Metabolism of new drug modalities research advances - 2023 year in review.

With contributions from colleagues across academia and industry, we have put together the annual reviews of research advances on drug biotransformation and bioactivation since 2016 led by Cyrus Khojasteh. While traditional small molecules and biologics are still predominant in drug discovery, we start to notice a paradigm shift toward new drug modalities (NDMs) including but not limited to peptide and oligonucleotide therapeutics, protein degraders (heterobifunctional degraders and molecule glues), conjugated drugs and covalent inhibitors. The readers can learn more on each new drug modality from several recent comprehensive reviews (Blanco et al.

Challenges and Opportunities for In Vitro-In Vivo Extrapolation of Aldehyde Oxidase-Mediated Clearance: Toward a Roadmap for Quantitative Translation.

Underestimation of aldehyde oxidase (AO)-mediated clearance by current in vitro assays leads to uncertainty in human dose projections, thereby reducing the likelihood of success in drug development. In the present study we first evaluated the current drug development practices for AO substrates. Next, the overall predictive performance of in vitro-in vivo extrapolation of unbound hepatic intrinsic clearance (CL) and unbound hepatic intrinsic clearance by AO (CL) was assessed using a comprehensive literature database of in vitro (human cytosol/S9/hepatocytes) and in vivo (intravenous/oral) data collated for 22 AO substrates (total of 100 datapoints from multiple studies).

Biotransformation research advances - 2022 year in review.

This annual review is the eighth of its kind since 2016 (Baillie et al. 2016, Khojasteh et al. 2017, Khojasteh et al.

GTP1 metabolic stability assessment: A study of the tau PET tracer [F]GTP1.

Tau PET imaging using the tau specific PET tracer [F]GTP1 has been and is part of therapeutic trials in Alzheimer's disease to monitor the accumulation of tau aggregates in the brain. Herein, we examined the metabolic processes of GTP1 and assessed the influence of smoking on its metabolism through in vitro assays. The tracer metabolic profile was assessed by incubating GTP1 with human liver microsomes (HLM) and human hepatocytes.

A one-pot Pd- and P450-catalyzed chemoenzymatic synthesis of a library of oxyfunctionalized biaryl alkanoic acids leveraging a substrate anchoring approach.

A one-pot chemoenzymatic approach was developed by combining Palladium-catalysis with selective cytochrome P450 enzyme oxyfunctionalization. Various iodophenyl alkanoic acids could be coupled with alkylphenyl boronic acids to generate a series of alkyl substituted biarylalkanoic acids in overall high yield. The identity of the products could be confirmed by various analytical and chromatographic techniques.

Promoting P450 BM3 heme domain dimerization with a tris(5-iodoacetamido-1,10-phenanthroline)Ru(II) complex.

Protein dimerization often occurs in many biological systems as to provide structural and functional advantages. A tris(5-iodoacetamido-1,10-phenanthroline)Ruthenium(II) complex was shown to promote the covalent dimerization of a P450 BM3 heme domain mutant containing a surface exposed non-native single cysteine residue. The formation of homodimeric species was confirmed by protein gel electrophoresis, mass spectrometry and UV-Vis spectroscopy.

Ru(II)-diimine complexes and cytochrome P450 working hand-in-hand.

With a growing interest in utilizing visible light to drive biocatalytic processes, several light-harvesting units and approaches have been employed to harness the synthetic potential of heme monooxygenases and carry out selective oxyfunctionalization of a wide range of substrates. While the fields of cytochrome P450 and Ru(II) photochemistry have separately been prolific, it is not until the turn of the 21st century that they converged. Non-covalent and subsequently covalently attached Ru(II) complexes were used to promote rapid intramolecular electron transfer in bacterial P450 enzymes.

Coupling efficiency in light-driven hybrid P450BM3 and CYP119 enzymes.

The light-driven hybrid P450 enzyme approach utilizing the photochemical properties of a covalently attached Ru(II)-diimine photosensitizer was extended to the archaeal Sulfolobus acidocaldarius CYP119 enzyme leading to high photocatalytic activity in the hydroxylation of the chromogenic substrate, 11-nitrophenoxyundecanoic acid. The determined k was greater than those reported with various natural redox partners. In addition, the sacrificial electron donor, diethyldithiocarbamate, used in the photocatalytic reaction is shown to play a dual role.

The hydrodynamic motion of Nanodiscs.

We present a fluorescence-based methodology for monitoring the rotational dynamics of Nanodiscs. Nanodiscs are nano-scale lipid bilayers surrounded by a helical membrane scaffold protein (MSP) that have found considerable use in studying the interactions between membrane proteins and their lipid bilayer environment. Using a long-lifetime Ruthenium label covalently attached to the Nanodiscs, we find that Nanodiscs of increasing diameter, made by varying the number of helical repeats in the MSP, display increasing rotational correlation times.

Cross-linked cytochrome P450 BM3 aggregates promoted by Ru(II)-diimine complexes bearing aldehyde groups.

Cross-linked enzyme aggregate (CLEA) methodology has been applied to immobilize cytochrome P450 BM3 variants (F87A and 21B3) with peroxygenase activity. Several Ru(II)-diimine complexes were found to be suitable cross-linking agents, surpassing the traditional glutaraldehyde and dextran aldehyde. They offer modular numbers of aldehyde functionalities and a more rigid framework than their organic counterparts.

Keeping the spotlight on cytochrome P450.

This review describes the recent advances utilizing photosensitizers and visible light to harness the synthetic potential of P450 enzymes. The structures of the photosensitizers investigated to date are first presented along with their photophysical and redox properties. Functional photosensitizers range from organic and inorganic complexes to nanomaterials as well as the biological photosystem I complex.

Correlating the para-Substituent Effects on Ru(II)-Polypyridine Photophysical Properties and on the Corresponding Hybrid P450 BM3 Enzymes Photocatalytic Activity.

Ru(II)-diimine complexes covalently attached near the heme active site of P450 BM3 enzymes have been used to rapidly inject electrons and drive selective C-H functionalization upon visible light irradiation. Herein, we have generated a series of hybrid P450 BM3 enzymes containing a photosensitizer of general formula [Ru(4,4'-Xbpy)(PhenA)] where X = Cl, H, tBu, Me OPhe, OMe, or NMe, bpy = 2,2'-bipyridine, and PhenA = 5-acetamido-1,10-phenanthroline. We then probed the effect of electron-withdrawing and -donating groups at the para position of the 4,4'-Xbpy ligands on the corresponding hybrid enzymes photocatalytic activity.

Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies.

Background: In order to perform selective CH functionalization upon visible light irradiation, Ru(II)-diimine functionalized P450 heme enzymes have been developed. The sL407C-1 enzyme containing the Ru(bpy)PhenA (bpy=2,2'-bipyridine and PhenA=5-acetamido-1,10-phenanthroline) photosensitizer (1) covalently attached to the non-native single cysteine L407C of the P450BM3 heme domain mutant, displays high photocatalytic activity in the selective CH bond hydroxylation of several substrates.

Methods: A combination of X-ray crystallography, site-directed mutagenesis, transient absorption measurements and enzymatic assays was used to gain insights into its photocatalytic activity and electron transfer pathway.

Chromogenic nitrophenolate-based substrates for light-driven hybrid P450 BM3 enzyme assay.

The incorporation of a p-nitrophenoxy moiety in substrates has enabled the development of colorimetric assays to rapidly screen for O-demethylation activity of P450 enzymes. For the light-driven hybrid P450 BM3 enzymes, where a Ru(II) photosensitizer powers the enzyme upon visible light irradiation, we have investigated a family of p-nitrophenoxy derivatives as useful chromogenic substrates compatible with the light-driven approach. The validation of this assay and its adaptability to a 96-well plate format will enable the screening of the next generation of hybrid P450 BM3 enzymes towards C-H bond functionalization of non-natural substrates.

Ru(II)-diimine functionalized metalloproteins: From electron transfer studies to light-driven biocatalysis.

The unique photochemical properties of Ru(II)-diimine complexes have helped initiate a series of seminal electron transfer studies in metalloenzymes. It has thus been possible to experimentally determine rate constants for long-range electron transfers. These studies have laid the foundation for the investigation of reactive intermediates in heme proteins and for the design of light-activated biocatalysts.

Regio- and stereoselective hydroxylation of 10-undecenoic acid with a light-driven P450 BM3 biocatalyst yielding a valuable synthon for natural product synthesis.

We report herein the selective hydroxylation of 10-undecenoic acid with a light-activated hybrid P450 BM3 enzyme. Under previously developed photocatalytic reaction conditions, only a monohydroxylated product is detected by gas chromatography. Hydroxylation occurs exclusively at the allylic position as confirmed from a synthesized authentic standard.

A facile and versatile methodology for cysteine specific labeling of proteins with octahedral polypyridyl d⁶ metal complexes.

We have synthesized and characterized four octahedral polypyridyl d(6) metal complexes bearing the 5,6-epoxy-5,6-dihydro-[1,10]phenanthroline ligand (L1) as cysteine specific labeling reagents. The proposed synthetic pathways allow the preparation of the metal complexes containing Re(I), Ru(II), Os(II) and Ir(III) while preserving the epoxide functionality. The complexes were characterized by (1)H and (13)C NMR, mass spectrometry, UV-visible and luminescence spectroscopies as well as cyclic voltammetry.

An efficient light-driven P450 BM3 biocatalyst.

P450s are heme thiolate enzymes that catalyze the regio- and stereoselective functionalization of unactivated C-H bonds using molecular dioxygen and two electrons delivered by the reductase. We have developed hybrid P450 BM3 heme domains containing a covalently attached Ru(II) photosensitizer in order to circumvent the dependency on the reductase and perform P450 reactions upon visible light irradiation. A highly active hybrid enzyme with improved stability and a modified Ru(II) photosensitizer is able to catalyze the light-driven hydroxylation of lauric acid with total turnover numbers of 935 and initial reaction rate of 125 mol product/(mol enzyme/min).

Light-triggered modulation of cellular electrical activity by ruthenium diimine nanoswitches.

Ruthenium diimine complexes have previously been used to facilitate light-activated electron transfer in the study of redox metalloproteins. Excitation at 488 nm leads to a photoexcited state, in which the complex can either accept or donate an electron, respectively, in the presence of a soluble sacrificial reductant or oxidant. Here, we describe a novel application of these complexes in mediating light-induced changes in cellular electrical activity.

A series of hybrid P450 BM3 enzymes with different catalytic activity in the light-initiated hydroxylation of lauric acid.

We have developed a series of hybrid P450 BM3 enzymes to perform the light-activated hydroxylation of lauric acid. These enzymes contain a Ru(II)-diimine photosensitizer covalently attached to single cysteine residues of mutant P450 BM3 heme domains. The library of hybrid enzymes includes four non-native single cysteine mutants (K97C, Q397C, Q109C and L407C).

Light-initiated hydroxylation of lauric acid using hybrid P450 BM3 enzymes.

We have developed hybrid P450 BM3 enzymes consisting of a Ru(II)-diimine photosensitizer covalently attached to non-native single cysteine residues of P450 BM3 heme domain mutants. These enzymes are capable, upon light activation, of selectively hydroxylating lauric acid with 40 times higher total turnover numbers compared to the peroxide shunt.