Anti-TNF Thioester Glucocorticoid Antibody-Drug Conjugate Fully Inhibits Inflammation with Minimal Effect on Systemic Corticosterone Levels in a Mouse Arthritis Model.

We describe the discovery of a thioester-containing glucocorticoid receptor modulator (GRM) payload and the corresponding antibody-drug conjugate (ADC). Payload 6 was designed for rapid hepatic inactivation to minimize systemic exposure of nonconjugated GRM. Mouse PK indicated that 6 is cleared 10-fold more rapidly than a first-generation GRM payload, resulting in 10-fold lower exposure and 3-fold decrease in Cmax. The anti-mTNF conjugate ADC5 fully inhibited inflammation in mouse contact hypersensitivity with minimal effects on corticosterone, a biomarker for systemic GRM effects, at doses up to and including 100 mg/kg. Concomitant inhibition of P1NP suggests potential delivery to cells involved in the remodeling of bone, which may be a consequence of TNF-targeting or bystander payload effects. Furthermore, ADC5 fully suppressed inflammation in collagen-induced arthritis mouse model after one 10 mg/kg dose for 21 days. The properties of the anti-hTNF conjugate were suitable for liquid formulation and may enable subcutaneous dosing.

The Impact of Metagenomics on Biocatalysis.

In the ever-growing demand for sustainable ways to produce high-value small molecules, biocatalysis has come to the forefront of greener routes to these chemicals. As such, the need to constantly find and optimise suitable biocatalysts for specific transformations has never been greater. Metagenome mining has been shown to rapidly expand the toolkit of promiscuous enzymes needed for new transformations, without requiring protein engineering steps. If protein engineering is needed, the metagenomic candidate can often provide a better starting point for engineering than a previously discovered enzyme on the open database or from literature, for instance. In this review, we highlight where metagenomics has made substantial impact on the area of biocatalysis in recent years. We review the discovery of enzymes in previously unexplored or 'hidden' sequence space, leading to the characterisation of enzymes with enhanced properties that originate from natural selection pressures in native environments.

An anti-TNF-glucocorticoid receptor modulator antibody-drug conjugate is efficacious against immune-mediated inflammatory diseases.

Glucocorticoids (GCs) are efficacious drugs used for treating many inflammatory diseases, but the dose and duration of administration are limited because of severe side effects. We therefore sought to identify an approach to selectively target GCs to inflamed tissue. Previous work identified that anti-tumor necrosis factor (TNF) antibodies that bind to transmembrane TNF undergo internalization; therefore, an anti-TNF antibody-drug conjugate (ADC) would be mechanistically similar, where lysosomal catabolism could release a GC receptor modulator (GRM) payload to dampen immune cell activity. Consequently, we have generated an anti-TNF-GRM ADC with the aim of inhibiting pro-inflammatory cytokine production from stimulated human immune cells. In an acute mouse model of contact hypersensitivity, a murine surrogate anti-TNF-GRM ADC inhibited inflammatory responses with minimal effect on systemic GC biomarkers. In addition, in a mouse model of collagen-induced arthritis, single-dose administration of the ADC, delivered at disease onset, was able to completely inhibit arthritis for greater than 30 days, whereas an anti-TNF monoclonal antibody only partially inhibited disease. ADC treatment at the peak of disease was also able to attenuate the arthritic phenotype. Clinical data for a human anti-TNF-GRM ADC (ABBV-3373) from a single ascending dose phase 1 study in healthy volunteers demonstrated antibody-like pharmacokinetic profiles and a lack of impact on serum cortisol concentrations at predicted therapeutic doses. These data suggest that an anti-TNF-GRM ADC may provide improved efficacy beyond anti-TNF alone in immune mediated diseases while minimizing systemic side effects associated with standard GC treatment.

Regiodivergent Radical Termination for Intermolecular Biocatalytic C-C Bond Formation.

Radical hydrofunctionalizations of electronically unbiased dienes are challenging to render regioselective, because the products are nearly identical in energy. Here, we report two engineered FMN-dependent "ene"-reductases (EREDs) that catalyze regiodivergent hydroalkylations of cyclic and linear dienes. While previous studies focused exclusively on the stereoselectivity of alkene hydroalkylation, this work highlights that EREDs can control the regioselectivity of hydrogen atom transfer, providing a method for selectively preparing constitutional isomers that would be challenging to prepare using traditional synthetic methods. Engineering the ERED from Gluconabacter sp. (GluER) furnished a variant that favors the γ,δ-unsaturated ketone, while an engineered variant from a commercial ERED panel favors the δ,ε-unsaturated ketone. The effect of beneficial mutations has been investigated using substrate docking studies and the mechanism probed by isotope labeling experiments. A variety of α-bromo ketones can be coupled with cyclic and linear dienes. These interesting building blocks can also be further modified to generate difficult-to-access heterocyclic compounds.

Engineering Biocatalysts for the C-H Activation of Fatty Acids by Ancestral Sequence Reconstruction.

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids, but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the subterminal positions of fatty acids. Herein we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multidomain, self-sufficient P450 monooxygenases. We resurrected 11 catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from subterminal in the older ancestors to mid-chain in the lineage leading to the extant, P450-TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multidomain enzymes to develop active, self-sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation.

Biocatalysis using Thermostable Cytochrome P450 Enzymes in Bacterial Membranes - Comparison of Metabolic Pathways with Human Liver Microsomes and Recombinant Human Enzymes.

Detailed structural characterization of small molecule metabolites is desirable during all stages of drug development, and often relies on the synthesis of metabolite standards. However, introducing structural changes into already complex, highly functionalized small molecules both regio- and stereo-selectively can be challenging using purely chemical approaches, introducing delays into the drug pipeline. An alternative is to use the cytochrome P450 enzymes (P450s) that produce the metabolites in vivo, taking advantage of the enzyme's inherently chiral active site to achieve regio- and stereoselectivity. Importantly, biotransformations are more sustainable: they proceed under mild conditions and avoid environmentally damaging solvents and transition metal catalysts. Recombinant enzymes avoid the need to use animal liver microsomes. However, native enzymes must be stabilized to work for extended periods or at elevated temperatures, and stabilizing mutations can alter catalytic activity. Here we assessed a set of novel, thermostable P450s in bacterial membranes, a format analogous to liver microsomes, for their ability to metabolize drugs through various pathways and compared them to human liver microsomes. Collectively, the thermostable P450s could replicate the metabolic pathways seen with human liver microsomes, including bioactivation to protein-reactive intermediates. Novel metabolites were found, suggesting the possibility of obtaining metabolites not produced by human or rodent liver microsomes. Importantly, no alteration in assay conditions from standard protocols for microsomal incubations was necessary. Thus, such bacterial membranes represent an analogous metabolite generation system to liver microsomes in terms of metabolites produced and ease of use, but which provides access to more diversity of metabolite structures. SIGNIFICANCE STATEMENT: In drug development it is often chemically challenging, to synthesize authentic metabolites of drug candidates for structural identification and evaluation of activity and safety. Biosynthesis using microsomes or recombinant human enzymes is confounded by the instability of the enzymes. Here we show that thermostable ancestral cytochrome P450 enzymes derived from P450 families responsible for human drug metabolism offer advantages over the native human forms in being more robust and over microbial enzymes in faithfully reflecting human drug metabolism.

A Universal, Continuous Assay for SAM-dependent Methyltransferases.

Enzyme-catalyzed late-stage functionalization (LSF), such as methylation of drug molecules and lead structures, enables direct access to more potent active pharmaceutical ingredients (API). S-adenosyl-l-methionine-dependent methyltransferases (MTs) can play a key role in the development of new APIs, as they catalyze the chemo- and regioselective methylation of O-, N-, S- and C-atoms, being superior to traditional chemical routes. To identify suitable MTs, we developed a continuous fluorescence-based, high-throughput assay for SAM-dependent methyltransferases, which facilitates screening using E. coli cell lysates. This assay involves two enzymatic steps for the conversion of S-adenosyl-l-homocysteine into H2 S to result in a selective fluorescence readout via reduction of an azidocoumarin sulfide probe. Investigation of two O-MTs and an N-MT confirmed that this assay is suitable for the determination of methyltransferase activity in E. coli cell lysates.

Biocatalysis in Drug Design: Engineered Reductive Aminases (RedAms) Are Used to Access Chiral Building Blocks with Multiple Stereocenters.

Novel building blocks are in constant demand during the search for innovative bioactive small molecule therapeutics by enabling the construction of structure-activity-property-toxicology relationships. Complex chiral molecules containing multiple stereocenters are an important component in compound library expansion but can be difficult to access by traditional organic synthesis. Herein, we report a biocatalytic process to access a specific diastereomer of a chiral amine building block used in drug discovery. A reductive aminase (RedAm) was engineered following a structure-guided mutagenesis strategy to produce the desired isomer. The engineered RedAm (IR-09 W204R) was able to generate the (S,S,S)-isomer 3 in 45% conversion and 95% ee from the racemic ketone 2. Subsequent palladium-catalyzed deallylation of 3 yielded the target primary amine 4 in a 73% yield. This engineered biocatalyst was used at preparative scale and represents a potential starting point for further engineering and process development.

Self-Immolative Carbamate Linkers for CD19-Budesonide Antibody-Drug Conjugates.

Antibody-drug conjugates consist of potent small-molecule payloads linked to a targeting antibody. Payloads must possess a viable functional group by which a linker for conjugation can be attached. Linker-attachment options remain limited for the connection to payloads via hydroxyl groups. A releasing group based on 2-aminopyridine was developed to enable stable attachment of para-aminobenzyl carbamate (PABC) linkers to the C21-hydroxyl group of budesonide, a glucocorticoid receptor agonist. Payload release involves a cascade of two self-immolative events that are initiated by the protease-mediated cleavage of the dipeptide-PABC bond. Budesonide release rates were determined for a series of payload-linker intermediates in buffered solution at pH 7.4 and 5.4, leading to the identification of 2-aminopyridine as the preferred releasing group. Addition of a poly(ethylene glycol) group improved linker hydrophilicity, thereby providing CD19-budesonide ADCs with suitable properties. ADC23 demonstrated targeted delivery of budesonide to CD19-expressing cells and inhibited B-cell activation in mice.

Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases.

Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases.

The Role of Cytochrome†P450 AbyV in the Final Stages of Abyssomicin†C Biosynthesis.

Abyssomicin C and its atropisomer are potent inhibitors of bacterial folate metabolism. They possess complex polycyclic structures, and their biosynthesis has been shown to involve several unusual enzymatic transformations. Using a combination of synthesis and in vitro assays we reveal that AbyV, a cytochrome P450 enzyme from the aby gene cluster, catalyses a key late-stage epoxidation required for the installation of the characteristic ether-bridged core of abyssomicin C. The X-ray crystal structure of AbyV has been determined, which in combination with molecular dynamics simulations provides a structural framework for our functional data. This work demonstrates the power of combining selective carbon-13 labelling with NMR spectroscopy as a sensitive tool to interrogate enzyme-catalysed reactions in vitro with no need for purification.

A Phase II, Two-Stage Study of Letrozole and Abemaciclib in Estrogen Receptor-Positive Recurrent Endometrial Cancer.

PURPOSE: Estrogen receptor (ER)-positive endometrial cancers (ECs) are characterized by phosphatidylinositol 3-kinase (PI3K) and receptor tyrosine kinase (RTK)/RAS/ß-catenin (CTNNB1) pathway alterations in approximately 90% and 80% of cases, respectively. Extensive cross-talk between ER, PI3K, and RTK/RAS/CTNNB1 pathways leads to both ligand-dependent and ligand-independent ER transcriptional activity as well as upregulation of cyclin D1 which, in complex with cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), is a critical regulator of cell cycle progression and a key mediator of resistance to hormonal therapy. We hypothesized that the combination of the aromatase inhibitor letrozole and CDK4/6 inhibitor abemaciclib would demonstrate promising activity in this setting. METHODS: We conducted a phase II, two-stage study of letrozole/abemaciclib in recurrent ER-positive EC. Eligibility criteria included measurable disease, no limit on prior therapies, and all EC histologies; prior hormonal therapy was allowed. Primary end points were objective response rate by RECIST 1.1 and progression-free survival (PFS) rate at 6 months. RESULTS: At the data cutoff date (December 03, 2021), 30 patients (28 with endometrioid EC) initiated protocol therapy; 15 (50%) patients had prior hormonal therapy. There were nine total responses (eight confirmed), for an objective response rate of 30% (95% CI, 14.7 to 49.4), all in endometrioid adenocarcinomas. Median PFS was 9.1 months, PFS at 6 months was 55.6% (95% CI, 35.1 to 72), and median duration of response was 7.4 months. Most common ≥ grade 3 treatment-related adverse events were neutropenia (20%) and anemia (17%). Responses were observed regardless of grade, prior hormonal therapy, mismatch repair, and progesterone receptor status. Exploratory tumor profiling revealed several mechanistically relevant candidate predictors of response (CTNNB1, KRAS, and CDKN2A mutations) or absence of response (TP53 mutations), which require independent validation. CONCLUSION: Letrozole/abemaciclib demonstrated encouraging and durable evidence of activity in recurrent ER positive endometrioid EC.

The Role of Cytochrome†P450 AbyV in the Final Stages of Abyssomicin†C Biosynthesis.

Abyssomicin C and its atropisomer are potent inhibitors of bacterial folate metabolism. They possess complex polycyclic structures, and their biosynthesis has been shown to involve several unusual enzymatic transformations. Using a combination of synthesis and in vitro assays we reveal that AbyV, a cytochrome P450 enzyme from the aby gene cluster, catalyses a key late-stage epoxidation required for the installation of the characteristic ether-bridged core of abyssomicin C. The X-ray crystal structure of AbyV has been determined, which in combination with molecular dynamics simulations provides a structural framework for our functional data. This work demonstrates the power of combining selective carbon-13 labelling with NMR spectroscopy as a sensitive tool to interrogate enzyme-catalysed reactions in vitro with no need for purification.

Discovery of ABBV-3373, an Anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody Drug Conjugate.

Using a convergent synthetic route to enable multiple points of diversity, a series of glucocorticoid receptor modulators (GRM) were profiled for potency, selectivity, and drug-like properties in vitro. Despite covering a large range of diversity, profiling the nonconjugated small molecule was suboptimal and they were conjugated to a mouse antitumor necrosis factor (TNF) antibody using the MP-Ala-Ala linker. Screening of the resulting antibody drug conjugates (ADCs) provided a better assessment of efficacy and physical properties, reinforcing the need to conduct structure-activity relationship studies on the complete ADC. DAR4 ADCs were screened in an acute mouse contact hypersensitivity model measuring biomarkers to ensure a sufficient therapeutic window. In a chronic mouse arthritis model, mouse anti-TNF GRM ADCs were efficacious after a single dose of 10 mg/kg i.p. for over 30 days. Data on the unconjugated payloads and mouse surrogate anti-TNF ADCs identified payload 17 which was conjugated to a human anti-TNF antibody and advanced to the clinic as ABBV-3373.

Oxalate Oxidase for In Situ H2 O2 -Generation in Unspecific Peroxygenase-Catalysed Drug Oxyfunctionalisations.

H2 O2 -driven enzymes are of great interest for industrial biotransformations. Herein, we show for the first time that oxalate oxidase (OXO) is an efficient in situ source of H2 O2 for one of these biocatalysts, which is known as unspecific peroxygenase (UPO). OXO is reasonably robust, produces only CO2 as a by-product and uses oxalate as a cheap sacrificial electron donor. UPO has significant potential as an industrial catalyst for selective C-H oxyfunctionalisations, as we confirm herein by testing a diverse drug panel using miniaturised high-throughput assays and mass spectrometry. 33 out of 64 drugs were converted in 5 µL-scale reactions by the UPO with OXO (conversion >70 % for 11 drugs). Furthermore, oxidation of the drug tolmetin was achieved on a 50 mg scale (TONUPO 25 664) with 84 % yield, which was further improved via enzyme immobilization. This one-pot approach ensures adequate H2 O2 levels, enabling rapid access to industrially relevant molecules that are difficult to obtain by other routes.

Use of engineered cytochromes P450 for accelerating drug discovery and development.

Numerous steps in drug development, including the generation of authentic metabolites and late-stage functionalization of candidates, necessitate the modification of often complex molecules, such as natural products. While it can be challenging to make the required regio- and stereoselective alterations to a molecule using purely chemical catalysis, enzymes can introduce changes to complex molecules with a high degree of stereo- and regioselectivity. Cytochrome P450 enzymes are biocatalysts of unequalled versatility, capable of regio- and stereoselective functionalization of unactivated CH bonds by monooxygenation. Collectively they catalyze over 60 different biotransformations on structurally and functionally diverse organic molecules, including natural products, drugs, steroids, organic acids and other lipophilic molecules. This catalytic versatility and substrate range makes them likely candidates for application as potential biocatalysts for industrial chemistry. However, several aspects of the P450 catalytic cycle and other characteristics have limited their implementation to date in industry, including: their lability at elevated temperature, in the presence of solvents, and over lengthy incubation times; the typically low efficiency with which they metabolize non-natural substrates; and their lack of specificity for a single metabolic pathway. Protein engineering by rational design or directed evolution provides a way to engineer P450s for industrial use. Here we review the progress made to date toward engineering the properties of P450s, especially eukaryotic forms, for industrial application, and including the recent expansion of their catalytic repertoire to include non-natural reactions.

Biocatalytic conversion of sunlight and carbon dioxide to solar fuels and chemicals.

This review discusses the progress in the assembly of photosynthetic biohybrid systems using enzymes and microbes as the biocatalysts which are capable of utilising light to reduce carbon dioxide to solar fuels. We begin by outlining natural photosynthesis, an inspired biomachinery to develop artificial photosystems, and the rationale and motivation to advance and introduce biological substrates to create more novel, and efficient, photosystems. The case studies of various approaches to the development of CO2-reducing microbial semi-artificial photosystems are also summarised, showcasing a variety of methods for hybrid microbial photosystems and their potential. Finally, approaches to investigate the relatively ambiguous electron transfer mechanisms in such photosystems are discussed through the presentation of spectroscopic techniques, eventually leading to what this will mean for the future of microbial hybrid photosystems.

Oxygenating Biocatalysts for Hydroxyl Functionalisation in Drug Discovery and Development.

C-H oxyfunctionalisation remains a distinct challenge for synthetic organic chemists. Oxygenases and peroxygenases (grouped here as "oxygenating biocatalysts") catalyse the oxidation of a substrate with molecular oxygen or hydrogen peroxide as oxidant. The application of oxygenating biocatalysts in organic synthesis has dramatically increased over the last decade, producing complex compounds with potential uses in the pharmaceutical industry. This review will focus on hydroxyl functionalisation using oxygenating biocatalysts as a tool for drug discovery and development. Established oxygenating biocatalysts, such as cytochrome P450s and flavin-dependent monooxygenases, have widely been adopted for this purpose, but can suffer from low activity, instability or limited substrate scope. Therefore, emerging oxygenating biocatalysts which offer an alternative will also be covered, as well as considering the ways in which these hydroxylation biotransformations can be applied in drug discovery and development, such as late-stage functionalisation (LSF) and in biocatalytic cascades.