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Proteases are an important class of drug targets that continue to drive inhibitor discovery. These enzymes are prone to resistance mutations, yet their promise for treating viral diseases and other disorders continues to grow. This study develops a general approach for detecting microbially synthesized protease inhibitors and uses it to screen terpenoid pathways for inhibitory compounds. The detection scheme relies on a bacterial two-hybrid (B2H) system that links protease inactivation to the transcription of a swappable reporter gene. This system, which can accomodate multiple biochemical outputs (i.e., luminescence and antibiotic resistance), permitted the facile incorporation of four disease-relevant proteases. A B2H designed to detect the inactivation of the main protease of severe acute respiratory syndrome coronavirus 2 enabled the identification of a terpenoid inhibitor of modest potency. An analysis of multiple pathways that make this terpenoid, however, suggested that its production was necessary but not sufficient to confer a survival advantage in growth-coupled assays. This finding highlights an important challenge associated with the use of genetic selection to search for inhibitors─notably, the influence of pathway toxicity─and underlines the value of including multiple pathways with overlapping product profiles in pathway screens. This study provides a detailed experimental framework for using microbes to screen libraries of biosynthetic pathways for targeted protease inhibitors.

Terpenoids, the largest and most structurally diverse group of natural products, include a striking variety of biologically active compounds, from flavors to medicines. Despite their well-documented biochemical versatility, the evolutionary processes that generate new functional terpenoids are poorly understood and difficult to recapitulate in engineered systems. This study uses a synthetic biochemical objective─a transcriptional system that links the inhibition of protein tyrosine phosphatase 1B (PTP1B), a human drug target, to the expression of a gene for antibiotic resistance in Escherichia coli ( E. coli )─to evolve a terpene synthase to produce enzyme inhibitors. Site saturation mutagenesis of poorly conserved residues on γ-humulene synthase (GHS), a promicuous enzyme, yielded mutants that improved fitness (i.e., the antibiotic resistance of E. coli ) by reducing GHS toxicity and/or by increasing inhibitor production. Intriguingly, a combination of two mutations enhanced the titer of a minority product─a terpene alcohol that inhibits PTP1B─by over 50-fold, and a comparison of similar mutants enabled the identification of a site where mutations permit efficient hydroxylation. Findings suggest that the plasticity of terpene synthases enables an efficient sampling of structurally distinct starting points for building new functional molecules and provide an experimental framework for exploiting this plasticity in activity-guided screens.

Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling. The lead chimeric mAb, 21B3, had high affinity and specificity for both human and mouse sFlt-1 and inhibited VEGF binding to sFlt-1 in a competitive manner. Proof-of-concept studies in the mdx mouse model of Duchenne muscular dystrophy showed that intravenous administration of 21B3 led to elevated VEGF levels, increased vascularization and blood flow to muscles, and decreased fibrosis after 6-12 weeks of treatment. Greater muscle strength was also observed after 4 weeks of treatment. A humanized form of the mAb, 27H6, was engineered and demonstrated a comparable pharmacologic effect. Overall, administration of anti-Flt-1 mAbs in mdx mice inhibited the VEGF:Flt-1 interaction, promoted angiogenesis, and improved muscle function. These studies suggest a potential therapeutic benefit of Flt-1 inhibition for patients with Duchenne muscular dystrophy., Competing Interests: J.B., Z.Z., N.L., B.K.M., S. Gu, D.M.L., Y.H., E.B., M.M., N.C., and H.S.D. are full-time employees of Shire Human Genetic Therapies, a Takeda company, and own stock/options in Takeda. S.J., M.J.T., and D.K. were employees of Shire Human Genetic Therapies, a Takeda company, at the time of these studies. S. Gabriëls, N.D.J., C.B., and H.d.H. are full-time employees of and own stock/options in argenx. A.A. received research support from Shire Human Genetic Therapies. M.V. declares no competing interests., (© 2021 Shire Human Genetic Therapies.)

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b 5 enhanced the k cat for p-nitroanisole dealkylation approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min -1 ) with little effect on K M (13 ± 1 vs 10 ± 1 μM). Inhibition studies and difference spectroscopy revealed that the organochlorine compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding spectra with spectral dissociation constants in the micromolar range suggesting that they may be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate, diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral assays, with a Kd value of 23 ± 3 μM CYP6G3 metabolised diazinon to the diazoxon and hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and neonicotinoid insecticides in other species., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Development of biopharmaceutical production cell lines requires efficient screening methods to select the host cell line and final production clone. This is often complicated by an incomplete understanding of the relationship between protein heterogeneity and function at early stages of product development. LC-MS/MS peptide mapping is well suited to the discovery and quantitation of protein heterogeneity; however, the intense hands-on time required to generate and analyze LC-MS/MS data typically accommodates only smaller sample sets at later stages of clone selection. Here we describe a simple approach to peptide mapping designed for large sample sets that includes higher-throughput sample preparation and automated data analysis. This approach allows for the inclusion of orthogonal protease digestions and multiple replicates of an assay control that encode an assessment of accuracy and precision into the data, significantly simplifying the identification of true-positive annotations in the LC-MS/MS results. This methodology was used to comprehensively identify and quantify glycosylation, degradation, unexpected post-translational modifications, and three types of sequence variants in a previously uncharacterized non-mAb protein therapeutic expressed in approximately 100 clones from three host cell lines. Several product quality risks were identified allowing for a more informed selection of the production clone. Moreover, the variability inherent in this unique sample set provides important structure/function information to support quality attribute identification and criticality assessments, two key components of Quality by Design.

Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the diverse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D. melanogaster and re-engineered for heterologous expression in Escherichia coli. Approximately 460 nmol L⁻¹ of P450 holoenzyme were obtained in 500 mL cultures. The recombinant enzyme was located predominantly within the bacterial cytosol. A two-step purification protocol using Ni-chelate affinity chromatography followed by removal of detergent on a hydroxyapatite column produced essentially homogenous enzyme from both soluble and membrane fractions. Recombinant CYP6G1 exhibited p-nitroanisole O-dealkylation activity but was not active against eleven other typical P450 marker substrates. Substrate-induced binding spectra and IC₅₀ values for inhibition of p-nitroanisole O-dealkylation were obtained for a wide selection of pesticides, namely DDT, imidacloprid, chlorfenvinphos, malathion, endosulfan, dieldrin, dicyclanil, lufenuron and carbaryl, supporting previous in vivo and in vitro studies on Drosophila that have suggested that the enzyme is involved in multi-pesticide resistance in insects., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Metabolic stability measurements are a critical component of preclinical drug development. Available measurement strategies rely on chromatography and mass spectrometry, which are expensive and labor intensive. We have developed a general method to determine the metabolic stability of virtually any compound by quantifying cofactors in the mechanism of cytochrome P450 enzymes using fluorescence intensity measurements. While many previous studies have shown that simple measurements of cofactor depletion do not correlate with substrate conversion (i.e., metabolic stability) in P450 systems, the present work employs a reaction engineering approach to simplify the overall rate equation, thus allowing the accurate and quantitative determination of substrate depletion from simultaneous measurements of NADPH and oxygen depletion. This method combines the accuracy and generality of chromatography with the ease, throughput, and real-time capabilities of fluorescence.

The cytochrome P450 (CYP) reaction mechanism often yields a broad array of coupled and uncoupled products from a single substrate. While it is well known that reaction conditions can drastically affect the rate of P450 catalysis, their effects on regioselectivity and coupling are not well characterized. To investigate such effects, the CYP1A2 oxidation of 7-ethoxymethoxy-3-cyanocoumarin (EOMCC) was examined as a function of buffer type, buffer concentration, pH, and temperature. A high-throughput, optical method was developed to simultaneously measure the rate of substrate depletion, NADPH depletion, and generation of the O-dealkylated product. Increasing the phosphate buffer concentration and temperature increased both the NADPH and EOMCC depletion rates by 6-fold, whereas coupling was constant at 7.9% and the regioselectivity of O-dealkylation to other coupled pathways was constant at 21.7%. Varying the buffer type and pH increased NADPH depletion by 2.5-fold and EOMCC depletion by 3.5-fold; however, neither coupling nor regioselectivity was constant, with variations of 14.4% and 21.6%, respectively. Because the enzyme-substrate binding interaction is a primary determinant of both coupling and regioselectivity, it is reasonable to conclude that ionic strength, as varied by the buffer concentration, and temperature alter the rate without affecting binding while buffer type and pH alter both., (Copyright © 2010 Elsevier Inc. All rights reserved.)