Design of Therapeutic Pronucleotides

Design of Therapeutic Pronucleotides (ProTides)

Nucleosides and nucleotide analogs have shown great therapeutic potential and utility for the treatment of cancer, comprising 20% of FDA approved anti-cancer drugs and 50% of the FDA approved anti-viral drugs, Currently, fourteen nucleosides are clinically used for the treatment of cancer, with several others under clinical development. In general, nucleosides must be transported into cells by nucleoside transporters where they are phosphorylated sequentially by nucleoside kinases to the mono-, di- and triphosphate. In most cases, the triphosphorylated derivatives function as substrates or inhibitors of DNA polymerases in transformed cells. Several of these nucleosides, such as floxuridine (5-FdU) and gemcitabine (dFdC), are also multi-target drug, since their 5’-monophosphate or 5’-diphosphate metabolites directly inhibit thymidine synthase or ribonucleotide reductase, respectively.

Because nucleosides generally target DNA synthesis and nucleoside metabolism, they can have serious toxicities toward normal proliferating tissues, such as hematopoietic cells (i.e., white and red blood cells). The results can be severe, such as the loss of B-cells, T-cells, neutrophils, platelets and erythrocytes during treatment, leaving the patient susceptible to infections, anemia and bleeding.

For example, in a recent study of 196 Hairy Cell Leukemia patients receiving cladribine, 70% developed neutropenia, 37% exhibited anemia, 12% developed thrombocytopenia, with 26% developing infections. In addition, the number of CD4 and CD8 T-cells was suppressed for 4-6 months post treatment and liver and kidney toxicity were also observed. The development of resistance by either reduced nucleoside transport or phosphorylation also hampers the effectiveness of nucleoside anticancer therapy.

Among the three successive activating phosphorylation steps, the first step is the most vulnerable, since cellular resistance is associated with alterations in intracellular monophosphate levels resulting in reduced cellular kinase activity. For example, resistance to gemcitabine and clofarabine has been attributed to reduced expression of the equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK). Using monophosphorylated nucleosides has been considered as a way to overcome resistance. However, the charged and polar nature of nucleotides prevents them from crossing intestinal or cellular membranes. In addition, they are rapidly dephosphorylated by ubiquitous extracellular phosphatases present in cell media, plasma and tissues.

To address the issues prohibiting the delivery of nucleoside mono-phosphate, we proposed that nucleoside phosphoramidates could be used as pronucleotides. Through our work, we demonstrated that phosphoramidate based pronucleotides could serve as potent antiviral and anticancer agents by undergoing intracellular P-N bond cleavage. We demonstrated that they were highly stable in plasma and exhibited good pharmacokinetic properties. In addition, our group identified Histidine Triad Nucleotide Binding Protein 1 (HINT1) as the intracellular enzyme responsible for the metabolism of nucleotide phosphoramidates. We have since mapped the substrate specificities, both kinetically and structurally, resulting in a pharmacophore map that can be used for pronucleotide design. These insights have been employed for the design of the first curative antiviral, Sofosbuvir (Gilead, 2013), for the treatment of Hepatitis C virus. The use of phosphoramidate based pronucleotides as potent drugs was further demonstrated by the recent FDA approval of Remdesivir (Gilead, 2020) for the treatment of SAR2.

The antiviral activity of pronucleotides, such as Sofosbuvir and Remdesivir, depends on carboxyesterase-catalyzed hydrolysis, followed by mixed anhydride formation and histidine triad nucleotide binding protein (HINT1) P-N bond hydrolysis, resulting in the intracellular release of the active nucleoside monophosphate. We refer to these as carboxyesterase-anhydride-HINT1 ProTides (CAH ProTides). Due to the high carboxyesterase activity in the liver, >90% of these ProTides are extracted from circulation by the liver during oral dosing. While advantageous for the treatment of HCV by sofosbuvir, high first pass metabolism has limited the use of remdesivir to IV dosing. In addition, due to the high level of carboxyesterase activity in their blood, it can be difficult for this class of ProTides to undergo pre-clinical evaluation with rodent disease or biodistribution models. To address the inherent issues surrounding the current pronucleotide approach, our group has designed and developed

To address the inherent issues surrounding current ProTide approaches, our group has designed a novel class of stable, orally bioavailable anchimerically HINT1 activated ProTides (AHA-ProTides). Because AHA-ProTide activation depends on a water activated non-enzymatic step followed by HINT phosphoramidase activity, AHA-ProTides have a greater potential for broad tissue distribution and antiviral activity upon either IV or oral dosing. Recently, we have prepared an AHA-ProTide and demonstrated that it has potent in vitro anti-Dengue (DENV) and anti-ZIKV activity. Significantly, the AHA-ProTide was shown to have potent anti-Zika activity with a mouse model. with no observable toxicity. In addition, we have developed the first cell permeable cap-dependet antagonists of cap- translation translation by eIF4E and demonstrated their ability to chemosensitize cancer cells to anti-cancer drugs, such as gemcitabine, with little toxicity to normal tissues. These probe molecules have been deployed as tools to study cap-dependent translation in a variety of biological contexts. Consequently, our group looks to expand the development of AHA-ProTides as a novel approach for the design of anti-viral and anti-cancer pronucleotides with the ability to be administered through either IV or oral dosing.

Key Accomplishments:

· Demonstration that nucleotide phosphoramidates can serve as pronucleotides for the delivery of nucleoside mono-phosphates.

· Demonstration that phosphoramidate pronucleotides undergo P-N bond hydrolysis intracellularly through the enzymatic activity of HINT1.

· Development of an in depth kinetic and structural substrate specificity map to guide pronucleotide design.

· Development of anchimerically assisted pronucleotides that can be dosed either IV or orally.

• Li, Shui, Jia, Yan, Jacobson, Blake, McCauley, Joel, Kratzke, Robert, Bitterman, Peter E., and Wagner, Carston R., “Treatment of Breast and Lung Cancer Cells with a N-7 Benzyl Guanosine Monophosphate Tryptamine Phosphoramidate Pronucleotide (4Ei-1) Results in Chemosensitization to Gemcitabine and Induced eIF4E Proteasomal Degradation”, Molecular Pharmaceutics 10, 523–531 (2013)

• Smith, Karen A., Zhou, Bo, Avdulov, Svetlana, Benyumov, Alexey, Peterson, Mark, Liu, Yi, Okon, Aniekan, Hergert, Paul, Braziunas, Jeffrey, Wagner, Carston R., Borok, Zeda and Bitterman, Peter B., “Transforming Growth Factor-1 Induced Epithelial Mesenchymal Transition Is Blocked By A Chemical Antagonist Of Translation Factor EIF4E”, Scientific Reports, 5, 18233, doi:10.1038/srep18233 (2015)

• Okon, Aniekan, Romário Matos de Souza, Marcos, Shah, Rachit, Amorim, Raquel Jesus da Costa, Luciana and Wagner, Carston, R., Anchimerically Activated Antiviral ProTides, ACS Medicinal Chemistry Letters, ASAP (2017).