Metabolism and mechanism of action of cordycepin

Ammar Sabah Karim Khamis

Metabolism and mechanism of action of cordycepin

Publication Date

July 29, 2026

Creator

Ammar Sabah Karim Khamis

University of Nottingham, UK

Abstract

Cordycepin (3'-deoxyadenosine) is a nucleoside analogue with broad anti-inflammatory and antiproliferative effects whose therapeutic development has been limited by rapid deamination to 3'-deoxyinosine (3DI) and an incomplete understanding of how its active nucleotides couple cleavage and polyadenylation (CPA) to growth-factor and inflammatory signalling. This thesis investigates how cordycepin and its metabolites, particularly cordycepin triphosphate (CoTP), modulate mRNA 3'-end processing, poly(A)-tail homeostasis and PI3K/Akt/mTOR signalling, and explores the hypothesis that the effects on polyadenylation mediate the effects on signal transduction through one or more RNA targets of cordycepin (RToC). Deamination of cordycepin to 3'-deoxyinosine (3DI) is known to occur rapidly and limit its potency. To explore the metabolism of cordycepin, siRNA knockdown of enzymes that could recycle 3DI to cordycepin were used. The data showed that, surprisingly, cordycepin activity in tissue culture is dependent on a previously unsuspected rescue pathway involving adenine phosphoribosyltransferase (APRT) and, in macrophages, the enzyme FAMIN/LACC1. These enzymes appear to channel adenine-linked 3'-deoxyribose metabolism, enabling generation of CoTP and repression of LPS-induced inflammatory transcripts. Data for 3DI suggested some differences between 3DI and cordycepin dependence on rescue pathways, but were difficult to interpret because contamination of the 3DI preparations with cordycepin was detected. Second, a haploid piggyBac survival screen in eHAP cells identified three broad classes of genetic determinants of cordycepin resistance. Loss-of-function insertions in ADK were the most consistent and dose-dependent hits, confirming that phosphorylation to cordycepin mono-, di- and triphosphates is essential for cytotoxicity. A second class comprised genes involved in RNA 3'-end formation, deadenylation and RNA maturation (including CNOT6, PARN and Integrator components), indicating that the state of mRNA 3'-end processing modulates sensitivity to CPA inhibition. A third class encompassed genes in AMPK–mTOR signalling, autophagy, mitophagy, ER–mitochondrial coupling and ubiquitin-dependent proteostasis, highlighting integrated stress-response and quality-control pathways that shape survival under cordycepin. Within this screen, the long non-coding RNA locus LINC03121 (Lnc-PLA2G4A-4) showed pronounced enrichment in one platform/dose combination; in light of independent evidence that LINC03121 promotes p-EGFR/p-AKT signalling via a miR-23b-3p/versican axis, this makes it a biologically credible candidate RToC. Third, comparative transcriptomics in LPS-stimulated RAW264.7 macrophages demonstrated that natural fermentation-derived and synthetic cordycepin produced highly similar gene-expression and pathway signatures. Both preparations repressed a broad panel of inflammatory transcripts and down-regulated pathways linked to NF-κB, interferon signalling and PI3K/Akt/mTOR-dependent translation. The ProTide NUC-7738 reproduced these core features when used at exposures that generate comparable intracellular 3'-deoxyadenosine nucleotide levels, whereas 3DI showed a weaker but directionally concordant profile. These findings argue that the shared active species is intracellular CoTP rather than preparation-specific contaminants. Fourth, Oxford Nanopore long-read sequencing revealed that cordycepin and NUC-7738 induced rapid, genome-wide poly(A)-tail shortening at 120 minutes (median shifts of ≈ 25 nt) with thousands of transcripts showing significant tail reduction and concomitant decreases in abundance, particularly among inflammatory and growth-factor-linked mRNAs. A smaller subset of transcripts exhibited tail lengthening and relative sparing of abundance, often enriched for mitochondrial and metabolic functions, suggesting selective stabilisation of messages that support bioenergetic adaptation under stress. Early-timepoint (15-minute) analyses showed that cordycepin already triggers coordinated shifts in poly(A)-tail length and expression in transcripts associated with translation control, signal transduction and growth-factor pathways, before wholesale transcriptional re-programming is established. Taken together, these strands support a model in which cordycepin acts principally as an intracellular nucleoside analogue that must be transported into cells, phosphorylated to CoTP, and then perturbs CPA and poly(A)-tail homeostasis to selectively restrain inflammatory and PI3K/Akt/mTOR outputs. Integrating these results with the literature on protein synthesis inhibition and Akt activation, the thesis advances the hypothesis that this signalling restraint is mediated by one or more non-coding RNAs whose polyadenylation-dependent function is cordycepin-sensitive—an RNA target of cordycepin (RToC). The convergence of strong loss-of-function enrichment for LINC03121 in the haploid screen, its established role as a positive regulator of Akt signalling, and the global evidence for cordycepin-induced CPA disruption leads this work to nominate LINC03121 as a leading candidate RToC, framed as a testable mechanistic hypothesis for future studies.

Item Type

ethesis

Thesis Type

PhD

Supervisors

de Moor, Cornelia

Univesity of Nottingham

Gershkovich Pavel

University of Nottingham