique. It was found that 3H-alaproclate bound with high affinity to three or four different sites and to one low affinity site. The binding to two of these sites was displaceable by 1 microM proadifen (SKF 525A), an inhibitor of drug metabolism. From iterative nonlinear regression analysis the KD-values of these sites were calculated to about 1 and 28 nM and the Bmax values 1.5 and 19 pmol/g wet tissue, respectively. The high affinity binding that was not displaceable by proadifen but by 10 microM alaproclate had KD-values of 1 nM and 6 nM and Bmax-values of 0.4 and 2 pmol/g wet tissue. The low affinity binding that was not displaceable by proadifen had a KD-value of about 200 nM and a Bmax-value of about 90 pmol/g tissue. The possible relationship between the proadifen sensitive high affinity binding of 3H-alaproclate and the brain cytochrome P-450 is discussed.     

Naturally occurring 2'-hydroxyl-substituted flavonoids as high-affinity benzodiazepine site ligands

Screening of traditional medicines has proven invaluable to drug development and discovery. Utilizing activity-guided purification, we previously reported the isolation of a list of flavonoids from the medicinal herb Scutellaria baicalensis Georgi, one of which manifested an affinity for the benzodiazepine receptor (BDZR) comparable to that of the synthetic anxiolytic diazepam (K(i)=6.4 nM). In the present study, this high-affinity, naturally occurring flavonoid derivative, 5,7,2'-trihydroxy-6,8-dimethoxyflavone (K36), was chosen for further functional and behavioral characterization. K36 inhibited [3H]flunitrazepam binding to native BDZR with a K(i) value of 6.05 nM. In electrophysiological experiments K36 potentiated currents mediated by rat recombinant alpha(1)beta(2)gamma(2) GABA(A) receptors expressed in Xenopus oocytes. This potentiation was characterized by a threshold (1 nM) and half-maximal stimulation (24 nM) similar to diazepam. This enhancement was demonstrated to act via the BDZR, since co-application of 1 microM of the BDZR antagonist Ro15-1788 reversed the potentiation. Oral administration of K36 produced significant BDZR-mediated anxiolysis in the mice elevated plus-maze, which was abolished upon co-administration of Ro15-1788. Sedation, myorelaxation and motor incoordination were not observed in the chosen dosage regimen. Structure-activity relationships utilizing synthetic flavonoids with different 2' substituents on the flavone backbone supported that 2'-hydroxyl-substitution is a critical moiety on flavonoids with regard to BDZR affinities. These results further underlined the potential of flavonoids as therapeutics for the treatment of BDZR-associated syndromes.     

Arylpiperazine derivatives as high-affinity 5-HT1A serotonin ligands

Although simple arylpiperazines are commonly considered to be moderately selective for 5-HT1B serotonin binding sites, N4-substitution of such compounds can enhance their affinity for 5-HT1A sites and/or decrease their affinity for 5-HT1B sites. A small series of 4-substituted 1-arylpiperazines was prepared in an attempt to develop agents with high affinity for 5-HT1A sites. Derivatives where the aryl portion is phenyl, 2-methoxyphenyl, or 1-naphthyl, and the 4-substituent is either a phthalimido or benzamido group at a distance of four methylene units away from the piperazine 4-position, display high affinity for these sites. One of these compounds, 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine (18), possesses a higher affinity than 5-HT and represents the highest affinity (Ki = 0.6 nM) agent yet reported for 5-HT1A sites.     

Discovery of high-affinity ligands of sigma1 receptor, ERG2, and emopamil binding protein by pharmacophore modeling and virtual screening

ERG2, emopamil binding protein (EBP), and sigma-1 receptor (sigma(1)) are enzymes of sterol metabolism and an enzyme-related protein, respectively, that share high affinity for various structurally diverse compounds. To discover novel high-affinity ligands, pharmacophore models were built with Catalyst based upon a series of 23 structurally diverse chemicals exhibiting K(i) values from 10 pM to 100 microM for all three proteins. In virtual screening experiments, we retrieved drugs that were previously reported to bind to one or several of these proteins and also tested 11 new hits experimentally, of which three, among them raloxifene, had affinities for sigma(1) or EBP of <60 nM. When used to search a database of 3525 biochemicals of intermediary metabolism, a slightly modified ERG2 pharmacophore model successfully retrieved 10 substrate candidates among the top 28 hits. Our results indicate that inhibitor-based pharmacophore models for sigma(1), ERG2, and EBP can be used to screen drug and metabolite databases for chemically diverse compounds and putative endogenous ligands.     

Structural basis of binding and inhibition of novel tarantula toxins in mammalian voltage-dependent potassium channels

Voltage-dependent potassium channel Kv2.1 is widely expressed in mammalian neurons and was suggested responsible for mediating the delayed rectifier (I(K)) currents. Further investigation of the central role of this channel requires the development of specific pharmacology, for instance, the utilization of spider venom toxins. Most of these toxins belong to the same structural family with a short peptide reticulated by disulfide bridges and share a similar mode of action. Hanatoxin 1 (HaTx1) from a Chilean tarantula was one of the earliest discussed tools regarding this and has been intensively applied to characterize the channel blocking not through the pore domain. Recently, more related novel toxins from African tarantulas such as heteroscordratoxins (HmTx) and stromatoxin 1 (ScTx1) were isolated and shown to act as gating modifiers such as HaTx on Kv2.1 channels with electrophysiological recordings. However, further interaction details are unavailable due to the lack of high-resolution structures of voltage-sensing domains in such mammalian Kv channels. Therefore, in the present study, we explored structural observation via molecular docking simulation between toxins and Kv2.1 channels based upon the solution structures of HaTx1 and a theoretical basis of an individual S3(C) helical channel fragment in combination with homology modeling for other novel toxins. Our results provide precise chemical details for the interactions between these tarantula toxins and channel, reasonably correlating the previously reported pharmacological properties to the three-dimensional structural interpretation. In addition, it is suggested that certain subtle structural variations on the interaction surface of toxins may discriminate between the related toxins with different affinities for Kv channels. Evolutionary links between spider peptide toxins and a "voltage sensor paddles" mechanism most recently found in the crystal structure of an archaebacterial K(+) channel, KvAP, are also delineated in this paper.     

Benz[f]isoquinoline analogues as high-affinity sigma ligands.

This paper describes the synthesis of some conformationally restricted 4-phenylpiperidine analogues and their affinities for the guinea pig cerebellum sigma recognition site ([3H]-DTG) and the rat striatum dopamine D2 receptor ([3H]-(-)-sulpiride) in order to develop potent selective sigma ligands as tools in the investigation of this site in psychosis. It was found that both hexa- and octahydrobenz[f]isoquinolines with lipophilic N-substituents had high affinities for the sigma site. Notably, trans-3-cyclohexyl-1,2,3,4,4a,5,6,10b-octahydrobenz[f]isoquinoline (26) had an affinity of 0.25 nM making it the highest affinity sigma ligand reported to date. Moreover, it is at least 10,000-fold selective over the D2 receptor and could prove to be a valuable tool in the study of sigma sites. Other analogues such as 1H-indeno[2,1-c]pyridines and 1H-benzo[3,4]cyclohepta[1,2-c]pyridines also displayed high sigma site affinity.     

Structural basis for understanding structure-activity relationships for the glutamate binding site of the NMDA receptor

We present new homology-based models of the glutamate binding site (in closed and open forms) of the NMDA receptor NR2B subunit derived from X-ray structures of the water soluble AMPA sensitive glutamate receptor. The models were used for revealing binding modes of agonists and competitive antagonists, as well as for rationalizing known experimental facts concerning structure-activity relationships: (i) the switching between the agonist and the antagonist modes of action upon lengthening the chain between the distal acidic group and the amino acid moiety, (ii) the preference for the methyl group attached to the alpha-amino group of ligands, (iii) the preference for the D-configuration of agonists and antagonists, and (iv) the existence of "superacidic" agonists.