Triphosphates of nucleoside-based antiviral drugs

Nucleoside analogues are the largest class of antiviral agents based on small molecules. Nowadays, they are the cornerstone of chemotherapy for several viral chronic infections, including infections by HIV, hepatitis B, or C viruses and herpes viruses. These drugs consist of chemically modified synthetic nucleosides that generally mimic their natural counterparts and target viral polymerase: a key enzyme in the virus replication cycle.

The active form of nucleoside analogues is their relative 5’-O-triphosphate (TP). Polymerases use nucleoside triphosphates to extend nucleic acids during replication, the biosynthesis of DNA, or transcription, the process by which RNA is synthesized. Viral polymerases do not distinguish between natural nucleotides and the active triphosphate form of synthetic ones. These enzymes incorporate the nucleotide analogue within the sequence of the growing strand instead of the natural nucleotide while synthesizing DNA/RNA.

Nucleoside triphosphates- mechanism of action

Although several inhibition mechanisms are employed, analogues are often designed to cause chain termination, thus blocking the viral replication once they are incorporated into the nucleic acid. Alternatively, nucleoside analogues can cause inhibition of cellular or viral enzymes involved in nucleoside/tide metabolism without being incorporated into DNA/RNA.

Although triphosphates are the active form of the antiviral drugs, direct delivery of the triphosphate into cells is not possible, as these multi charged molecules cannot permeate the lipophilic cell membranes. Thus, nucleosides or nucleotide prodrugs - generally consisting of nucleoside 5’-O-monophosphates (MPs) where charges on the phosphate group are masked with lipophilic substituents – see our article on nucleotide prodrugs) - must be employed for therapy or in vivo studies. However, the efficiency of the drug relies on intracellular phosphorylation by kinases to reach the final active form, the triphosphate.

Importance of triphosphates

Even though synthetic triphosphates cannot be directly used as drugs, their use in biochemical research labs is of outstanding importance. As they are the active form of the drug, they are used in in vitro experiments for testing their affinity to the virus-encoded or host polymerases, as well as the ability of the enzyme to incorporate the triphosphate into nucleic acids, resulting in suppression of the DNA/RNA synthesis by chain termination or other mechanisms.

Moreover, when a new virus emerges, it is essential to have a rapidly available first-line defence until vaccines, and virus-specific antivirals are developed (as we have seen with COVID-19 and nucleoside drug Remdesivir). In many cases, nucleoside analogues that already showed high antiviral activity or are already used to treat acute infections caused by medically important RNA and DNA viruses are a first-choice test, as their cytotoxicity and pharmacokinetics are already known. Having in hand triphosphates of important nucleoside-based drugs allows thus for a fast in vitro screening of antiviral activity against newly emerging viruses.

Santiago lab and triphosphates

At Santiago lab, we are intensively working on the synthesis of novel nucleoside triphosphates with potential antiviral activity. During the COVID-19 pandemic, we successfully developed our own innovative synthetic pathway for the drug Remdesivir and its phosphate derivatives (mono-, di- and triphosphate).

Lately, we have also successfully prepared triphosphates of other pharmaceutically important nucleosides (eg. Islatravir or Lamivudine, you can read more here) that have already been enlisted in our catalogue.

We have gained many experiences in this field, and therefore, we are happy to offer our knowledge and skills to other researchers. If you are interested in our services (custom synthesis, contract research, custom synthesis of nucleoside triphosphates), do not hesitate to contact us by email to Krystof Sigut at krystof.sigut@santiago-lab.com or reach him on the phone +420 776 750 591.

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