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Remdesivir (RDV; GS-5734, Veklury), the first FDA-approved antiviral to treat COVID-19, is a single-diastereomer monophosphoramidate prodrug of an adenosine analogue. RDV is taken up in the target cells and metabolized in multiple steps to form the active nucleoside triphosphate (TP) (GS-443902), which, in turn, acts as a potent and selective inhibitor of multiple viral RNA polymerases. In this report, we profiled the key enzymes involved in the RDV metabolic pathway with multiple parallel approaches: (i) bioinformatic analysis of nucleoside/nucleotide metabolic enzyme mRNA expression using public human tissue and lung single-cell bulk mRNA sequence (RNA-seq) data sets, (ii) protein and mRNA quantification of enzymes in human lung tissue and primary lung cells, (iii) biochemical studies on the catalytic rate of key enzymes, (iv) effects of specific enzyme inhibitors on the GS-443902 formation, and (v) the effects of these inhibitors on RDV antiviral activity against SARS-CoV-2 in cell culture. Our data collectively demonstrated that carboxylesterase 1 (CES1) and cathepsin A (CatA) are enzymes involved in hydrolyzing RDV to its alanine intermediate MetX, which is further hydrolyzed to the monophosphate form by histidine triad nucleotide-binding protein 1 (HINT1). The monophosphate is then consecutively phosphorylated to diphosphate and triphosphate by cellular phosphotransferases. Our data support the hypothesis that the unique properties of RDV prodrug not only allow lung-specific accumulation critical for the treatment of respiratory viral infection such as COVID-19 but also enable efficient intracellular metabolism of RDV and its MetX to monophosphate and successive phosphorylation to form the active TP in disease-relevant cells.

Momelotinib (MMB), a small-molecule inhibitor of Janus kinase (JAK)1/2 and of activin A receptor type 1 (ACVR1), is in clinical development for the treatment of myeloproliferative neoplasms. The pharmacokinetics and disposition of [14C]MMB were characterized in a single-dose, human mass-balance study. Metabolism and the pharmacologic activity of key metabolites were elucidated in multiple in vitro and in vivo experiments. MMB was rapidly absorbed following oral dosing with approximately 97% of the radioactivity recovered, primarily in feces with urine as a secondary route. Mean blood-to-plasma [14C] area under the plasma concentration-time curve ratio was 0.72, suggesting low association of MMB and metabolites with blood cells. [14C]MMB-derived radioactivity was detectable in blood for ≤48 hours, suggesting no irreversible binding of MMB or its metabolites. The major circulating human metabolite, M21 (a morpholino lactam), is a potent inhibitor of JAK1/2 and ACVR1 in vitro. Estimation of pharmacological activity index suggests M21 contributes significantly to the pharmacological activity of MMB for the inhibition of both JAK1/2 and ACVR1. M21 was observed in disproportionately higher amounts in human plasma than in rat or dog, the rodent and nonrodent species used for the general nonclinical safety assessment of this molecule. This discrepancy was resolved with additional nonclinical studies wherein the circulating metabolites and drug-drug interactions were further characterized. The human metabolism of MMB was mediated primarily by multiple cytochrome P450 enzymes, whereas M21 formation involved initial P450 oxidation of the morpholine ring followed by metabolism via aldehyde oxidase.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in young children. Presatovir (previously GS-5806) is a novel, orally administered RSV fusion inhibitor with a favorable safety profile and proven antiviral efficacy in preclinical and clinical studies. In vitro, presatovir is a substrate of the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) and hepatic uptake transporters organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 and is slowly metabolized by cytochrome P450 (CYP) 3A4 and CYP3A5. This study enrolled 64 healthy subjects to evaluate the effect of cyclosporine, a P-gp, BCRP, and OATP1B1/1B3 inhibitor; rifampin, a strong CYP3A4 and P-gp inducer; efavirenz, a moderate CYP3A4 inducer; and cobicistat, a potent CYP3A inhibitor, on presatovir pharmacokinetics. Presatovir plasma exposures (maximum observed plasma concentration [Cmax ] and area under the plasma concentration-time curve from time 0 extrapolated to infinity [AUCinf ]) were not affected by coadministration of cyclosporine, suggesting presatovir is not a sensitive substrate of P-gp, BCRP, or OATP1B1/1B3. As expected, based on the role of CYP3A in presatovir metabolism, presatovir exposure was increased by cobicistat (122% in AUCinf ), and decreased by rifampin (40.3% in Cmax and 82.5% in AUCinf ) and efavirenz (55.7% in AUCinf ). These data support coadministration of presatovir with inhibitors of P-gp, BCRP, OATP1B1/1B3, or CYP3A, but not with moderate or strong CYP3A4 inducers. Presatovir was well-tolerated with the most common drug-related adverse events of dizziness (n = 12) and somnolence (n = 4) reported during efavirenz treatment.

The HIV protease inhibitor (PI) ritonavir (RTV) has been widely used as a pharmacoenhancer for other PIs, which are substrates of cytochrome P450 3A (CYP3A). However the potent anti-HIV activity of ritonavir may limit its use as a pharmacoenhancer with other classes of anti-HIV agents. Ritonavir is also associated with limitations such as poor physicochemical properties. To address these issues a series of compounds with replacements at the P2 and/or P3 region was designed and evaluated as novel CYP3A inhibitors. Through these efforts, a potent and selective inhibitor of CYP3A, GS-9350 (cobicistat) with improved physiochemical properties was discovered.

Ritonavir (RTV), an HIV-1 protease inhibitor (PI), is also a potent mechanism-based inhibitor of human cytochrome P450 3A (CYP3A) and has been widely prescribed as a pharmacoenhancer. As a boosting agent for marketed PIs, it reduces pill burden, and improves compliance. Removal of the hydroxyl group from RTV reduces, but does not eliminate HIV PI activity and does not affect CYP3A inhibition. Herein we report the discovery of a novel series of CYP3A inhibitors that are devoid of antiviral activity. The synthesis and evaluation of analogs with extensive modifications of the 1,4-diamine core along with the structure activity relationships with respect to anti-HIV activity, CYP3A inhibitory activity, selectivity against other CYP enzymes and the human pregnane X receptor (PXR) will be discussed.

Investigation of thiophene-2-carboxylic acid HCV NS5B site II inhibitors, guided by measurement of cell culture medium binding, revealed the structure–activity relationships for intrinsic cellular potency. The pharmacokinetic profile was enhanced through incorporation of heterocyclic ethers on the N-alkyl substituent. Hydroxyl groups were incorporated to modulate protein binding. Intrinsic potency was further improved through enantiospecific introduction of an olefin in the N-acyl motif, resulting in the discovery of the phase 2 clinical candidate GS-9669. The unexpected activity of this compound against the clinically relevant NS5B M423T mutant, relative to the wild type, was shown to arise from both the N-alkyl substituent and the N-acyl group.

GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC 50 ) of ≤11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4. The M423T mutation is readily generated clinically upon monotherapy with the thumb site II inhibitors filibuvir and lomibuvir, and it is notable that GS-9669 exhibited only a 3-fold loss in potency against this variant in the genotype 1b replicon. Rather than M423T, resistance predominantly tracks to residues R422K and L419M and residue I482L in GT 1b and 1a replicons, respectively. GS-9669 exhibited at least additive activity in combination with agents encompassing four other direct modes of action (NS3 protease, NS5A, NS5B via an alternative allosteric binding site, and NS5B nucleotide) as well as with alpha interferon or ribavirin in replicon assays. It exhibited high metabolic stability in in vitro human liver microsomal assays, which, in combination with its pharmacokinetic profiles in rat, dog, and two monkey species, is predictive of good human pharmacokinetics. GS-9669 is well suited for combination with other orally active, direct-acting antiviral agents in the treatment of genotype 1 chronic HCV infection. (This study has been registered at ClinicalTrials.gov under registration number NCT01431898.)

The activation of heterocyclic amines to mutagenic products by hepatic microsomal fractions from cynomolgus monkey, marmoset monkey and man was compared with the respective levels of cytochrome P450 enzymes CYP1A1 and CYP1A2. The rate of activation of 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to mutagens by hepatic microsomal fraction from cynomolgus monkey was very low. This was associated with a lack of constitutive expression of CYP1A1 and CYP1A2. In contrast, human hepatic microsomal fraction readily activates these heterocyclic amines and this is associated with constitutive expression of CYP1A2. Treatment of cynomolgus monkey with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a very modest induction of CYP1A2, and a small increase in the activation of MeIQx and IQ. However, there was marked induction of CYP1A1 which was accompanied by > 10-fold increases in PhIP activation and 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD) and aryl hydrocarbon hydroxylase activities. Following treatment of cynomolgus monkey with 3-methylcholanthrene, induction of CYP1A1, but not CYP1A2, was evident. In untreated marmoset monkey the activations of MeIQx and PhIP, as well as phenacetin O-deethylase, EROD, MROD and aryl hydrocarbon hydroxylase activities, are similar to those in man, although the activations of IQ and coumarin 7-hydroxylase activity are lower than in man. The presence of constitutive CYP1A2, and the absence of CYP1A1, in the liver of this species correspond to the situation in man. Treatment of marmoset monkey with TCDD results in increased CYP1A2 levels (4-fold), accompanied by proportional increases in the activation of MeIQx and IQ and phenacetin O-deethylase, EROD and MROD activities. The activation of PhIP is increased disproportionately, by 8-fold, most likely due to the activity of CYP1A1 which is also induced by TCDD in this species. Overall, the hepatic metabolism of heterocyclic amines by CYP1A enzymes in the untreated marmoset monkey resembles that in human more closely than that in the cynomolgus monkey. Therefore, marmoset monkey may be a more suitable model than the cynomolgus monkey for carcinogenicity studies involving MeIQx and PhIP, but not IQ.

A novel series of HCV replication inhibitors based on a pyrido[3,2-d]pyrimidine core were optimized for pharmacokinetics (PK) in rats. Several associations between physicochemical properties and PK were identified and exploited to guide the design of compounds. In addition, a simple new metric that may aid in the prediction of bioavailability for compounds with higher polar surface area is described (3*HBD-cLogP).

The liver cytosolic enzyme tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidation of L-tryptophan to formylkynurenine and controls the physiological flux of tryptophan into both the serotonergic and kynureninic pathways. This hemoprotein enzyme is composed of four noncovalently bound subunits of equivalent mass and contains two heme moieties per molecule. Electron paramagnetic resonance analyses have indicated that a histidyl nitrogen is involved in heme ligation [Henry et al., (1976) J. Biol. Chem. 251, 1578], but the identity of the His residue(s) is unknown. In an attempt to characterize the active site of the enzyme we have substituted each of the 12 His residues in the rat TDO subunit with Ala, to determine their relative importance in heme binding. Sequence alignment of the rat liver protein with that of known or putative TDO sequences from other organisms reveals that four of the His residues are conserved in eukaryotes, two of which are also conserved in prokaryotes. Our findings indicate that replacement of the evolutionarily conserved His 76 and 328 residues resulted in a dramatic reduction of TDO activity, whereas that of the eukaryotically conserved His70 resulted in a significant reduction relative to that of the wild-type enzyme. On the other hand, replacement of the other eukaryotically conserved His273 residue, while affecting the relative expression of the enzyme, had little effect on its specific activity. Size-exclusion analyses revealed that the His76Ala and His328Ala mutants retained little or no heme, suggesting that these may be key residues in ligating the prosthetic heme moieties. Whether these His residues are both provided by the same TDO subunit or a different TDO subunit remains to be determined.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant of the heterocyclic aromatic amines formed during the cooking of meat, is genotoxic and carcinogenic in rodents. MeIQx requires metabolic activation by P450 before it can exert these effects. Whilst there is indirect evidence that the mutagenic product is N-hydroxy-MeIQx (-OHMeIQx), we have now identified this unequivocally following incubation of the amine with human hepatic microsomal fraction. A mixture of unlabelled MeIQx, [C-13, N-15(2)]MeIQx and [C-14]MeIQx was used as substrate and the products analysed by HPLC - thermospray mass spectrometry. Characteristic doublet ions, 3 mass units apart, were found at m/z 214/217 ([M + H]+) from the parent compound, MeIQx and at 230/233 ([M + H]+) from N-OHMeIQx. The presence of a doublet ion at m/z 214/217 with the doublet at 230/233 [M + H+] provided additional evidence that this was N-OHMeIQx, as facile loss of 'O' is characteristic of N-hydroxylamines. Further evidence for the identity of the major metabolite, which accounted for approximately 90% of all microsomal metabolism, was obtained by comparing the mutagenicity of the HPLC eluate using Salmonella typhimurium YG1024, which is particularly sensitive to N-hydroxylamines, and TA98/1,8-DNP6 which is resistant to most N-hydroxylamines. Ninety-five per cent of direct-acting mutagenicity present in the reaction mixture was associated with a single peak, which co-eluted with N-OHMeIQx, as indicated by mass spectrometry. In the presence of a metabolic activation system, only one additional mutagenic peak, corresponding to unchanged MeIQx, could be detected. MeIQx (5 muM was N-hydroxylated at a rate of 77 +/- 11 pmol/mg/min (mean +/- SEM, n = 4) by human liver microsomes. The specific inhibitor of human CYP1A2, furafylline (5 muM) inhibited the N-hydroxylation of MeIQx by >90%. These data show that N-OHMeIQx is both the major oxidation product and the major genotoxic product of MeIQx generated by microsomal fractions of human liver and that the reaction is catalysed almost exclusively by CYP1A2.

The monoclonal antibody, 3/4/2, which was raised against purified rat cytochrome P450 isoenzyme 1A1 (CYP1A1) binds to cytochromes P4501A in many species. It was shown by immunoblotting that the antibody binds to CYP1A1 in microsomal fractions prepared from rat, mouse, rabbit, hamster and human. The antibody also binds to cytochrome P450 isoenzyme 1A2 in microsomal fractions prepared from rabbit and human, but not rat or mouse. Using purified isoenzymes in an enzyme-linked immunosorbent assay it was found that the affinity of binding to the two rabbit hydrocarbon-inducible isoenzymes is reduced compared with that for rat CYP1A1. Binding is not affected by denaturation of the antigens. The effects of chemical and enzymatic treatments on rat CYP1A1 showed that the epitope contains a trypsin-sensitive site that includes arginine, but lacks lysine. The epitope does not contain methionine, cysteine, aspartic acid or glutamic acid residues. In addition, digestion of the protein with cyanogen bromide produces a fragment of M r 20,000 which contains the antibody binding site. By comparing the cross-reactivity of the antibody with the primary structures of CYP1A1 and 1A2 from the rat, mouse, rabbit and human, and by considering the results of the chemical and enzymatic treatments, it was possible to deduce the likely location and structure of the binding site of 3/4/2 on members of the CYP1A subfamily. It is concluded that the epitope for this antibody is Phe-Arg-His-Ser-Ser-Phe, which lies at positions 380–385 in rat CYP1A1. Further, it is predicted from a model of the tertiary structure of eukaryotic cytochrome P450 that a part of this binding site lies within a helix in the native protein.

The orientation of eukaryotic cytochromes P450, with respect to the membrane of the endoplasmic reticulum, has been investigated. There is now good evidence that the tertiary structure of these proteins is essentially the same as that of the soluble bacterial isoenzyme cytochrome P450CI, with the exception of an extension at the N-terminus which is thought to form a membrane-anchoring sequence. The remainder of the molecule protrudes from the cytosolic face of the membrane so that it can interact with substrates and electron-donating proteins. Two models based on this structure have been considered, in which the plane of the heme of cytochrome P450 is oriented either parallel with or perpendicular to the plane of the membrane of the endoplasmic reticulum. The validity of these models has been assessed from the results of studies involving the binding of antipeptide antibodies directed toward known regions of cytochromes P450, modeling of the interaction of cytochrome P450 with cytochrome b5, proposed intramolecular movements of cytochrome P450 during its catalytic cycle, and the partitioning of substrates for cytochrome P450 between the cytosol and membrane. It is concluded that cytochrome P450 is most likely oriented such that the heme is not fixed horizontal to the plane of the membrane of the endoplasmic reticulum and may well lie with the heme perpendicular to the membrane.

In all recent surveys of the aetiology of human cancer, environment has been identified as a major factor (Wynder and Gori, 1977; Higginson and Muir, 1979; Doll and Peto, 1981). In most studies, diet has proven to be the single most important determinant, associated with more cancer deaths than even cigarette smoking. Indeed, in some studies as many as 35% of all cancer deaths have been attributed to dietary factors which exclude alcohol and food additives (Doll and Peto, 1981). There are numerous mechanisms whereby diet might influence the incidence of cancer. However, the identification of mutagenic chemicals in food formed during its processing and/or cooking (Sugimura et al., 1977) has focussed considerable attention on the possible role of these compounds in human cancer. Recently, a number of extremely potent mutagens arising from the Maillard reaction, involving sugars, amino acids and creatine, have been isolated from cooked meat. These are all heterocyclic aromatic amines amongst which the most potent and abundant are 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) (Felton et al., 1986).

Many xenobiotics including the pharmacoenhancer cobicistat increase serum creatinine by inhibiting its renal active tubular secretion without affecting the glomerular filtration rate. This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects. The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine. At physiologic creatinine concentrations, the specific activity of OAT2 transport was over twofold higher than OCT2 or OCT3, establishing OAT2 as a likely relevant creatinine transporter and further challenging the traditional view that creatinine is solely transported by a cationic pathway. The apical multidrug and toxin extrusion transporters MATE1 and MATE2-K demonstrated low-affinity and high-capacity transport. All drugs known to affect creatinine inhibited OCT2 and MATE1. Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 μM for creatinine transport. Trimethoprim potently inhibited MATE2-K, whereas dolutegravir preferentially inhibited OCT2. Cimetidine was unique, inhibiting all transporters that interact with creatinine. Thus, the clinical observation of elevated serum creatinine in patients taking cobicistat is likely a result of OCT2 transport, facilitating intracellular accumulation, and MATE1 inhibition.

1. Furafylline (1,8-dimethyl-3-(2'-furfuryl)methylxanthine) is a methylxanthine derivative that was introduced as a long-acting replacement for theophylline in the treatment of asthma. Administration of furafylline was associated with an elevation in plasma levels of caffeine, due to inhibition of caffeine oxidation, a reaction catalysed by one or more hydrocarbon-inducible isoenzymes of P450. We have now investigated the selectivity of inhibition of human monooxygenase activities by furafylline. 2. Furafylline was a potent, non-competitive inhibitor of high affinity phenacetin O-deethylase activity of microsomal fractions of human liver, a reaction catalysed by P450IA2, with an IC50 value of 0.07 microM. 3. Furafylline had either very little or no effect on human monooxygenase activities catalysed by other isoenzymes of P450, including P450IID1, P450IIC, P450IIA. Of particular interest, furafylline did not inhibit P450IA1, assessed from aryl hydrocarbon hydroxylase activity of placental samples from women who smoked cigarettes. 4. It is concluded that furafylline is a highly selective inhibitor of P450IA2 in man. 5. Furafylline was a potent inhibitor of the N3-demethylation of caffeine and of a component of the N1- and N7-demethylation. This confirms earlier suggestions that caffeine is a selective substrate of a hydrocarbon-inducible isoenzyme of P450 in man, and identifies this as P450IA2. Thus, caffeine N3-demethylation should provide a good measure of the activity of P450IA in vivo in man. 6. Although furafylline selectively inhibited P450IA2, relative to P450IA1, in the rat, this was at 1000-times the concentration required to inhibit the human isoenzyme, suggesting a major difference in the active site geometry between the human and the rat orthologues of P50IA2.