Phytochemicals in Drug Discovery-A Confluence of Tradition and Innovation.

Phytochemicals have a long and successful history in drug discovery. With recent advancements in analytical techniques and methodologies, discovering bioactive leads from natural compounds has become easier. Computational techniques like molecular docking, QSAR modelling and machine learning, and network pharmacology are among the most promising new tools that allow researchers to make predictions concerning natural products' potential targets, thereby guiding experimental validation efforts. Additionally, approaches like LC-MS or LC-NMR speed up compound identification by streamlining analytical processes. Integrating structural and computational biology aids in lead identification, thus providing invaluable information to understand how phytochemicals interact with potential targets in the body. An emerging computational approach is machine learning involving QSAR modelling and deep neural networks that interrelate phytochemical properties with diverse physiological activities such as antimicrobial or anticancer effects.

Antibiotic origami: selective formation of spirotetronates in abyssomicin biosynthesis.

The abyssomicins are a structurally intriguing family of bioactive natural products that include compounds with potent antibacterial, antitumour and antiviral activities. The biosynthesis of the characteristic abyssomicin spirotetronate core occurs via an enzyme-catalysed intramolecular Diels-Alder reaction, which proceeds via one of two distinct stereochemical pathways to generate products differing in configuration at the C15 spirocentre. Using the purified spirotetronate cyclases AbyU (from abyssomicin C/atrop-abyssomicin C biosynthesis) and AbmU (from abyssomicin 2/neoabyssomicin biosynthesis), in combination with synthetic substrate analogues, here we show that stereoselectivity in the spirotetronate-forming [4 + 2]-cycloaddition is controlled by a combination of factors attributable to both the enzyme and substrate. Furthermore, an achiral substrate was enzymatically cyclised to a single enantiomer of a spirocyclic product. X-ray crystal structures, molecular dynamics simulations, and assessment of substrate binding affinity and reactivity in both AbyU and AbmU establish the molecular determinants of stereochemical control in this important class of biocatalysts.

Delineation of the complete reaction cycle of a natural Diels-Alderase.

The Diels-Alder reaction is one of the most effective methods for the synthesis of substituted cyclohexenes. The development of protein catalysts for this reaction remains a major priority, affording new sustainable routes to high value target molecules. Whilst a small number of natural enzymes have been shown capable of catalysing [4 + 2] cycloadditions, there is a need for significant mechanistic understanding of how these prospective Diels-Alderases promote catalysis to underpin their development as biocatalysts for use in synthesis. Here we present a molecular description of the complete reaction cycle of the bona fide natural Diels-Alderase AbyU, which catalyses formation of the spirotetronate skeleton of the antibiotic abyssomicin C. This description is derived from X-ray crystallographic studies of AbyU in complex with a non-transformable synthetic substrate analogue, together with transient kinetic analyses of the AbyU catalysed reaction and computational reaction simulations. These studies reveal the mechanistic intricacies of this enzyme system and establish a foundation for the informed reengineering of AbyU and related biocatalysts.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Exploring the enzyme-catalyzed synthesis of isotope labeled cyclopropanes.

Cyclopropanes are commonly employed structural moieties in drug design since their incorporation is often associated with increased target affinity, improved metabolic stability, and increased rigidity to access bioactive conformations. Robust chemical cyclopropanation procedures have been developed which proceed with high yield and broad substrate scope, and have been applied to labeled substrates. Recently, engineered enzymes have been shown to perform cyclopropanations with remarkable diastereoselectivity and enantioselectivity, but this biocatalytic approach has not been applied to labeled substrates to date. In this study, the use of enzyme catalysis for the synthesis of labeled cyclopropanes was investigated. Two readily available enzymes, a modified CYP450 enzyme and a modified Aeropyrum pernix protoglobin, were investigated for the cyclopropanation of a variety of substituted styrenes. For this biocatalytic transformation, the enzymes required the use of ethyl diazoacetate. Due to the highly energetic nature of this molecule, alternatives were investigated. The final optimized cyclopropanation was successfully demonstrated using n-hexyl diazoacetate, resulting in moderate to high enantiomeric excess. The optimized procedure was used to generate labeled cyclopropanes from 13 C-glycine, forming all four labeled stereoisomers of phosphodiesterase type-IV inhibitor, MK0952. These reactions provide a convenient and effective biocatalytic route to stereoselective 13 C-labeled cyclopropanes and serve as a proof-of-concept for generating stereoselective labeled cyclopropanes.

Iridium-catalyzed C-H methylation and d 3-methylation of benzoic acids with application to late-stage functionalizations.

Late-stage functionalization (LSF) has over the past years emerged as a powerful approach in the drug discovery process. At its best, it allows for rapid access to new analogues from a single drug-like molecule, bypassing the need for de novo synthesis. To be successful, methods able to tolerate the diverse functional groups present in drug-like molecules that perform under mild conditions are required. C-H methylation is of particular interest due to the magic methyl effect in medicinal chemistry. Herein we report an iridium-catalyzed carboxylate-directed ortho C-H methylation and d 3-methylation of benzoic acids. The method uses commercially available reagents and precatalyst and requires no inert atmosphere or exclusion of moisture. Substrates bearing electron-rich and electron-poor groups were successfully methylated, including compounds with competing directing/coordinating groups. The method was also applied to the LSF of several marketed drugs, forming analogues with increased metabolic stability compared with the parent drug.

A Perspective on Synthetic Biology in Drug Discovery and Development-Current Impact and Future Opportunities.

The global impact of synthetic biology has been accelerating, because of the plummeting cost of DNA synthesis, advances in genetic engineering, growing understanding of genome organization, and explosion in data science. However, much of the discipline's application in the pharmaceutical industry remains enigmatic. In this review, we highlight recent examples of the impact of synthetic biology on target validation, assay development, hit finding, lead optimization, and chemical synthesis, through to the development of cellular therapeutics. We also highlight the availability of tools and technologies driving the discipline. Synthetic biology is certainly impacting all stages of drug discovery and development, and the recognition of the discipline's contribution can further enhance the opportunities for the drug discovery and development value chain.

Enzymatic Late-Stage Modifications: Better Late Than Never.

Enzyme catalysis is gaining increasing importance in synthetic chemistry. Nowadays, the growing number of biocatalysts accessible by means of bioinformatics and enzyme engineering opens up an immense variety of selective reactions. Biocatalysis especially provides excellent opportunities for late-stage modification often superior to conventional de novo synthesis. Enzymes have proven to be useful for direct introduction of functional groups into complex scaffolds, as well as for rapid diversification of compound libraries. Particularly important and highly topical are enzyme-catalysed oxyfunctionalisations, halogenations, methylations, reductions, and amide bond formations due to the high prevalence of these motifs in pharmaceuticals. This Review gives an overview of the strengths and limitations of enzymatic late-stage modifications using native and engineered enzymes in synthesis while focusing on important examples in drug development.