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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Article
  • Peer Reviewed
UC Irvine Previously Published Works (2018)
Structure-function relationships of nine rationally designed ritonavir-like compounds were investigated to better understand the ligand binding and inhibitory mechanism in human drug-metabolizing cytochrome P450 3A4 (CYP3A4). The analogs had a similar backbone and pyridine and tert-butyloxycarbonyl (Boc) as the heme-ligating and terminal groups, respectively. N-Isopropyl, N-cyclopentyl, or N-phenyl were the R1-side group substituents alone (compounds 5a-c) or in combination with phenyl or indole at the R2 position (8a-c and 8d-f subseries, respectively). Our experimental and structural data indicate that (i) for all analogs, a decrease in the dissociation constant (Ks) coincides with a decrease in IC50, but no relation with other derived parameters is observed; (ii) an increase in the R1 volume, hydrophobicity, and aromaticity markedly lowers Ks and IC50, whereas the addition of aromatic R2 has a more pronounced positive effect on the inhibitory potency than the binding strength; (iii) the ligands association mode is strongly influenced by the mutually dependent R1-R2 interplay, but the R1-mediated interactions are dominant and define the overall conformation in the active site; (iv) formation of a strong H-bond with Ser119 is a prerequisite for potent CYP3A4 inhibition; and (v) the strongest inhibitor in the series, the R1-phenyl/R2-indole containing 8f (Ks and IC50 of 0.08 and 0.43 μM, respectively), is still less potent than ritonavir, even under conditions that prevent the mechanism based inactivation of CYP3A4. Crystallographic data were essential for better understanding and interpretation of the experimental results, and suggested how the inhibitor design could be further optimized.
    Cover page: Inhibition of Human CYP3A4 by Rationally Designed Ritonavir-Like Compounds: Impact and Interplay of the Side Group Functionalities.
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2019)
    In a continuing effort to identify structural attributes required for strong binding and potent inhibition of human drug-metabolizing CYP3A4, we designed ten ritonavir-like analogues differing in the side-group stereochemistry, backbone atomic composition, and headgroup spacing. All analogues had pyridine and tert-butyloxycarbonyl (Boc) as the heme-ligating head and tail groups, respectively, phenyl side groups, and either a methyl- or ethyl-pyridyl linker. Each linker subseries had S/ R, R/ S, R/ R, and S/S side-group conformers (4a-d and 4e-h, respectively), and one S/S stereoisomer with the backbone S-to-N-heteroatom substitution (6a and 6b). To elucidate structure-activity relationships, ligand-dependent changes in optical spectra, dissociation constant ( Ks), inhibitory potency (IC50), thermostability, and heme ligation and reduction kinetics were analyzed. Comparison of the subseries and individual compounds showed that CYP3A4 only weakly discriminates between side-group configurations, associates more tightly with the pyridyl-ethyl-linker analogues, and strongly disfavors the N-containing backbone. Ks and IC50 for the pyridyl-ethyl R/ R conformer, 4g, were the lowest and close to those for ritonavir: 0.04 and 0.31 μM versus 0.02 and 0.13 μM, respectively. Determination of the X-ray structures of the inhibitory complexes was critical for experimental data interpretation, especially for the uniquely oriented 4a and 4e. Based on structural analysis, we conclude that, for this series of analogues, the ligand-mediated interactions near the heme are dominant and define the binding mode and that fine-tuning of these interactions as well as the backbone spacing could further improve the affinity and inhibitory strength.
      Cover page: Structure-Activity Relationships of Rationally Designed Ritonavir Analogues: Impact of Side-Group Stereochemistry, Headgroup Spacing, and Backbone Composition on the Interaction with CYP3A4.
      • Thesis
      • Peer Reviewed
      UC Irvine Electronic Theses and Dissertations (2018)

      PART I

      The amidohydrolase urease is critical for the survival and pathogenesis of Helicobacter pylori. Urease catalyzes the hydrolysis of one molecule of urea to two molecules of ammonia. It is through the generation and excretion of basic ammonia that H. pylori can survive the acidic gut. It has been well established that inhibition of the enzyme renders the pathogen defenseless against the harsh human stomach. Therefore it is not surprising that urease has become an ideal target for H. pylori eradication. However, despite considerable research efforts, few practical urease inhibitors exist. Additionally, increasing antibiotic resistance has generated a need for effective therapies against H. pylori. Urease’s unique ability to generate a basic product simplifies activity detection using pH indicators such as phenol red. The first phenol red-based (pH-based) high-throughput screening (HTS) assay for small molecule inhibitors of H. pylori urease has been developed and optimized. The phenol-red based assay, which allows for rapid screening at room temperature, has exhibited high quality and reproducibility, with an OD Z’- factor = 0.6109 ± 0.2123. The assay has been utilized to screen upwards of 200,000 compounds against recombinant H. pylori urease, with 255 primary hits (0.13%), 61 reconfirmed hits, and 26 valid inhibitors. Additionally several novel compounds that exhibit low and submicromolar inhibition against H. pylori urease have been identified. Further characterization of these inhibitors is ongoing, including co-crystal structures of the inhibitors with H. pylori urease. Overall, the phenol red assay unlocks opportunities to discover and develop highly effective, direct treatment against H. pylori urease that will eradicate the chronic bacterial infection in humans.

      PART II

      Cytochrome P450 represents a superfamily of heme-containing oxidase enzymes responsible for transmuting xenobiotics into more water-soluble compounds. Amongst the cytochrome P450 isoforms, CYP3A4 is the most profuse, and one of the most clinically relevant xenobiotic metabolizing enzymes in humans. Moreover, CYP3A4 is the most chemically versatile P450, with the ability to oxidize a variety of molecules ranging in size and chemical structure. In addition to drugs, CYP3A4 is involved in the transformation of toxins, environmental pollutants, and carcinogens. Variations in expression and activity of CYP3A4 can greatly affect drug efficacy or toxicity. Additionally, various synthetic or naturally derived agents such as furanocoumarin derivatives can either enhance or inhibit CYP3A4 activity. Although inhibition of CYP3A4 can result in undesirable drug toxicity and/or drug-drug interactions, carefully controlled inhibition can be exploited to increase the bioavailability of drugs that would otherwise be rapidly metabolized. Controlled inhibition is already being exploited in HIV and HCV combination treatment with ritonavir. Initially designed as an HIV protease inhibitor, ritonavir was later found to be an effective CYP3A4 inhibitor. However, neither ritonavir, nor its derivatives were developed using pharmacophore models, rational design, or CYP3A4 crystal structures. Therefore, little is understood on the mechanism of inhibition. Using the ritonavir-CYP3A4 crystal structure as a guide, a preliminary pharmacophore model of CYP3A4 inhibition was proposed. Through scaffold modification and rational design, the requirements for successful CYP3A4 inhibition are now more clearly understood. To date, several modest, yet highly potent CYP3A4 inhibitors have been developed. Ultimately, these rationally designed pharmacoenhancers may assist in more effective combination treatment and lower drug costs.

        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2020)
        Identification of structural determinants required for potent inhibition of drug-metabolizing cytochrome P450 3A4 (CYP3A4) could help develop safer drugs and more effective pharmacoenhancers. We utilize a rational inhibitor design to decipher structure-activity relationships in analogues of ritonavir, a highly potent CYP3A4 inhibitor marketed as pharmacoenhancer. Analysis of compounds with the R1 side-group as phenyl or naphthalene and R2 as indole or naphthalene in different stereo configuration showed that (i) analogues with the R2-naphthalene tend to bind tighter and inhibit CYP3A4 more potently than the R2-phenyl/indole containing counterparts; (ii) stereochemistry becomes a more important contributing factor, as the bulky side-groups limit the ability to optimize protein-ligand interactions; (iii) the relationship between the R1/R2 configuration and preferential binding to CYP3A4 is complex and depends on the side-group functionality/interplay and backbone spacing; and (iv) three inhibitors, 5a-b and 7d, were superior to ritonavir (IC50 of 0.055-0.085 μM vs. 0.130 μM, respectively).
          Cover page: An increase in side-group hydrophobicity largely improves the potency of ritonavir-like inhibitors of CYP3A4
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2021)
          Inhibition of the major human drug-metabolizing cytochrome P450 3A4 (CYP3A4) by pharmaceuticals and other xenobiotics could lead to toxicity, drug-drug interactions and other adverse effects, as well as pharmacoenhancement. Despite serious clinical implications, the structural basis and attributes required for the potent inhibition of CYP3A4 remain to be established. We utilized a rational inhibitor design to investigate the structure-activity relationships in the analogues of ritonavir, the most potent CYP3A4 inhibitor in clinical use. This study elucidated the optimal length of the head-group spacer using eleven (series V) analogues with the R1/R2 side-groups as phenyls or R1-phenyl/R2-indole/naphthalene in various stereo configurations. Spectral, functional and structural characterization of the inhibitory complexes showed that a one-atom head-group linker elongation, from pyridyl-ethyl to pyridyl-propyl, was beneficial and markedly improved Ks, IC50 and thermostability of CYP3A4. In contrast, a two-atom linker extension led to a multi-fold decrease in the binding and inhibitory strength, possibly due to spatial and/or conformational constraints. The lead compound, 3h, was among the best inhibitors designed so far and overall, the strongest binder (Ks and IC50 of 0.007 and 0.090 µM, respectively). 3h was the fourth structurally simpler inhibitor superior to ritonavir, which further demonstrates the power of our approach.
            Cover page: Rational Design of CYP3A4 Inhibitors: A One-Atom Linker Elongation in Ritonavir-Like Compounds Leads to a Marked Improvement in the Binding Strength
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2022)
            Controlled inhibition of drug-metabolizing cytochrome P450 3A4 (CYP3A4) is utilized to boost bioavailability of anti-viral and immunosuppressant pharmaceuticals. We investigate structure-activity relationships (SARs) in analogues of ritonavir, a potent CYP3A4 inhibitor marketed as pharmacoenhancer, to determine structural elements required for potent inhibition and whether the inhibitory potency can be further improved via a rational structure-based design. This study investigated eight (series VI) inhibitors differing in head- and end-moieties and their respective linkers. SAR analysis revealed the multifactorial regulation of inhibitory strength, with steric constraints imposed on the tethered heme-ligating moiety being a key factor. Minimization of these constraints by changing the linkers' length/flexibility and N-heteroatom position strengthened heme coordination and markedly improved binding and/or inhibitory strength. Impact of the end-pyridine attachment was not uniform due to influence of other determinants controlling the ligand-binding mode. This interplay between pharmacophoric determinants and the end-group enlargement can be used for further inhibitor optimization.
              Cover page: Interaction of CYP3A4 with Rationally Designed Ritonavir Analogues: Impact of Steric Constraints Imposed on the Heme-Ligating Group and the End-Pyridine Attachment
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2021)
              We report the synthesis and photochemical and biological characterization of the first selective and potent metal-based inhibitors of cytochrome P450 3A4 (CYP3A4), the major human drug metabolizing enzyme. Five Ru(II)-based derivatives were prepared from two analogs of the CYP3A4 inhibitor ritonavir, 4 and 6: [Ru(tpy)(L)(6)]Cl2 (tpy = 2,2:6,2″-terpyridine) with L = 6,6-dimethyl-2,2-bipyridine (Me2bpy; 8), dimethylbenzo[i]dipyrido[3,2-a:2,3-c]phenazine (Me2dppn; 10) and 3,6-dimethyl-10,15-diphenylbenzo[i]dipyrido[3,2-a:2,3-c]phenazine (Me2Ph2dppn; 11), [Ru(tpy)(Me2bpy)(4)]Cl2 (7) and [Ru(tpy)(Me2dppn)(4)]Cl2 (9). Photochemical release of 4 or 6 from 7-11 was demonstrated, and the spectrophotometric evaluation of 7 showed that it behaves similarly to free 4 (type II heme ligation) after irradiation with visible light but not in the dark. Unexpectedly, the intact Ru(II) complexes 7 and 8 were found to inhibit CYP3A4 potently and specifically through direct binding to the active site without heme ligation. Caged inhibitors 9-11 showed dual action properties by combining photoactivated dissociation of 4 or 6 with efficient 1O2 production. In prostate adenocarcinoma DU-145 cells, compound 9 had the best synergistic effect with vinblastine, the anticancer drug primarily metabolized by CYP3A4 in vivo. Thus, our study establishes a new paradigm in CYP inhibition using metalated complexes and suggests possible utilization of photoactive CYP3A4 inhibitory compounds in clinical applications, such as enhancement of therapeutic efficacy of anticancer drugs.
                Cover page: Photosensitive Ru(II) Complexes as Inhibitors of the Major Human Drug Metabolizing Enzyme CYP3A4.
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