Biotechnological production of cyclic dinucleotides—Challenges and opportunities

Abstract

Cyclic dinucleotides (CDNs) are widely used secondary signaling molecules in prokaryotic and eukaryotic cells. As strong agonists of the stimulator of interferon genes, they are of great interest for pharmaceutical applications. In particular, cyclic-GMP-AMP and related synthetic CDNs are promising candidates in preclinical work and even some in clinical phase 1 and 2 studies. The comparison of chemical and biocatalytic synthesis routes elucidated that biological CDN synthesis offers some advantages, such as shorter synthesis time, avoiding complex protective group chemistry, and the access to a new spectrum of CDNs. However, the synthesis of CDNs in preparative quantities is still a challenge, since the chemical synthesis of CDNs suffers from low yields and complex synthetic routes and the enzymatically catalyzed synthesis is limited by low product titers and process stability. We aim to review the latest discoveries and recent trends in chemical and biocatalytic synthesis of CDNs with a focus on the synthesis of a huge variety of CDN derivatives. We furthermore consider the most promising biotechnological processes for CDN production by evaluating key figures of the currently known processes.

Design, Synthesis, and Biochemical and Biological Evaluation of Novel 7-Deazapurine Cyclic Dinucleotide Analogues as STING Receptor Agonists

“…Considering the results of the initial trials, we decided to focus mainly on 7-deazaadenine derivatives due to the fact that the presence of 7-deazaguanine in compound 1e caused a substantial drop in affinity to wt hSTING . When possible, compounds were prepared enzymatically by mouse cGAS (mcGAS) as previously shown or with bacterial dinucleotide cyclase from Vibrio cholerae (DncV) and diadenylate cyclase from Bacillus thuringiensis (DisA). ,− Otherwise, the compounds were prepared by chemical synthesis, either by arylation of an enzymatically prepared precursor or by total synthesis. All compounds were tested by differential scanning fluorimetry (DSF) and in cell-based assays.…”

“… 20 When possible, compounds were prepared enzymatically by mouse cGAS (mcGAS) as previously shown or with bacterial dinucleotide cyclase from Vibrio cholerae (DncV) and diadenylate cyclase from Bacillus thuringiensis (DisA). 20 , 33 − 35 Otherwise, the compounds were prepared by chemical synthesis, either by arylation of an enzymatically prepared precursor or by total synthesis. All compounds were tested by differential scanning fluorimetry (DSF) and in cell-based assays.…”

Biocatalytic Synthesis of Antiviral Nucleosides, Cyclic Dinucleotides, and Oligonucleotide Therapies

“…5,56 As such, cGAMP analogues are being evaluated as immunotherapy cancer treatments and antiviral agents. [4][5][6]57 A prominent example is the cGAMP analogue MK-1454, developed by Merck as an immuno-oncology treatment. 12,58,59 MK-1454 contains a 2′-fluoro-modified AMP unit, a 3′-fluoromodified GMP, and two R p phosphorothioate linkages, designed to improve metabolic stability, bioavailability, and potency.…”

Cascades of Evolved Enzymes for the Synthesis of Complex Molecules

“…This is a remarkable synthesis yield, especially when compared to chemical synthesis, where a yield of < 30 % has been reported. [6] The cyclization of phosphorothioate nucleotides was performed using a nucleotidyltransferase, which was optimized by nine rounds of protein engineering to improve reactivity. [5] The enzyme was evolved in seven rounds for improved reactivity towards the non-natural substrates, and in two additional rounds with Zn 2 + and Co 2 + as cofactors.…”