Application of rational enzyme engineering in a new route to etonogestrel and levonorgestrel: carbonyl reductase bioreduction of ethyl secodione.

Women in developing countries still face enormous challenges when accessing reproductive health care. Access to voluntary family planning empowers women allowing them to complete their education and join the paid workforce. This effectively helps to end poverty, hunger and promotes good health for all. According to the United Nations (UN) organization, in 2022, an estimated 257 million women still lacked access to safe and effective family planning methods globally. One of the main barriers is the associated cost of modern contraceptive methods. Funded by the Bill & Melinda Gates Foundation, Almac Group worked on the development of a novel biocatalytic route to etonogestrel and levonorgestrel, two modern contraceptive APIs, with the goal of substantially decreasing the cost of production and so enabling their use in developing nations. This present work combines the selection and engineering of a carbonyl reductase (CRED) enzyme from Almac's selectAZyme™ panel, with process development, to enable efficient and economically viable bioreduction of ethyl secodione to (13R,17S)-secol, the key chirality introducing intermediate en route to etonogestrel and levonorgestrel API. CRED library screening returned a good hit with an Almac CRED from Bacillus weidmannii, which allowed for highly stereoselective bioreduction at low enzyme loading of less than 1% w/w under screening assay conditions. However, the only co-solvent tolerated was DMSO up to ∼30% v/v, and it was impossible to achieve reaction completion with any enzyme loading at substrate titres of 20 g L-1 and above, due to the insolubility of the secodione. This triggered a rapid enzyme engineering program fully based on computational mutant selection. A small panel of 93 CRED mutants was rationally designed to increase the catalytic activity as well as thermal and solvent stability. The best mutant, Mutant-75, enabled a reaction at 45 °C to go to completion at 90 g L-1 substrate titre in a buffer/DMSO/heptane reaction medium fed over 6 h with substrate DMSO stock solution, with a low enzyme loading of 3.5% w/w wrt substrate. In screening assay conditions, Mutant-75 also showed a 2.2-fold activity increase. Our paper shows which computations and rational decisions enabled this outcome.

Convergent Biocatalytic Mediated Synthesis of siRNA.

New technologies are required to combat the challenges faced with manufacturing commercial quantities of oligonucleotide drug substances which are required for treating large patient populations. Herein we report a convergent biocatalytic synthesis strategy for an Alnylam model siRNA. The siRNA chemical structure includes several of the unnatural modifications and conjugations typical of siRNA drug substances. Using Almac's 3-2-3-2 hybrid RNA ligase enzyme strategy that sequentially ligates short oligonucleotide fragments (blockmers), the target siRNA was produced to high purity at 1 mM concentration. Additional strategies were investigated including the use of polynucleotide kinase phosphorylation and the use of crude blockmer starting materials without chromatographic purification. These findings highlight a path toward a convergent synthesis of siRNAs for large-scale manufacture marrying both enzymatic liquid and classical solid-phase synthesis.

Discovery and Rational Mutagenesis of Methionine Sulfoxide Reductase Biocatalysts To Expand the Substrate Scope of the Kinetic Resolution of Chiral Sulfoxides.

Methionine sulfoxide reductase A (MsrA) enzymes have recently found applications as nonoxidative biocatalysts in the enantioselective kinetic resolution of racemic sulfoxides. This work describes the identification of selective and robust MsrA biocatalysts able to catalyze the enantioselective reduction of a variety of aromatic and aliphatic chiral sulfoxides at 8-64 mM concentration with high yields and excellent ees (up to 99%). Moreover, with the aim to expand the substrate scope of MsrA biocatalysts, a library of mutant enzymes has been designed via rational mutagenesis utilizing in silico docking, molecular dynamics, and structural nuclear magnetic resonance (NMR) studies. The mutant enzyme MsrA33 was found to catalyze the kinetic resolution of bulky sulfoxide substrates bearing non-methyl substituents on the sulfur atom with ees up to 99%, overcoming a significant limitation of the currently available MsrA biocatalysts.

Production of recombinant proteins in Escherichia coli using an N-terminal tag derived from sortase.

The use of Escherichia coli protein expression systems has many benefits, including the ease of propagation, amounts of protein that can be generated and cost. However, this host has some drawbacks due to difficulties in the production of soluble foreign proteins with their alternate codon usage bias, reductive cytoplasmic environment and lack of complex post-translational modifications. We have designed a novel fusion protein tag derived from the sequence of sortase (SrtA) which we have named Solubility 'eNhancing'Ubiquitous Tag (SNUT). Here we demonstrate its application and effectiveness as an N-terminal fusion tag for the expression and purification of proteins that could not be effectively produced with other tags. We show this tag can be utilized for the purification of proteins through both native and refolding immobilized metal ion chromatography and in combination with an anti-SNUT monoclonal antibody, can also be used as a detection tag. This tag may prove useful in circumventing expression and purification issues with the production of proteins in bacterial expression hosts.

Engineering and application of P450 monooxygenases in pharmaceutical and metabolite synthesis.

Cytochrome P450 monoxygenase (P450s or CYPs) allow access to drug metabolites, necessary for approval of new therapeutics in one step, with increased success being demonstrated using bacterial and fungal P450s. Moreover, 12 of the 13 products of the human metabolism of verapamil can be accessed through engineered and chimeric bacterial P450s. These P450s are also used in the synthesis of pharmaceuticals themselves, including the semi-synthetic production of artemisinin in an engineered cell. The integration of new technologies including ultrasound and polyfluorinated hydrocarbon solvents offers an attractive means by the true synthetic potential of ubiquitous P450s can be fully realised.

Recent advances in the application of antibodies as therapeutics.

The application of antibodies as therapeutic agents in the treatment of cancer now represents a significant proportion of the oncology drug arena. Despite this success, the ability to engineer and exploit antibodies in many different formats is ensuring that new avenues for their therapeutic application are constantly being examined. This review examines a selection of novel antibody-based therapeutic strategies that are currently in late preclinical and clinical evaluation.