Marcel J. de Groot

Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels

Significance Voltage-gated sodium (Nav) channels contribute to physiological and pathophysiological electrical signaling in nerve and muscle cells. Because Nav channel isoforms exhibit tissue-specific expression, subtype selective modulation of this channel family provides important drug development opportunities. However, most available Nav channel modulators are unable to distinguish between Nav channel subtypes, which limits their therapeutic utility because of cardiac or nervous system toxicity. This study describes a new class of subtype selective Nav channel inhibitors that interact with a region of the channel that controls voltage sensitivity. This interaction site may enable development of selective therapeutic interventions with reduced potential for toxicity. Voltage-gated sodium (Nav) channels play a fundamental role in the generation and propagation of electrical impulses in excitable cells. Here we describe two unique structurally related nanomolar potent small molecule Nav channel inhibitors that exhibit up to 1,000-fold selectivity for human Nav1.3/Nav1.1 (ICA-121431, IC50, 19 nM) or Nav1.7 (PF-04856264, IC50, 28 nM) vs. other TTX-sensitive or resistant (i.e., Nav1.5) sodium channels. Using both chimeras and single point mutations, we demonstrate that this unique class of sodium channel inhibitor interacts with the S1–S4 voltage sensor segment of homologous Domain 4. Amino acid residues in the “extracellular” facing regions of the S2 and S3 transmembrane segments of Nav1.3 and Nav1.7 seem to be major determinants of Nav subtype selectivity and to confer differences in species sensitivity to these inhibitors. The unique interaction region on the Domain 4 voltage sensor segment is distinct from the structural domains forming the channel pore, as well as previously characterized interaction sites for other small molecule inhibitors, including local anesthetics and TTX. However, this interaction region does include at least one amino acid residue [E1559 (Nav1.3)/D1586 (Nav1.7)] that is important for Site 3 α-scorpion and anemone polypeptide toxin modulators of Nav channel inactivation. The present study provides a potential framework for identifying subtype selective small molecule sodium channel inhibitors targeting interaction sites away from the pore region.

Pharmacophore modeling of cytochromes P450.

Understanding the binding of ligands in the active site of a membrane-bound protein is difficult in the absence of a crystal structure. When these proteins are the enzymes involved in drug metabolism, it leaves little option but to use site-directed mutagenesis and in vitro studies to provide critical information relating to determinants of binding affinity. Pharmacophore models and three-dimensional quantitative structure-activity relationships have been used either alone or in combination with protein homology models to provide this information for cytochrome P450s. At present, their application has been directed to the major enzymes but this may escalate in future as more in vitro data are generated for other P450s. The following review outlines the methodologies and models as well as future prospects for applying these technologies to P450s in the hope that future drugs will be selected with increased metabolic stability and fewer incidences of undesirable drug-drug interactions.

Subtype‐selective targeting of voltage‐gated sodium channels

Voltage‐gated sodium channels are key to the initiation and propagation of action potentials in electrically excitable cells. Molecular characterization has shown there to be nine functional members of the family, with a high degree of sequence homology between the channels. This homology translates into similar biophysical and pharmacological properties. Confidence in some of the channels as drug targets has been boosted by the discovery of human mutations in the genes encoding a number of them, which give rise to clinical conditions commensurate with the changes predicted from the altered channel biophysics. As a result, they have received much attention for their therapeutic potential. Sodium channels represent well‐precedented drug targets as antidysrhythmics, anticonvulsants and local anaesthetics provide good clinical efficacy, driven through pharmacology at these channels. However, electrophysiological characterization of clinically useful compounds in recombinant expression systems shows them to be weak, with poor selectivity between channel types. This has led to the search for subtype‐selective modulators, which offer the promise of treatments with improved clinical efficacy and better toleration. Despite developments in high‐throughput electrophysiology platforms, this has proven very challenging. Structural biology is beginning to offer us a greater understanding of the three‐dimensional structure of voltage‐gated ion channels, bringing with it the opportunity to do real structure‐based drug design in the future. This discipline is still in its infancy, but developments with the expression and purification of prokaryotic sodium channels offer the promise of structure‐based drug design in the not too distant future.

Genetic Basis for Differential Activities of Fluconazole and Voriconazole against Candida krusei

ABSTRACT Invasive infections caused by Candida krusei are a significant concern because this organism is intrinsically resistant to fluconazole. Voriconazole is more active than fluconazole against C. krusei in vitro. One mechanism of fluconazole resistance in C. krusei is diminished sensitivity of the target enzyme, cytochrome P450 sterol 14α-demethylase (CYP51), to inhibition by this drug. We investigated the interactions of fluconazole and voriconazole with the CYP51s of C. krusei (ckCYP51) and fluconazole-susceptible Candida albicans (caCYP51). We found that voriconazole was a more potent inhibitor of both ckCYP51 and caCYP51 in cell extracts than was fluconazole. Also, the ckCYP51 was less sensitive to inhibition by both drugs than was caCYP51. These results were confirmed by expressing the CYP51 genes from C. krusei and C. albicans in Saccharomyces cerevisiae and determining the susceptibility of the transformants to voriconazole and fluconazole. We constructed homology models of the CYP51s of C. albicans and C. krusei based on the crystal structure of CYP51 from Mycobacterium tuberculosis. These models predicted that voriconazole is a more potent inhibitor of both caCYP51 and ckCYP51 than is fluconazole, because the extra methyl group of voriconazole results in a stronger hydrophobic interaction with the aromatic amino acids in the substrate binding site and more extensive filling of this site. Although there are multiple differences in the predicted amino acid sequence of caCYP51 and ckCYP51, the models of the two enzymes were quite similar and the mechanism for the relative resistance of ckCYP51 to the azoles was not apparent.

Quantum Mechanics/Molecular Mechanics Modeling of Regioselectivity of Drug Metabolism in Cytochrome P450 2C9

Cytochrome P450 enzymes (P450s) are important in drug metabolism and have been linked to adverse drug reactions. P450s display broad substrate reactivity, and prediction of metabolites is complex. QM/MM studies of P450 reactivity have provided insight into important details of the reaction mechanisms and have the potential to make predictions of metabolite formation. Here we present a comprehensive study of the oxidation of three widely used pharmaceutical compounds (S-ibuprofen, diclofenac, and S-warfarin) by one of the major drug-metabolizing P450 isoforms, CYP2C9. The reaction barriers to substrate oxidation by the iron-oxo species (Compound I) have been calculated at the B3LYP-D/CHARMM27 level for different possible metabolism sites for each drug, on multiple pathways. In the cases of ibuprofen and warfarin, the process with the lowest activation energy is consistent with the experimentally preferred metabolite. For diclofenac, the pathway leading to the experimentally observed metabolite is not the one with the lowest activation energy. This apparent inconsistency with experiment might be explained by the two very different binding modes involved in oxidation at the two competing positions. The carboxylate of diclofenac interacts strongly with the CYP2C9 Arg108 side chain in the transition state for formation of the observed metabolite—but not in that for the competing pathway. We compare reaction barriers calculated both in the presence and in the absence of the protein and observe a marked improvement in selectivity prediction ability upon inclusion of the protein for all of the substrates studied. The barriers calculated with the protein are generally higher than those calculated in the gas phase. This suggests that active-site residues surrounding the substrate play an important role in controlling selectivity in CYP2C9. The results show that inclusion of sampling (particularly) and dispersion effects is important in making accurate predictions of drug metabolism selectivity of P450s using QM/MM methods.

In silico methods for predicting ligand binding determinants of cytochromes P450.

A large number of computational methodologies have been used to predict, and thus help explain, the metabolism catalysed by the enzymes of the cytochrome P450 superfamily (P450s). A summary of the methodologies and resulting models is presented. This shows that investigations so far have focused on just a few of the many P450s, mainly those that are involved in drug metabolism. The models have evolved from simple comparisons of known substrates to more elaborate models requiring considerable computer power. These help to explain and, more importantly, predict the involvement of P450s in the metabolism of specific compounds.

The Discovery of CCR5 Receptor Antagonists for the Treatment of HIV Infection: Hit-to-Lead Studies

Infection with HIV leads, in the vast majority of cases, to progressive disease and ultimately death. By 2004, just 23 years after AIDS was first recognised, the Joint United Nations Programme on HIV/AIDS estimated that 42 million people worldwide were infected with HIV, with more than 20 million dead since the beginning of the epidemic. Furthermore, rates of infection are once again on the increase in the developed world. Despite the undoubted achievements of highly active antiretroviral therapy (HAART) using cocktails of reverse transcriptase and protease inhibitors, there is still a high unmet medical need for better tolerated, conveniently administered agents to treat HIV and AIDS. New mechanisms of action are particularly attractive to avoid issues of viral resistance. HIV enters the host cell by fusing the lipid membrane of the virus with the host cell membrane. This fusion is triggered by the interaction of proteins on the surface of the HIV envelope with specific cell surface receptors. One of these is CD4, the main receptor for HIV-1 that binds to gp120, a surface protein on the virus particle. However, CD4 alone is not sufficient to permit HIV fusion and cell entry, an additional coreceptor from the chemokine family of G-protein coupled receptors (GPCRs) is required. The chemokine receptor CCR5 has been demonstrated to be the major coreceptor for the fusion and entry of macrophage tropic (R5-tropic) HIV-1 into cells. R5-tropic strains are prevalent in the early asymptomatic stages of infection. Indeed, the CCR5 monoclonal antibody PRO140 has been demonstrated to potently inhibit a broad range of HIV-1 strains from infecting their target cells. Shifts in tropism do occur during progression, mainly to X4 viruses that use CXCR4 as coreceptor, however, approximately 50% of individuals are infected with strains that maintain their requirement for CCR5. Currently the cause of the switch in tropism in late stage disease is unknown. There is evidence that homozygotes possessing a 32 base pair deletion in the CCR5 coding region are resistant to infection with R5-tropic HIV-1. These homozygotes do not express functional CCR5 receptors on the cell surface. Individuals who are heterozygous for the 32 base pair deletion display significantly longer progression times to the symptomatic stages of infection and evidence is emerging that they respond better to HAART. Moreover, CCR5 deficient individuals are apparently fully immunocompetent, indicating that absence of CCR5 function may not be detrimental and that a CCR5 antagonist should be well tolerated. Currently, some clinical trials involving CCR5 ligands are being halted increasing the focus on this promising mechanism for the treatment of HIV. In particular, aplaviroc was halted due to liver toxicity developed in a Phase IIb clinical trial that was also observed in a subsequent Phase III trial. Herein, the discovery of hits, design and synthesis, structure–activity relationships (SAR), and biological evaluation of UK-374,503 (1) and related compounds are described.

Understanding CYP2D6 interactions

Owing to the polymorphic nature of CYP2D6, clinically significant issues can arise when drugs rely on that enzyme either for clearance, or metabolism to an active metabolite. Available screening methods to determine if the compound is likely to cause drug-drug interactions, or is likely to be a victim of inhibition of CYP2D6 by other compounds will be described. Computational models and examples will be given on strategies to design out the CYP2D6 liabilities for both heme-binding compounds and non-heme-binding compounds.

Overcoming hERG Affinity in the Discovery of Maraviroc; A CCR5 Antagonist for the Treatment of HIV

Avoiding cardiac liability associated with blockade of hERG (human ether a go-go) is key for successful drug discovery and development. This paper describes the work undertaken in the discovery of a potent CCR5 antagonist, maraviroc 34, for the treatment of HIV. In particular the use of a pharmacophore model of the hERG channel and a high throughput binding assay for the hERG channel are described that were critical to elucidate SAR to overcome hERG liabilities. The key SAR involves the introduction of polar substituents into regions of the molecule where it is postulated to undergo hydrophobic interactions with the ion channel. Within the CCR5 project there appeared to be no strong correlation between hERG affinity and physiochemical parameters such as pKa or lipophilicity. It is believed that chemists could apply these same strategies early in drug discovery to remove hERG interactions associated with lead compounds while retaining potency at the primary target.

The Discovery of Tropane‐derived CCR5 Receptor Antagonists

The development of compound 1, a piperidine‐based CCR5 receptor antagonist with Type I CYP2D6 inhibition, into the tropane‐derived analogue 5, is described. This compound, which is devoid of CYP2D6 liabilities, is a highly potent ligand for the CCR5 receptor and has broad‐spectrum activity against a range of clinically relevant HIV isolates. The identification of human ether a‐go‐go‐related gene channel inhibition within this series is described and the potential for QTc interval prolongation discussed. Furthermore, structure activity relationship (SAR) around the piperidine moiety is also described.