Chemoenzymatic approaches to plant natural product inspired compounds

Complex molecules produced by plants have provided us with a range of medicines, flavour and fragrance compounds and pesticides. However, there are challenges associated with accessing these in an economically viable manner, including low natural abundance and the requirement for complex multi-step synthetic strategies. Chemoenzymatic approaches provide a valuable alternative strategy by combining traditional synthetic methods with biocatalysis. This review highlights recent chemoenzymatic syntheses towards plant natural products and analogues, focusing on the advantages of incorporating biocatalysts into a synthetic strategy

Methyltransferases: Functions and Applications

In this review the current state of the art of S-adenosylmethionine (SAM)-dependent methyltransferases and SAM are evaluated. Their structural classification and diversity is introduced and key mechanistic aspects presented which are then detailed further. Then, catalytic SAM as a target for drugs and approaches to utilise SAM as a cofactor in synthesis is introduced with different recycling approaches evaluated. The use of SAM analogues are also described. Finally O-, N-, C- and S-MTs, their synthetic applications and potential for compound diversification is given

Multienzyme one-pot cascades incorporating methyltransferases for the strategic diversification of tetrahydroisoquinoline alkaloids

The tetrahydroisoquinoline (THIQ) ring system is present in a large variety of structurally diverse natural products exhibiting a wide range of biological activities. Routes to mimic the biosynthetic pathways to such alkaloids, by building cascade reactions in vitro, represents a successful strategy and can offer better stereoselectivities than traditional synthetic methods. S-Adenosylmethionine (SAM)-dependent methyltransferases are crucial in the biosynthesis and diversification of THIQs; however, their application is often limited in vitro by the high cost of SAM and low substrate scope. In this study, we describe the use of methyltransferases in vitro in multi-enzyme cascades, including for the generation of SAM in situ. Up to seven enzymes were used for the regioselective diversification of natural and non-natural THIQs on an enzymatic preparative scale. Regioselectivites of the methyltransferases were dependent on the group at C-1 and presence of fluorine in the THIQs. An interesting dual activity was also discovered for the catechol methyltransferases used, which were found to be able to regioselectively methylate two different catechols in a single molecule

Multienzyme One‐Pot Cascades Incorporating Methyltransferases for the Strategic Diversification of Tetrahydroisoquinoline Alkaloids

The tetrahydroisoquinoline (THIQ) ring system is present in a large variety of structurally diverse natural products exhibiting a wide range of biological  activities. Routes  to mimic the biosynthetic pathways to such alkaloids, by building cascade reactions in vitro, represents a successful strategy and offers better stereoselectivities than traditional synthetic methods.  (S)-Adenosylmethionine (SAM)  dependent methyltransferases   are crucial in the biosynthesis and diversification of THIQs; however, their application is often limited in vitro by the high cost of SAM and low substrate scope. In this study, we describe  the use  of methyltransferases in vitro in multi-enzyme cascades,  including for the generation of SAM   in situ . Up to seven enzymes  were used  for the regioselective diversification of natural and non-natural THIQs on  an enzymatic  preparative scale.  Regioselectivites of the methyltransferases were dependent on the group at C-1 and presence of fluorine in the THIQs.   An interesting dual activity was also discovered for the catechol methyltransferases used, which were found to be able to regioselectively methylate two different catechols in a single molecule

Development of biocatalysts for the alkylation of complex molecules

The medicinal effects of compounds can be dramatically improved by even the smallest alkylations of their structures. Making those additions selectively, however, can be challenging for traditional synthesis, especially when molecules have many reactive sites. This has limited the ability to access and study derivatives of complex molecules, which may be hampering the discovery of new bioactives. Methyltransferases (MTs) are a vast, structurally divergent class of enzymes, responsible for catalysing nearly all alkylation reactions in cells. In many cases, the transferral of a methyl group by an MT is highly selective, and able to significantly alter the interactions of the target. MTs thus have the potential to access the sought-after alkylated derivatives of medicinally promising structures. In doing so, they may allow the study and manufacture of molecules that would otherwise be difficult to produce. This thesis documents work towards the goal of realising that potential. A threeenzyme cascade was used to generate a reactive cofactor in situ, use that cofactor in MT reactions and break down the inhibitory side product. With this system, selective alkylation of compounds featuring the privileged tetrahydroisoquinoline scaffold were explored through small- and preparative-scale assays, with primary analysis by HPLC. The first efforts were towards methylation, comprising investigations using the more established biocatalyst catechol-O-MT alongside a search for novel MTs. The work then evolved into the development of capacity for other alkylations. Analogues of the alkyl donor methionine, sourced commercially and later through syntheses, were integrated into the cascades. The challenges encountered during this effort prompted computational investigations, directed mutagenesis to improve enzyme performance and ultimately a high-throughput random mutagenesis screen to search for improved enzyme variants

Methyltransferases: Functions and Applications

In this review the current state-of-the-art of S-adenosylmethionine (SAM)-dependent methyltransferases and SAM are evaluated. Their structural classification and diversity is introduced and key mechanistic aspects presented which are then detailed further. Then, catalytic SAM as a target for drugs, and approaches to utilise SAM as a cofactor in synthesis are introduced with different supply and regeneration approaches evaluated. The use of SAM analogues are also described. Finally O-, N-, C- and S-MTs, their synthetic applications and potential for compound diversification is given.BT/Biocatalysi

Combining Immune Checkpoint Inhibitors: Established and Emerging Targets and Strategies to Improve Outcomes in Melanoma

The immune system employs several checkpoint pathways to regulate responses, maintain homeostasis and prevent self-reactivity and autoimmunity. Tumor cells can hijack these protective mechanisms to enable immune escape, cancer survival and proliferation. Blocking antibodies, designed to interfere with checkpoint molecules CTLA-4 and PD-1/PD-L1 and counteract these immune suppressive mechanisms, have shown significant success in promoting immune responses against cancer and can result in tumor regression in many patients. While inhibitors to CTLA-4 and the PD-1/PD-L1 axis are well-established for the clinical management of melanoma, many patients do not respond or develop resistance to these interventions. Concerted efforts have focused on combinations of approved therapies aiming to further augment positive outcomes and survival. While CTLA-4 and PD-1 are the most-extensively researched targets, results from pre-clinical studies and clinical trials indicate that novel agents, specific for checkpoints such as A2AR, LAG-3, IDO and others, may further contribute to the improvement of patient outcomes, most likely in combinations with anti-CTLA-4 or anti-PD-1 blockade. This review discusses the rationale for, and results to date of, the development of inhibitory immune checkpoint blockade combination therapies in melanoma. The clinical potential of new pipeline therapeutics, and possible future therapy design and directions that hold promise to significantly improve clinical prognosis compared with monotherapy, are discussed