International Journal of Molecular Sciences Article Rational Design of CYP3A4 Inhibitors: A One-Atom Linker Elongation in Ritonavir-Like Compounds Leads to a Marked Improvement in the Binding Strength Eric R. Samuels 1 and Irina F. Sevrioukova 2,* 1 Department of Pharmaceutical Sciences, University of California, Irvine, CA 92697-3900, USA; [email protected] 2 Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA * Correspondence: [email protected]; Tel.: +1-949-824-1953 Abstract: 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 K , s 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 Citation: Samuels, E.R.; constraints. The lead compound, 3h, was among the best inhibitors designed so far and overall, the Sevrioukova, I.F. Rational Design of CYP3A4 Inhibitors: A One-Atom strongest binder (Ks and IC50 of 0.007 and 0.090 µM, respectively). 3h was the fourth structurally Linker Elongation in Ritonavir-Like simpler inhibitor superior to ritonavir, which further demonstrates the power of our approach. Compounds Leads to a Marked Improvement in the Binding Strength. Keywords: CYP3A4; ligand binding; inhibitor design; crystal structure; structure–activity relations; Int. J. Mol. Sci. 2021, 22, 852. surface mutation https://doi.org/10.3390/ijms22020852 Academic Editor: Patrick M. Dansette Received: 15 December 2020 1. Introduction Accepted: 12 January 2021 Cytochrome P450 (CYP) enzymes are monooxygenases that mediate xenobiotic metabolism Published: 16 January 2021 and the synthesis of steroids, vitamins and fatty acids [1,2]. In humans, CYP3A4 is the major liver and intestinal P450 isoform with a large and malleable active site which can Publisher’s Note: MDPI stays neutral accommodate chemically diverse compounds. As a result, CYP3A4 clears the majority of with regard to jurisdictional claims in administered drugs along with other foreign compounds, such as environmental pollu- published maps and institutional affil- tants, insecticides and pesticides [3–5]. Drugs may serve not only as substrates but also iations. as inducers and inhibitors of CYP3A4 [6]. The inhibition of CYP3A4 is usually undesired, because it could lead to drug toxicity, drug–drug interactions and other serious adverse effects. However, when carefully controlled, CYP3A4 inhibition can improve therapeutic efficacy of drugs by slowing down their conversion to more water-soluble, readily excreted Copyright: © 2021 by the authors. metabolites. This beneficial pharmacoenhancing (boosting) effect is currently used for Licensee MDPI, Basel, Switzerland. the treatment of HIV infection, where potent CYP3A4 inhibitors, ritonavir (Figure1A) This article is an open access article and its derivative cobicistat, are co-administered with anti-HIV drugs that otherwise are distributed under the terms and quickly metabolized by CYP3A4 [7,8]. The discovery and utilization of ritonavir as a conditions of the Creative Commons pharmacoenhancer for HIV-protease inhibitors was a paradigm shift in the treatment of Attribution (CC BY) license (https:// HIV and helped transform the deadly infection into a chronic and manageable disease [9]. creativecommons.org/licenses/by/ 4.0/). Int. J. Mol. Sci. 2021, 22, 852. https://doi.org/10.3390/ijms22020852 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 23 Int. J. Mol. Sci. 2021, 22, 852 paradigm shift in the treatment of HIV and helped transform the deadly2 of 22 infection into a chronic and manageable disease [9]. FigureFigure 1. Chemical1. Chemical structures structures of ritonavir of ritonavir (A), a highly (A), potent a highly CYP3A4 potent inhibitor CYP3A4 in clinical inhibitor use, and in clinical use, and thethe best best two two rationally rationally designed designed inhibitors inhibitors from series from IV [10 series](B,C ).IV The [10] R1 and(B,C R2).side-groups The R1 and are R2 side-groups are indicated.indicated. If the If centralthe central hydroxyl hydroxyl group of group ritonavir of is ritonavir removed, the is phenylremoved, side-groups the phenyl would side-groups be in would be R/Rin R/R configuration. configuration. Other areas that could benefit from in-depth inhibitory studies on CYP3A4 are the treatmentOther of chronic areas hepatitisthat could C virus benefit (HCV) from infection in-depth and the inhibitory development studies of CYP3A4- on CYP3A4 are the specifictreatment index inhibitors.of chronic HCV hepatitis infection isC a worldwidevirus (HCV) health infection issue with noand effective the ordevelopment of wellCYP3A4-specific tolerated treatments index available inhibitors. to date. ThroughHCV infection CYP3A4 inhibition,is a worldwide ritonavir health is able to issue with no ef- diminish the hepatic metabolism of co-administered anti-HCV drugs, enhance response rates,fective alleviate or well a pill tolerated burden, and treatments provide an available additional to barrier date. to Through the development CYP3A4 of inhibition, ri- resistance-associatedtonavir is able to variants diminish [11]. the The hepatic antifungal meta agentbolism ketoconazole, of co-administered on the other hand, anti-HCV drugs, wasenhance widely response used as an rates, index inhibitoralleviate of a CYP3A pill burden, enzymes and in in provide vivo and anin vitroadditionaldrug– barrier to the drugdevelopment interaction studies of resistance-associated but, due to high hepatotoxicity, variants its usage[11]. isThe now antifungal limited [12], agent with ketoconazole, ritonavir recommended as the best replacement [13]. on the other hand, was widely used as an index inhibitor of CYP3A enzymes in in vivo However, two major issues associated with ritonavir are off-target activities and poorand physicochemical in vitro drug–drug properties interaction [14]. Even thoughstudies cobicistat but, due has to higher high solubility, hepatotoxicity, better its usage is tolerabilitynow limited and fewer[12], with side effectsritonavir [8,15 ],recomm it inhibitsended CYP3A4 as the 2-fold best weaker replacement and has sim- [13]. ilar reactivityHowever, toward two other major drug-metabolizing issues associated CYPs [16with]. Thus, ritonavir the inhibitory are off-target studies activities and onpoor CYP3A4 physicochemical could help develop properties safer and [14]. more Even potent though pharmacoenhancers cobicistat has and higher index solubility, better inhibitors for in vivo and in vitro studies. Importantly, both ritonavir and cobicistat are chemicallytolerability complex and fewer molecules sidethat effects were [8,15], not developed it inhibits based CYP3A4 on the 2-fold 3D structure weaker of and has similar CYP3A4.reactivity Moreover, toward ritonavir other was drug-metabolizing originally designed asCY anPs HIV [16]. protease Thus, inhibitor, the inhibitory whose studies on abilityCYP3A4 to potently could inhibit help CYP3A4develop was safer purely and coincidental more potent [7]. Therefore, pharmacoenhancers we undertook a and index in- serieshibitors of studies for in to investigatevivo and whatin vitro structural studies. determinants Importantly, are needed both for ritonavir potent inhibi- and cobicistat are tion and whether the inhibitory potency of ritonavir, the most potent CYP3A4 inhibitor inchemically clinical use, complex could be further molecules improved that via were rational not structure-based developed based inhibitor on design. the 3D structure of DetailedCYP3A4. investigations Moreover, on theritonavir structure–activity was originally relationship designed (SAR) of ritonaviras an HIV analogues protease inhibitor, andwhose their ability interaction to withpotently CYP3A4 inhibit are also CYP3A4 important was from purely the basic coincidental science perspective, [7]. Therefore, we un- asdertook they could a clarifyseries the of inhibitory/ligand studies to investigate binding mechanism what structural and provide determinants deeper insight are needed for intopotent the plasticity inhibition and adaptabilityand whether of CYP3A4. the inhibitory potency of ritonavir, the most potent Our earlier studies on the analogues of desoxyritonavir (provided by Gilead Sci- ences)CYP3A4 demonstrated inhibitor that in clinical the association use, could of ritonavir-like be further molecules improved is driven via rational by heme structure-based coordinationinhibitor design. [17], identified Detailed pyridine investigations as the strongest on heme-ligatingthe structure–activity group [18] and relationship laid