Visceral leishmaniasis is responsible for up to 30,000 deaths every year. Current treatments have shortcomings that include toxicity and variable efficacy across endemic regions. Previously, we reported the discovery of GNF6702, a selective inhibitor of the kinetoplastid proteasome, which cleared parasites in murine models of leishmaniasis, Chagas disease, and human African trypanosomiasis. Here, we describe the discovery and characterization of LXE408, a structurally related kinetoplastid-selective proteasome inhibitor currently in Phase 1 human clinical trials. Furthermore, we present high-resolution cryo-EM structures of the Leishmania tarentolae proteasome in complex with LXE408, which provides a compelling explanation for the noncompetitive mode of binding of this novel class of inhibitors of the kinetoplastid proteasome.

Biocatalysis is an effective tool to access chiral molecules that are otherwise hard to synthesize or purify. Time-efficient processes are needed to develop enzymes that adequately perform the desired chemistry. We evaluated machine-directed evolution as an enzyme engineering strategy using a moderately stereoselective imine reductase as the model system. We compared machine-directed evolution approaches to deep mutational scanning (DMS) and error-prone PCR. Within one cycle, it was found that machine-directed evolution yielded a library of high-activity mutants with a dramatically shifted activity distribution compared to that of traditional directed evolution. Structure-guided analysis revealed that linear additivity might provide a simple explanation for the effectiveness of machine-directed evolution. The most active and selective enzyme mutant, which was identified through DMS and error-prone PCR, was used for the gram-scale synthesis of the H4 receptor antagonist ZPL389 with full conversion, > 99% ee (R), and a 72% yield.

Cytochrome P450 mono-oxygenases (P450) are versatile enzymes which play essential roles in C-source assimilation, secondary metabolism, and in degradations of endo- and exogenous xenobiotics. In humans, several P450 isoforms constitute the largest part of phase I metabolizing enzymes and catalyze oxidation reactions which convert lipophilic xenobiotics, including drugs, to more water soluble species. Recombinant human P450s and microorganisms are applied in the pharmaceutical industry for the synthesis of drug metabolites for pharmacokinetics and toxicity studies. Compared to the membrane-bound eukaryotic P450s, prokaryotic ones exhibit some advantageous features, such as high stability and generally easier heterologous expression. Here, we describe a novel P450 from Streptomyces platensis DSM 40041 classified as CYP107L that efficiently converts several commercial drugs of various size and properties. This P450 was identified by screening of actinobacterial strains for amodiaquine and ritonavir metabolizing activities, followed by genome sequencing and expression of the annotated S. platensis P450s in Escherichia coli. Performance of CYP107L in biotransformations of amodiaquine, ritonavir, amitriptyline, and thioridazine resembles activities of the main human metabolizing P450s, namely CYPs 3A4, 2C8, 2C19, and 2D6. For application in the pharmaceutical industry, an E. coli whole-cell biocatalyst expressing CYP107L was developed and evaluated for preparative amodiaquine metabolite production.

Supplemental informationSynthesis of M27.5: In a 400 mL polyethylene cup the reaction buffer [30 mL, 50 mM uridine 5'-diphosphoglucuronic acid trisodium salt, 25 mM magnesium cloride, 250 mM 2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethansulfonic acid, aq. pH 7.5] was mixed with bovine liver S9 preparation (15 mL; the liver S9 fractions were prepared as described in Kittellmann M. et al 2003). Substrate solution (4 mL, 55 mM asciminib in DMSO) was added. The reaction was incubated on an orbital shaker at 37°C and 230 rpm for 16 hours. The reaction was mixed with two volume equivalents of acetonitrile:methanol mixture (50:50) and stirred at room temperature for 15 minutes. The broth was centrifuged at 8600 g for 40 min and the supernatant was filtered through a paper filter. The filtrate was mixed with 900 mL of aqueous trifluoroacetic acid 0.05 % and pumped directly on the RP18 chromatography column. The conditions for preparative HPLC were: 250 x 21 mm Nucloeodur 100-10 C18 ec column (Macherey-Nagel, Düren, Germany); solvent A: aqueous trifluoroacetic acid 0.05 %; solvent B: acetonitrile; gradient: 0 -5 min 10 % B, 40 min 95 % B, flow rate of 40 mL/min; room temperature; detection at 220 nm; fraction size 40 mL. The product containing fractions were again combined, concentrated to about 50 mL and dried by lyophilization overnight. M27.5, 65 mg (40%), was obtained with > 97 % purity (HPLC/full DAD) as a trifluoroacetic acid salt and analyzed by NMR spectroscopy.

Fevipiprant is a novel oral prostaglandin D receptor 2 (DP; also known as CRTh2) antagonist, which is currently in development for the treatment of severe asthma and atopic dermatitis. We investigated the absorption, distribution, metabolism, and excretion properties of fevipiprant in healthy subjects after a single 200-mg oral dose of [C]-radiolabeled fevipiprant. Fevipiprant and metabolites were analyzed by liquid chromatography coupled to tandem mass spectrometry and radioactivity measurements, and mechanistic in vitro studies were performed to investigate clearance pathways and covalent plasma protein binding. Biotransformation of fevipiprant involved predominantly an inactive acyl glucuronide (AG) metabolite, which was detected in plasma and excreta, representing 28% of excreted drug-related material. The AG metabolite was found to covalently bind to human plasma proteins, likely albumin; however, in vitro covalent binding to liver protein was negligible. Excretion was predominantly as unchanged fevipiprant in urine and feces, indicating clearance by renal and possibly biliary excretion. Fevipiprant was found to be a substrate of transporters organic anion transporter 3 (OAT3; renal uptake), multidrug resistance gene 1 (MDR1; possible biliary excretion), and organic anion-transporting polypeptide 1B3 (OATP1B3; hepatic uptake). Elimination of fevipiprant occurs via glucuronidation by several uridine 5'-diphospho glucuronosyltransferase (UGT) enzymes as well as direct excretion. These parallel elimination pathways result in a low risk of major drug-drug interactions or pharmacogenetic/ethnic variability for this compound.

BackgroundGetting access to authentic human drug metabolites is an important issue during the drug discovery and development process. Employing recombinant microorganisms as whole-cell biocatalysts constitutes an elegant alternative to organic synthesis to produce these compounds. The present work aimed for the generation of an efficient whole-cell catalyst based on the flavin monooxygenase isoform 2 (FMO2), which is part of the human phase I metabolism.ResultsWe show for the first time the functional expression of human FMO2 in E. coli. Truncations of the C-terminal membrane anchor region did not result in soluble FMO2 protein, but had a significant effect on levels of recombinant protein. The FMO2 biocatalysts were employed for substrate screening purposes, revealing trifluoperazine and propranolol as FMO2 substrates. Biomass cultivation on the 100 L scale afforded active catalyst for biotransformations on preparative scale. The whole-cell conversion of trifluoperazine resulted in perfectly selective oxidation to 48 mg (46% yield) of the corresponding N1-oxide with a purity >98%.ConclusionsThe generated FMO2 whole-cell catalysts are not only useful as screening tool for human metabolites of drug molecules but more importantly also for their chemo- and regioselective preparation on the multi-milligram scale.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0262-0) contains supplementary material, which is available to authorized users.

KAE609 [(19R,39S)-5,79-dichloro-69-fluoro-39-methyl-29,39,49,99-tetrahydrospiro[indoline-3,19-pyridol[3,4-b]indol]-2-one] is a potent, fast-acting, schizonticidal agent being developed for the treatment of malaria. After oral dosing of KAE609 to rats and dogs, the major radioactive component in plasma was KAE609. An oxidative metabolite, M18, was the prominent metabolite in rat and dog plasma. KAE609 was well absorbed and extensively metabolized such that low levels of parent compound (£11% of the dose) were detected in feces. The elimination of KAE609 and metabolites was primarily mediated via biliary pathways ( ‡93% of the dose) in the feces of rats and dogs. M37 and M23 were the major metabolites in rat and dog feces, respectively. Among the prominent metabolites of KAE609, the isobaric chemical species, M37, was observed, suggesting the involvement of an isomerization or rearrangement during biotransformation. Subsequent structural elucidation of M37 revealed that KAE609, a spiroindolone, undergoes an unusual C-C bond cleavage, followed by a 1,2-acyl shift to form a ring expansion metabolite M37. The in vitro metabolism of KAE609 in hepatocytes was investigated to understand this novel biotransformation. The metabolism of KAE609 was qualitatively similar across the species studied; thus, further investigation was conducted using human recombinant cytochrome P450 enzymes. The ring expansion reaction was found to be primarily catalyzed by cytochrome P450 (CYP) 3A4 yielding M37. M37 was subsequently oxidized to M18 by CYP3A4 and hydroxylated to M23 primarily by CYP1A2. Interestingly, M37 was colorless, whereas M18 and M23 showed orange yellow color. The source of the color of M18 and M23 was attributed to their extended conjugated system of double bonds in the structures.

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