Tuning in to stereochemistry

LA JOLLA, Calif.—Knowing the 3D structures of molecules is important in helping to answer questions about crystallization or potential binding sites, but in those cases, such knowledge is needed to work with molecules’ natural structures. Thanks to a new tool developed by scientists at Scripps Research and Bristol-Myers Squibb, there’s now an option for bypassing such issues by controlling the 3D architecture—or stereochemistry—of molecules comprised of nucleotides to precisely create desired configurations. Their results were published in Science in a paper titled “Unlocking P(V): Reagents for chiral phosphorothioate synthesis.”

The technology in question has been named phosphorous-sulfur incorporation (PSI), which connects chains of nucleotides (also known as oligonucleotides) into preprogrammed structures. The collaborators tested PSI by using it to synthesize pure 3D forms of the molecular configurations used in drugs like Spinraza (the first approved spinal muscular atrophy therapy) and cyclic dinucleotides (CDNs), a new class of immunotherapy drugs. This approach is also of interest in accelerating efforts to develop single isomer thiophosphate-derived oligonucleotide drugs, molecules that can have hundreds of thousands of stereoisomers. While stereoisomers share the same molecular formula, the spatial configuration of their atoms is different.

“Molecular gene therapies such as emerging CDNs represent remarkable therapeutic potential, but their development and refinement has been hindered by the inability to effectively control the stereochemistry during drug synthesis,” explained Dr. Phil Baran, a Scripps Research professor and senior scientist on the study. “PSI provides a robust and stereocontrolled method of synthesizing oligonucleotide drugs, allowing us to create, analyze and manufacture stereoisomers of a drug candidate in ways that were never before possible.”

For some background on oligonucleotides and phosphorothioate, as explained by Millipore Sigma, “Several phosphate backbone variants have been developed in an attempt to alter the chemical properties of native-state DNA and therefore overcome the two major challenges involved with using oligonucleotides in vivo, including: 1) delivery to the interior of the cell through the plasma membrane, a lipid bilayer that, without transport proteins, is mostly impermeable to polar molecules; and 2) extension of the effective molecular lifetime by minimizing extra and intracellular nuclease degradation.

“One of the original and still most widely used backbone variants is phosphorothioate (commonly referred to as S-oligo when incorporated into an oligonucleotide). Phosphorothioate has been found to help alleviate the second major challenge associated with using oligonucleotides in vivo by reducing the activity of a variety of extra and intracellular nucleases.”

The method of synthesizing nucleotides has not changed much over the years. As noted in a supplementary blog post to this work on the Baran laboratory’s website, “We discovered that while the earliest reports of nucleotide synthesis relied on the natural phosphorous (V) oxidation state, the late 1970s saw the lethargic P(V) largely supplanted by P(III) in the form of phosphoramidites and H-phosphonates. Perhaps most striking was the apparent reliance on incremental modifications to advance the field; the P(III)-based phosphoramidite approach remains the standard mode of construction to this day.”

A major limitation to P(III) chemistry, however, is the lack of control over the shape of thiophosphorus-centered linkages—a lack that, in thiophosphate-based oligonucleotides, can lead to a product with more than 100,000 stereoisomers. In addition, Justine deGruyter, a Scripps Research graduate student and one of the first authors on the paper, reported that “Using P(III) chemistry to produce even a miniscule amount of the drug as a single stereoisomer form is extremely complicated, which means you can’t produce enough to test which shape is the most effective as a therapy or whether certain isomers might cause side effects.”

To try and find a way around the issues of P(III), the Scripps and Bristol-Myers Squibbs collaborators looked back at P(V), which is less reactive than P(III) but more stable. It took two years to engineer a solution for using P(V) to generate desired stereoisomers of molecules, and PSI was that solution. It links two nucleosides (nucleotides without a phosphorous atom) in a chosen 3D shape, and a benefit of the thiophosphate bond created with the PSI reagent is that it improves a drug candidate’s metabolic stability, and by association, its safety and efficacy.

In addition to CDNs, which play a role in the innate immune response, it’s thought that the PSI reagent approach will also enable a jump in research into antisense oligonucleotide (ASO) drugs. According to a Scripps Research press release, “the ability to synthesize a single stereoisomer will allow scientists to explore what shapes of the drugs are most therapeutically effective and generate those stereoisomers for clinical use.” ASOs, as noted in a Nature Reviews Neurology review article titled “Antisense oligonucleotides: the next frontier for treatment of neurological disorders,” are “short, synthetic, single-stranded oligodeoxynucleotides that can alter RNA and reduce, restore, or modify protein expression through several distinct mechanisms. By targeting the source of the pathogenesis, ASO-mediated therapies have an higher chance of success than therapies targeting downstream pathways.”

With a faster, more accurate way of precisely generating desired stereoisomers, the PSI solution from the Bristol-Myers Squibb and Scripps teams could enable researchers to start to truly explore—and perhaps harness—the potential of ASO and CDN drugs.