Functionalization of 2,4-Dichloropyrimidines by 2,2,6,6-Tetramethylpiperidyl Zinc Base Enables Modular Synthesis of Antimalarial Diaminopyrimidine P218 and Analogues.

Two routes to the antimalarial diaminopyrimidine P218 were developed based on the C-6 metalation of suitable 2,4-dichloro-5-alkoxy pyrimidines using (TMP)2Zn·2MgCl2·2LiCl base. One approach involves a late-stage modification of the C-6 position, while the other allows for tail fragment modification of P218. Both routes have proven reliable in synthesizing P218, as well as eight analogues. These innovative strategies have the potential to contribute to the search for new antimalarial drugs.

Design and Synthesis of Clinical Candidate PF-06751979: A Potent, Brain Penetrant, ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor Lacking Hypopigmentation.

A major challenge in the development of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors for the treatment of Alzheimer's disease is the alignment of potency, drug-like properties, and selectivity over related aspartyl proteases such as Cathepsin D (CatD) and BACE2. The potential liabilities of inhibiting BACE2 chronically have only recently begun to emerge as BACE2 impacts the processing of the premelanosome protein (PMEL17) and disrupts melanosome morphology resulting in a depigmentation phenotype. Herein, we describe the identification of clinical candidate PF-06751979 (64), which displays excellent brain penetration, potent in vivo efficacy, and broad selectivity over related aspartyl proteases including BACE2. Chronic dosing of 64 for up to 9 months in dog did not reveal any observation of hair coat color (pigmentation) changes and suggests a key differentiator over current BACE1 inhibitors that are nonselective against BACE2 in later stage clinical development.

Discovery and Characterization of (R)-6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates.

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance.

Aminomethyl-Derived Beta Secretase (BACE1) Inhibitors: Engaging Gly230 without an Anilide Functionality.

A growing subset of ß-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease (AD) utilizes an anilide chemotype that engages a key residue (Gly230) in the BACE1 binding site. Although the anilide moiety affords excellent potency, it simultaneously introduces a third hydrogen bond donor that limits brain availability and provides a potential metabolic site leading to the formation of an aniline, a structural motif of prospective safety concern. We report herein an alternative aminomethyl linker that delivers similar potency and improved brain penetration relative to the amide moiety. Optimization of this series identified analogues with an excellent balance of ADME properties and potency; however, potential drug-drug interactions (DDI) were predicted based on CYP 2D6 affinities. Generation and analysis of key BACE1 and CYP 2D6 crystal structures identified strategies to obviate the DDI liability, leading to compound 16, which exhibits robust in vivo efficacy as a BACE1 inhibitor.

Dihedral Angle-Based Sampling of Natural Product Polyketide Conformations: Application to Permeability Prediction.

Macrocycles pose challenges for computer-aided drug design due to their conformational complexity. One fundamental challenge is identifying all low-energy conformations of the macrocyclic ring, which is important for modeling target binding, passive membrane permeation, and other conformation-dependent properties. Macrocyclic polyketides are medically and biologically important natural products characterized by structural and functional diversity. Advances in synthetic biology and semisynthetic methods may enable creation of an even more diverse set of non-natural product polyketides for drug discovery and other applications. However, the conformational sampling of these flexible compounds remains demanding. We developed and optimized a dihedral angle-based macrocycle conformational sampling method for macrocycles of arbitrary structure, and here we apply it to diverse polyketide natural products. First, we evaluated its performance using a data set of 37 polyketides with available crystal structures, with 9-22 rotatable bonds in the macrocyclic ring. Our optimized protocol was able to reproduce the crystal structure of polyketides' aglycone backbone within 0.50 Å RMSD for 31 out of 37 polyketides. Consistent with prior structural studies, our analysis suggests that polyketides tend to have multiple distinct low-energy structures, including the bioactive (target-bound) conformation as well as others of unknown significance. For this reason, we also introduce a strategy to improve both efficiency and accuracy of the conformational search by utilizing torsional restraints derived from NMR vicinal proton couplings to restrict the conformational search. Finally, as a first application of the method, we made blinded predictions of the passive membrane permeability of a diverse set of polyketides, based on their predicted structures in low- and high-dielectric media.

Utilizing structures of CYP2D6 and BACE1 complexes to reduce risk of drug-drug interactions with a novel series of centrally efficacious BACE1 inhibitors.

In recent years, the first generation of ß-secretase (BACE1) inhibitors advanced into clinical development for the treatment of Alzheimer's disease (AD). However, the alignment of drug-like properties and selectivity remains a major challenge. Herein, we describe the discovery of a novel class of potent, low clearance, CNS penetrant BACE1 inhibitors represented by thioamidine 5. Further profiling suggested that a high fraction of the metabolism (>95%) was due to CYP2D6, increasing the potential risk for victim-based drug-drug interactions (DDI) and variable exposure in the clinic due to the polymorphic nature of this enzyme. To guide future design, we solved crystal structures of CYP2D6 complexes with substrate 5 and its corresponding metabolic product pyrazole 6, which provided insight into the binding mode and movements between substrate/inhibitor complexes. Guided by the BACE1 and CYP2D6 crystal structures, we designed and synthesized analogues with reduced risk for DDI, central efficacy, and improved hERG therapeutic margins.

Discovery of a series of efficient, centrally efficacious BACE1 inhibitors through structure-based drug design.

The identification of centrally efficacious ß-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease (AD) has historically been thwarted by an inability to maintain alignment of potency, brain availability, and desired absorption, distribution, metabolism, and excretion (ADME) properties. In this paper, we describe a series of truncated, fused thioamidines that are efficiently selective in garnering BACE1 activity without simultaneously inhibiting the closely related cathepsin D or negatively impacting brain penetration and ADME alignment, as exemplified by 36. Upon oral administration, these inhibitors exhibit robust brain availability and are efficacious in lowering central Amyloid ß (Aß) levels in mouse and dog. In addition, chronic treatment in aged PS1/APP mice effects a decrease in the number and size of Aß-derived plaques. Most importantly, evaluation of 36 in a 2-week exploratory toxicology study revealed no accumulation of autofluorescent material in retinal pigment epithelium or histology findings in the eye, issues observed with earlier BACE1 inhibitors.

Discovery and preclinical characterization of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo-[3,4-b]pyrazine (PF470): a highly potent, selective, and efficacious metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulator.

A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (1), a systematic structure-activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (PF470, 14) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound 14 demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of 14 to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-mediated type IV hypersensitivity in a 90-day NHP regulatory toxicology study.

Spirocyclic sulfamides as ß-secretase 1 (BACE-1) inhibitors for the treatment of Alzheimer's disease: utilization of structure based drug design, WaterMap, and CNS penetration studies to identify centrally efficacious inhibitors.

ß-Secretase 1 (BACE-1) is an attractive therapeutic target for the treatment and prevention of Alzheimer's disease (AD). Herein, we describe the discovery of a novel class of BACE-1 inhibitors represented by sulfamide 14g, using a medicinal chemistry strategy to optimize central nervous system (CNS) penetration by minimizing hydrogen bond donors (HBDs) and reducing P-glycoprotein (P-gp) mediated efflux. We have also taken advantage of the combination of structure based drug design (SBDD) to guide the optimization of the sulfamide analogues and the in silico tool WaterMap to explain the observed SAR. Compound 14g is a potent inhibitor of BACE-1 with excellent permeability and a moderate P-gp liability. Administration of 14g to mice produced a significant, dose-dependent reduction in central Aß(X-40) levels at a free drug exposure equivalent to the whole cell IC(50) (100 nM). Furthermore, studies of the P-gp knockout mouse provided evidence that efflux transporters affected the amount of Aß lowering versus that observed in wild-type (WT) mouse at an equivalent dose.

Oxidative metabolism of a quinoxaline derivative by xanthine oxidase in rodent plasma.

As part of efforts directed at the G protein-coupled receptor 119 agonist program for type 2 diabetes, a series of cyanopyridine derivatives exemplified by isopropyl-4-(3-cyano-5-(quinoxalin-6-yl)pyridine-2-yl)piperazine-1-carboxylate (1) were identified as novel chemotypes worthy of further hit-to-lead optimization. Compound 1, however, was found to be unstable in plasma (37 °C, pH 7.4) from rat (T(1/2) = 16 min), mouse (T(1/2) = 61 min), and guinea pig (T(1/2) = 4 min). Lowering the temperature of plasma incubations (4-25 °C) attenuated the degradation of 1, implicating the involvement of an enzyme-mediated process. Failure to detect any appreciable amount of 1 in plasma samples from protein binding and pharmacokinetic studies in rats was consistent with its labile nature in plasma. Instability noted in rodent plasma was not observed in plasma from dogs, monkeys, and humans (T(1/2) > 370 min at 37 °C, pH 7.4). Metabolite identification studies in rodent plasma revealed the formation of a single metabolite (M1), which was 16 Da higher than the molecular weight of 1 (compound 1, MH(+) = 403; M1, MH(+) = 419). Pretreatment of rat plasma with allopurinol, but not raloxifene, abolished the conversion of 1 to M1, suggesting that xanthine oxidase (XO) was responsible for the oxidative instability. Consistent with the known catalytic mechanism of XO, the source of oxygen incorporated in M1 was derived from water rather than molecular oxygen. The formation of M1 was also demonstrated in incubations of 1 with purified bovine XO. The structure of M1 was determined by NMR analysis to be isopropyl-4-(3-cyano-5-(3-oxo-3,4-dihydroquinoxalin-6-yl)pyridine-2-yl)piperazine-1-carboxylate. The regiochemistry of quinoxaline ring oxidation in 1 was consistent with ab initio calculations and molecular docking studies using a published crystal structure of bovine XO. A close-in analogue of 1, which lacked the quinoxaline motif (e.g., 5-(4-cyano-3-methylphenyl)-2-(4-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidin-1-yl)nicotinitrile (2)) was stable in rat plasma and possessed substantially improved GPR119 agonist properties. To the best of our knowledge, our studies constitute the first report on the involvement of rodent XO in oxidative drug metabolism in plasma.

Structural comparison of chromosomal and exogenous dihydrofolate reductase from Staphylococcus aureus in complex with the potent inhibitor trimethoprim.

Dihydrofolate reductase (DHFR) is the enzyme responsible for the NADPH-dependent reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential cofactor in the synthesis of purines, thymidylate, methionine, and other key metabolites. Because of its importance in multiple cellular functions, DHFR has been the subject of much research targeting the enzyme with anticancer, antibacterial, and antimicrobial agents. Clinically used compounds targeting DHFR include methotrexate for the treatment of cancer and diaminopyrimidines (DAPs) such as trimethoprim (TMP) for the treatment of bacterial infections. DAP inhibitors of DHFR have been used clinically for >30 years and resistance to these agents has become widespread. Methicillin-resistant Staphylococcus aureus (MRSA), the causative agent of many serious nosocomial and community acquired infections, and other gram-positive organisms can show resistance to DAPs through mutation of the chromosomal gene or acquisition of an alternative DHFR termed "S1 DHFR." To develop new therapies for health threats such as MRSA, it is important to understand the molecular basis of DAP resistance. Here, we report the crystal structure of the wild-type chromosomal DHFR from S. aureus in complex with NADPH and TMP. We have also solved the structure of the exogenous, TMP resistant S1 DHFR, apo and in complex with TMP. The structural and thermodynamic data point to important molecular differences between the two enzymes that lead to dramatically reduced affinity of DAPs to S1 DHFR. These differences in enzyme binding affinity translate into reduced antibacterial activity against strains of S. aureus that express S1 DHFR.

Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding.

Newly generated actin free barbed ends at the front of motile cells provide sites for actin filament assembly driving membrane protrusion. Growth factors induce a rapid biphasic increase in actin free barbed ends, and we found both phases absent in fibroblasts lacking H(+) efflux by the Na-H exchanger NHE1. The first phase is restored by expression of mutant cofilin-H133A but not unphosphorylated cofilin-S3A. Constant pH molecular dynamics simulations and nuclear magnetic resonance (NMR) reveal pH-sensitive structural changes in the cofilin C-terminal filamentous actin binding site dependent on His133. However, cofilin-H133A retains pH-sensitive changes in NMR spectra and severing activity in vitro, which suggests that it has a more complex behavior in cells. Cofilin activity is inhibited by phosphoinositide binding, and we found that phosphoinositide binding is pH-dependent for wild-type cofilin, with decreased binding at a higher pH. In contrast, phosphoinositide binding by cofilin-H133A is attenuated and pH insensitive. These data suggest a molecular mechanism whereby cofilin acts as a pH sensor to mediate a pH-dependent actin filament dynamics.

From docking false-positive to active anti-HIV agent.

Virtual screening of the Maybridge library of ca. 70 000 compounds was performed using a similarity filter, docking, and molecular mechanics-generalized Born/surface area postprocessing to seek potential non-nucleoside inhibitors of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (NNRTIs). Although known NNRTIs were retrieved well, purchase and assaying of representative, top-scoring compounds from the library failed to yield any active anti-HIV agents. However, the highest-ranked library compound, oxadiazole 1, was pursued as a potential "near-miss" with the BOMB program to seek constructive modifications. Subsequent synthesis and assaying of several polychloro-analogs did yield anti-HIV agents with EC50 values as low as 310 nM. The study demonstrates that it is possible to learn from a formally unsuccessful virtual-screening exercise and, with the aid of computational analyses, to efficiently evolve a false positive into a true active.

Search for non-nucleoside inhibitors of HIV-1 reverse transcriptase using chemical similarity, molecular docking, and MM-GB/SA scoring.

A virtual screening protocol has been applied to seek non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) and its K103N mutant. First, a chemical similarity search on the Maybridge library was performed using known NNRTIs as reference structures. The top-ranked molecules obtained from this procedure plus 26 known NNRTIs were then docked into the binding sites of the wild-type reverse transcriptase (HIV-RT) and its K103N variant (K103N-RT) using Glide 3.5. The top-ranked 100 compounds from the docking for both proteins were post-scored with a procedure using molecular mechanics and continuum solvation (MM-GB/SA). The validity of the virtual screening protocol was supported by (i) testing of the MM-GB/SA procedure, (ii) agreement between predicted and crystallographic binding poses, (iii) recovery of known potent NNRTIs at the top of both rankings, and (iv) identification of top-scoring library compounds that are close in structure to recently reported NNRTI HTS hits. However, purchase and assaying of selected top-scoring compounds from the library failed to yield active anti-HIV agents. Nevertheless, the highest-ranked database compound, S10087, was pursued as containing a potentially viable core. Subsequent synthesis and assaying of S10087 analogues proposed by further computational analysis yielded anti-HIV agents with EC50 values as low as 310 nM. Thus, with the aid of computational tools, it was possible to evolve a false positive into a true active.

Human Cytomegalovirus Protease: Why is the Dimer Required for Catalytic Activity?

Human cytomegalovirus (HCMV) is a pathogenic agent responsible for morbidity and mortality in immunocompromised and immunosuppressed individuals. HCMV encodes a serine protease that is essential for the production of infectious virions. In this work, we applied molecular dynamics (MD) simulations on HCMV protease models in order to investigate the experimentally observed (i) catalytic activity of the enzyme homodimer and (ii) induced-fit mechanism upon the binding of substrates and peptidyl inhibitors. Long and stable trajectories were obtained for models of the monomeric and dimeric states, free in solution and bound covalently and noncovalently to a peptidyl-activated carbonyl inhibitor, with very good agreement between theoretical and experimental results. The MD results suggest that HCMV protease indeed operates by an induced-fit mechanism. Also, our analysis indicates that the catalytic activity of the dimer is a result of more favorable interactions between the oxyanion in the covalently bound state and the backbone nitrogen of Arg165, resulting in a reaction that is 7.0 kcal/mol more exergonic and a more significant thermodynamic driving force. The incipient oxyanion in the transition state should also benefit from the stronger interactions with Arg165, reducing in this manner the intrinsic activation barrier for the reaction in the dimeric state.

Molecular dynamics simulations of the 136 unique tetranucleotide sequences of DNA oligonucleotides. I. Research design and results on d(CpG) steps.

We describe herein a computationally intensive project aimed at carrying out molecular dynamics (MD) simulations including water and counterions on B-DNA oligomers containing all 136 unique tetranucleotide base sequences. This initiative was undertaken by an international collaborative effort involving nine research groups, the "Ascona B-DNA Consortium" (ABC). Calculations were carried out on the 136 cases imbedded in 39 DNA oligomers with repeating tetranucleotide sequences, capped on both ends by GC pairs and each having a total length of 15 nucleotide pairs. All MD simulations were carried out using a well-defined protocol, the AMBER suite of programs, and the parm94 force field. Phase I of the ABC project involves a total of approximately 0.6 mus of simulation for systems containing approximately 24,000 atoms. The resulting trajectories involve 600,000 coordinate sets and represent approximately 400 gigabytes of data. In this article, the research design, details of the simulation protocol, informatics issues, and the organization of the results into a web-accessible database are described. Preliminary results from 15-ns MD trajectories are presented for the d(CpG) step in its 10 unique sequence contexts, and issues of stability and convergence, the extent of quasiergodic problems, and the possibility of long-lived conformational substates are discussed.

Molecular dynamics simulations of DNA curvature and flexibility: helix phasing and premelting.

Recent studies of DNA axis curvature and flexibility based on molecular dynamics (MD) simulations on DNA are reviewed. The MD simulations are on DNA sequences up to 25 base pairs in length, including explicit consideration of counterions and waters in the computational model. MD studies are described for ApA steps, A-tracts, for sequences of A-tracts with helix phasing. In MD modeling, ApA steps and A-tracts in aqueous solution are essentially straight, relatively rigid, and exhibit the characteristic features associated with the B'-form of DNA. The results of MD modeling of A-tract oligonucleotides are validated by close accord with corresponding crystal structure results and nuclear magnetic resonance (NMR) nuclear Overhauser effect (NOE) and residual dipolar coupling (RDC) structures of d(CGCGAATTCGCG) and d(GGCAAAAAACGG). MD simulation successfully accounts for enhanced axis curvature in a set of three sequences with phased A-tracts studied to date. The primary origin of the axis curvature in the MD model is found at those pyrimidine/purine YpR "flexible hinge points" in a high roll, open hinge conformational substate. In the MD model of axis curvature in a DNA sequence with both phased A-tracts and YpR steps, the A-tracts appear to act as positioning elements that make the helix phasing more precise, and key YpR steps in the open hinge state serve as curvature elements. Our simulations on a phased A-tract sequence as a function of temperature show that the MD simulations exhibit a premelting transition in close accord with experiment, and predict that the mechanism involves a B'-to-B transition within A-tracts coupled with the prediction of a transition in key YpR steps from the high roll, open hinge, to a low roll, closed hinge substate. Diverse experimental observations on DNA curvature phenomena are examined in light of the MD model with no serious discrepancies. The collected MD results provide independent support for the "non-A-tract model" of DNA curvature. The "junction model" is indicated to be a special case of the non-A-tract model when there is a Y base at the 5' end of an A-tract. In accord with crystallography, the "ApA wedge model" is not supported by MD.

Investigation of the induced-fit mechanism and catalytic activity of the human cytomegalovirus protease homodimer via molecular dynamics simulations.

Human cytomegalovirus (HCMV) is a highly species-specific DNA virus infecting up to 80% of the general population. The viral genome contains the open reading frame UL80, which encodes the full-length 80 kDa HCMV serine protease and its substrate. Full-length HCMV protease is composed of an N-terminal 256-amino-acid proteolytic domain, called assemblin, a linker region, and a C-terminal structural domain, the assembly protein precursor. Biochemical studies have shown that dimerization activates assemblin because of an induced stabilization of the oxyanion hole (Arg166). Thus, we performed here molecular dynamics (MD) simulations on HCMV protease models to study the induced-fit mechanism of the enzyme upon the binding of substrates and peptidyl inhibitors, and structural and energetic factors that are responsible for the catalytic activity of the enzyme dimer. Long and stable trajectories were obtained for the models of the monomeric and dimeric states, free in solution and bound to a peptidyl-activated carbonyl inhibitor, with very good agreement between theoretical and experimental results. Our results suggest that HCMV protease is indeed a novel example of serine protease that operates by an induced-fit mechanism. Also, in agreement with mutagenesis studies, our MD simulations suggest that the dimeric form is necessary to activate the enzyme because of an induced stabilization of the oxyanion hole.

Potential of mean force calculations on an L-type calcium channel model.

To understand the mechanisms of Na(+)/Li(+) permeation at submicromolar Ca(2+) concentrations, Na(+)/Li(+) blocking at higher Ca(2+) concentrations (10(-6)-10(-4) M) and Ca(2+) permeation at millimolar Ca(2+) concentrations, we used our recently described L-type calcium channel model. For this purpose, we obtained potential of mean force (pmf) curves for the position change of one Na(+) and one Ca(2+) ion inside the channel and for the position change of a second Ca(2+) ion when the EEEE locus is coordinated to Ca(2+). The pmf curves suggest that (i) at submicromolar Ca(2+) concentrations, because of the low velocity of Ca(2+) entry in the channel, monovalent ion flux occurs; (ii) at Ca(2+) concentrations between 10(-6) and 10(-4) M, thermodynamic equilibrium between the channel and Ca(2+) is achieved; as the coordination of Ca(2+) with the locus is more favorable than the coordination of Na(+), the monovalent ion flux is blocked; and (iii) to put a second Ca(2+) ion inside the channel at an appropriate rate, the Ca(2+) concentration should reach millimolar levels. Nevertheless, the entry of a second Ca(2+) is thermodynamically unfavorable, indicating that the competition of two Ca(2+) ions for the locus leads to Ca(2+) permeation.

A molecular dynamics study of an L-type calcium channel model.

In this work, we propose a molecular model of the L-type calcium channel pore from the human cardiac alpha1 subunit. Four glutamic acid residues, the EEEE locus, located at highly conserved P loops (also called SS1-SS2 segments) of the alpha1 subunit, molecularly express the calcium channel selectivity. The proposed alpha-helix structure for the SS1 segment, analyzed through molecular dynamics simulations in aqueous-phase, was validated by the plotting of Ramachandran diagrams for the averaged structures and by the analysis of i and i + 4 helical hydrogen bonding between the amino acid residues. The results of the simulation of the calcium channel model with one and two Ca2+ ions at the binding site are in accordance with mutation studies which suggest that the EEEE locus in the L-type calcium channel must form a single high-affinity binding site. These results suggest that the Ca2+ permeation through the channel would be derived from competition between two ions for the only high-affinity binding site. Furthermore, the experimentally observed blocking of the Na+ flux at micromolar Ca2+ concentrations, probably due to the occupancy of the single high-affinity binding site for one Ca2+, was also reproduced by our model.