RNA-methylation-dependent RNA processing controls the speed of the circadian clock.

The eukaryotic biological clock involves a negative transcription-translation feedback loop in which clock genes regulate their own transcription and that of output genes of metabolic significance. While around 10% of the liver transcriptome is rhythmic, only about a fifth is driven by de novo transcription, indicating mRNA processing is a major circadian component. Here, we report that inhibition of transmethylation reactions elongates the circadian period. RNA sequencing then reveals methylation inhibition causes widespread changes in the transcription of the RNA processing machinery, associated with m(6)A-RNA methylation. We identify m(6)A sites on many clock gene transcripts and show that specific inhibition of m(6)A methylation by silencing of the m(6)A methylase Mettl3 is sufficient to elicit circadian period elongation and RNA processing delay. Analysis of the circadian nucleocytoplasmic distribution of clock genes Per2 and Arntl then revealed an uncoupling between steady-state pre-mRNA and cytoplasmic mRNA rhythms when m(6)A methylation is inhibited.

N6-methyltubercidin gives sterile cure in a cutaneous Leishmania amazonensis mouse model.

Leishmania is a trypanosomatid parasite that causes skin lesions in its cutaneous form. Current therapies rely on old and expensive drugs, against which the parasites have acquired considerable resistance. Trypanosomatids are unable to synthesize purines relying on salvaging from the host, and nucleoside analogues have emerged as attractive antiparasitic drug candidates. 4-Methyl-7-ß-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine (CL5564), an analogue of tubercidin in which the amine has been replaced by a methyl group, demonstrates activity against Trypanosoma cruzi and Leishmania infantum. Herein, we investigated its in vitro and in vivo activity against L. amazonensis. CL5564 was 6.5-fold (P = 0.0002) more potent than milteforan™ (ML) against intracellular forms in peritoneal mouse macrophages, and highly selective, while combination with ML gave an additive effect. These results stimulated us to study the activity of CL5564 in mouse model of cutaneous Leishmania infection. BALB/c female and male mice infected by L. amazonensis treated with CL5564 (10 mg kg−1, intralesional route for five days) presented a >93% reduction of paw lesion size likely ML given orally at 40 mg kg−1, while the combination (10 + 40 mg kg−1 of CL5564 and ML, respectively) caused >96% reduction. The qPCR confirmed the suppression of parasite load, but only the combination approach reached 66% of parasitological cure. These results support additional studies with nucleoside derivatives.

Enhancing nicotinamide N-methyltransferase bisubstrate inhibitor activity through 7-deazaadenosine and linker modifications.

Nicotinamide N-methyltransferase (NNMT) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide (NAM) and other pyridine-related compounds and is involved in various metabolic processes in the human body. In addition, abnormal expression of NNMT occurs under various pathological conditions such as cancer, diabetes, metabolic disorders, and neurodegenerative diseases, making it a promising drug target worthy of in-depth research. Small-molecule NNMT inhibitors with high potency and selectivity are necessary chemical tools to test biological hypotheses and potential therapies. In this study, we developed a series of highly active NNMT inhibitors by modifying N7 position of adenine. Among them, compound 3-12 (IC50 = 47.9 ± 0.6 nM) exhibited potent inhibitory activity and also had an excellent selectivity profile over a panel of human methyltransferases. We showed that the N7 position of adenine in the NNMT bisubstrate inhibitor was a modifiable site, thus offering insights into the development of NNMT inhibitors.

Impact of 3-deazapurine nucleobases on RNA properties.

Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c7G, c7A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.

Cloning and Characterization of Trypanosoma congolense and T. vivax Nucleoside Transporters Reveal the Potential of P1-Type Carriers for the Discovery of Broad-Spectrum Nucleoside-Based Therapeutics against Animal African Trypanosomiasis.

African Animal Trypanosomiasis (AAT), caused predominantly by Trypanosoma brucei brucei, T. vivax and T. congolense, is a fatal livestock disease throughout Sub-Saharan Africa. Treatment options are very limited and threatened by resistance. Tubercidin (7-deazaadenosine) analogs have shown activity against individual parasites but viable chemotherapy must be active against all three species. Divergence in sensitivity to nucleoside antimetabolites could be caused by differences in nucleoside transporters. Having previously characterized the T. brucei nucleoside carriers, we here report the functional expression and characterization of the main adenosine transporters of T. vivax (TvxNT3) and T. congolense (TcoAT1/NT10), in a Leishmania mexicana cell line ('SUPKO') lacking adenosine uptake. Both carriers were similar to the T. brucei P1-type transporters and bind adenosine mostly through interactions with N3, N7 and 3'-OH. Expression of TvxNT3 and TcoAT1 sensitized SUPKO cells to various 7-substituted tubercidins and other nucleoside analogs although tubercidin itself is a poor substrate for P1-type transporters. Individual nucleoside EC50s were similar for T. b. brucei, T. congolense, T. evansi and T. equiperdum but correlated less well with T. vivax. However, multiple nucleosides including 7-halogentubercidines displayed pEC50>7 for all species and, based on transporter and anti-parasite SAR analyses, we conclude that nucleoside chemotherapy for AAT is viable.

Identification of natural compounds tubercidin and lycorine HCl against small-cell lung cancer and BCAT1 as a therapeutic target.

Although small-cell lung cancer (SCLC) accounts for a small fraction of lung cancer cases (~15%), the prognosis of patients with SCLC is poor with an average overall survival period of a few months without treatment. Current treatments include standard chemotherapy, which has minimal efficacy and a newly developed immunotherapy that thus far, benefits a limited number of patients. In the current study, we screened a natural product library and identified 5 natural compounds, in particular tubercidin and lycorine HCl, that display prominent anti-SCLC activities in vitro and in vivo. Subsequent RNA-sequencing and functional validation assays revealed the anti-SCLC mechanisms of these new compounds, and further identified new cellular factors such as BCAT1 as a potential therapeutic target with clinical implication in SCLC patients. Taken together, our study provides promising new directions for fighting this aggressive lung cancer.

Anomeric DNA: Functionalization of α-d Anomers of 7-Deaza-2'-deoxyadenosine and 2'-Deoxyuridine with Clickable Side Chains and Click Adducts in Homochiral and Heterochiral Double Helices.

Anomeric base pairs in heterochiral DNA with strands in the α-d and ß-d configurations and homochiral DNA with both strands in α-d configuration were functionalized. The α-d anomers of 2'-deoxyuridine and 7-deaza-2'-deoxyadenosine were synthesized and functionalized with clickable octadiynyl side chains. Nucleosides were protected and converted to phosphoramidites. Solid-phase synthesis furnished 12-mer oligonucleotides, which were hybridized. Pyrene click adducts display fluorescence, a few of them with excimer emission. Tm values and thermodynamic data revealed the following order of duplex stability α/α-d≫ß/ß-d≥α/ß-d. CD spectra disclosed that conformational changes occur during hybridization. Functionalized DNAs were modeled and energy minimized. Clickable side chains and bulky click adducts are well accommodated in the grooves of anomeric DNA. The investigation shows for the first time that anomeric DNAs can be functionalized in the same way as canonical DNA for potential applications in nucleic acid chemistry, chemical biology, and DNA material science.

Loss of haspin suppresses cancer cell proliferation by interfering with cell cycle progression at multiple stages.

Our recent studies have shown that haspin, a protein kinase imperative for mitosis, is engaged in the interphase progression of HeLa and U2OS cancer cells. In this investigation, we employed the Fucci reporter system and time-lapse imaging to examine the impact of haspin gene silencing on cell cycle progressions at a single-cell level. We found that the loss of haspin induced multiple cell cycle defects. Specifically, the S/G2 duration was greatly prolonged by haspin gene depletion or inhibition in synchronous HeLa cells. Haspin gene depletion in asynchronous HeLa and U2OS cells led to a similarly protracted S/G2 phase, followed by mitotic cell death or postmitotic G1 arrest. In addition, haspin deficiency resulted in robust induction of the p21CIP1/WAF1 checkpoint protein, a target of the p53 activation. Also, co-depleting haspin with either p21 or p53 could rescue U2OS cells from postmitotic G1 arrest and partially restore their proliferation. These results substantiate the haspin's capacity to regulate interphase and mitotic progression, offering a broader antiproliferative potential of haspin loss in cancer cells.

The Trypanosoma cruzi TcrNT2 Nucleoside Transporter Is a Conduit for the Uptake of 5-F-2'-Deoxyuridine and Tubercidin Analogues.

Among the scarce validated drug targets against Chagas disease (CD), caused by Trypanosoma cruzi, the parasite's nucleoside salvage system has recently attracted considerable attention. Although the trypanocidal activity of tubercidin (7-deazapurine) has long been known, the identification of a class of 7-substituted tubercidin analogs with potent in vitro and in vivo activity and much-enhanced selectivity has made nucleoside analogs among the most promising lead compounds against CD. Here, we investigate the recently identified TcrNT2 nucleoside transporter and its potential role in antimetabolite chemotherapy. TcrNT2, expressed in a Leishmania mexicana cell line lacking the NT1 nucleoside transporter locus, displayed very high selectivity and affinity for thymidine with a Km of 0.26 ± 0.05 µM. The selectivity was explained by interactions of 2-oxo, 4-oxo, 5-Me, 3'-hydroxy and 5'-hydroxy with the transporter binding pocket, whereas a hydroxy group at the 2' position was deleterious to binding. This made 5-halogenated 2'-deoxyuridine analogues good substrates but 5-F-2'-deoxyuridine displayed disappointing activity against T. cruzi trypomastigotes. By comparing the EC50 values of tubercidin and its 7-substituted analogues against L. mexicana Cas9, Cas9ΔNT1 and Cas9ΔNT1+TcrNT2 it was shown that TcrNT2 can take up tubercidin and, at a minimum, a subset of the analogs.

Halogen Bonding in Haspin-Halogenated Tubercidin Complexes: Molecular Dynamics and Quantum Chemical Calculations.

Haspin, an atypical serine/threonine protein kinase, is a potential target for cancer therapy. 5-iodotubercidin (5-iTU), an adenosine derivative, has been identified as a potent Haspin inhibitor in vitro. In this paper, quantum chemical calculations and molecular dynamics (MD) simulations were employed to identify and quantitatively confirm the presence of halogen bonding (XB), specifically halogen∙∙∙π (aromatic) interaction between halogenated tubercidin ligands with Haspin. Consistent with previous theoretical finding, the site specificity of the XB binding over the ortho-carbon is identified in all cases. A systematic increase of the interaction energy down Group 17, based on both quantum chemical and MD results, supports the important role of halogen bonding in this series of inhibitors. The observed trend is consistent with the experimental observation of the trend of activity within the halogenated tubercidin ligands (F < Cl < Br < I). Furthermore, non-covalent interaction (NCI) plots show that cooperative non-covalent interactions, namely, hydrogen and halogen bonds, contribute to the binding of tubercidin ligands toward Haspin. The understanding of the role of halogen bonding interaction in the ligand-protein complexes may shed light on rational design of potent ligands in the future.

Exploring the thermodynamic, kinetic and inhibitory mechanisms of 5-iTU targeting mitotic kinase haspin by integrated molecular dynamics.

As a human mitotic kinase, haspin is considered as a promising target for various diseases including cancers. However, no inhibitors targeting haspin have entered clinical trials presently. 5-iTU (5-iodotubercidin) is a useful and classical chemical probe for the investigation of haspin activity, but its inhibitory mechanism remains unclear. In this study, integrated molecular dynamics (MD) of conventional MD, extended adaptive biasing force (eABF), random acceleration MD and well-tempered metadynamics were applied to investigate the thermodynamic and kinetic features of 5-iTU and three derivatives targeting haspin. To emphasize the importance of gatekeeper Phe605, two haspin mutants (F605Y and F605T) were also built. The results showed that the binding affinity of 5-iTU and haspin was highest in all wild type (WT) systems, relying on the strong halogen aromatic π interaction between 5-iTU and gatekeeper Phe605. Gatekeeper mutations, because of damage to this interaction, led to the rearrangement of water distributions at the binding site and the decrease of 5-iTU residence times. Additionally, compared with the smaller 5-fTU, 5-iTU dissociated from WT haspin with more difficulty through distinct unbinding pathways. These findings will provide crucial guidance for the design and development of novel haspin inhibitors and the rational modification of existing inhibitors.

Uncoupling of nucleo-cytoplasmic RNA export and localization during stress.

Eukaryotic cells contain sub-cellular compartments that are not membrane bound. Some structures are always present, such as nuclear speckles that contain RNA-binding proteins (RBPs) and poly(A)+ RNAs. Others, like cytoplasmic stress granules (SGs) that harbor mRNAs and RBPs, are induced upon stress. When we examined the formation and composition of nuclear speckles during stress induction with tubercidin, an adenosine analogue previously shown to affect nuclear speckle composition, we unexpectedly found that it also led to the formation of SGs and to the inhibition of several crucial steps of RNA metabolism in cells, thereby serving as a potent inhibitor of the gene expression pathway. Although transcription and splicing persisted under this stress, RBPs and mRNAs were mislocalized in the nucleus and cytoplasm. Specifically, lncRNA and RBP localization to nuclear speckles was disrupted, exon junction complex (EJC) recruitment to mRNA was reduced, mRNA export was obstructed, and cytoplasmic poly(A)+ RNAs localized in SGs. Furthermore, nuclear proteins that participate in mRNA export, such as nucleoporins and mRNA export adaptors, were mislocalized to SGs. This study reveals structural aspects of granule assembly in cells, and describes how the flow of RNA from the nucleus to the cytoplasm is severed under stress.

5'-Iodotubercidin represses insulinoma-associated-1 expression, decreases cAMP levels, and suppresses human neuroblastoma cell growth.

Insulinoma-associated-1 (INSM1) is a key protein functioning as a transcriptional repressor in neuroendocrine differentiation and is activated by N-Myc in human neuroblastoma (NB). INSM1 modulates the phosphoinositide 3-kinase (PI3K)-AKT Ser/Thr kinase (AKT)-glycogen synthase kinase 3ß (GSK3ß) signaling pathway through a positive-feedback loop, resulting in N-Myc stabilization. Accordingly, INSM1 has emerged as a critical player closely associated with N-Myc in facilitating NB cell growth. Here, an INSM1 promoter-driven luciferase-based screen revealed that the compound 5'-iodotubercidin suppresses adenosine kinase (ADK), an energy pathway enzyme, and also INSM1 expression and NB tumor growth. Next, we sought to dissect how the ADK pathway contributes to NB tumor cell growth in the context of INSM1 expression. We also found that 5'-iodotubercidin inhibits INSM1 expression and induces an intra- and extracellular adenosine imbalance. The adenosine imbalance, which triggers adenosine receptor-3 signaling that decreases cAMP levels and AKT phosphorylation and enhances GSK3ß activity. We further observed that GSK3ß then phosphorylates ß-catenin and promotes the cytoplasmic proteasomal degradation pathway. 5'-Iodotubercidin treatment and INSM1 inhibition suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) activity and the AKT signaling pathways required for NB cell proliferation. The 5'-iodotubercidin treatment also suppressed ß-catenin, lymphoid enhancer-binding factor 1 (LEF-1), cyclin D1, N-Myc, and INSM1 levels, ultimately leading to apoptosis via caspase-3 and p53 activation. The identification of the signaling pathways that control the proliferation of aggressive NB reported here suggests new options for combination treatments of NB patients.

Haspin inhibition delays cell cycle progression through interphase in cancer cells.

Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a serine/threonine kinase pertinent to normal mitosis progression and mitotic phosphorylation of histone H3 at threonine 3 in mammalian cells. Different classes of small molecule inhibitors of haspin have been developed and utilized to investigate its mitotic functions. We report herein that applying haspin inhibitor CHR-6494 or 5-ITu at the G1/S boundary could delay mitotic entry in synchronized HeLa and U2OS cells, respectively, following an extended G2 or the S phase. Moreover, late application of haspin inhibitors at S/G2 boundary is sufficient to delay mitotic onset in both cell lines, thereby, indicating a direct effect of haspin on G2/M transition. A prolonged interphase duration is also observed with knockdown of haspin expression in synchronized and asynchronous cells. These results suggest that haspin can regulate cell cycle progression at multiple stages at both interphase and mitosis.

First Total Synthesis of 5'-O-α-d-Glucopyranosyl Tubercidin.

The first total synthesis of 5'-O-α-d-glucopyranosyl tubercidin was successfully developed. It is a structurally unique disaccharide 7-deazapurine nucleoside exhibiting fungicidal activity, and was isolated from blue-green algae. The total synthesis was accomplished in eight steps with 27% overall yield from commercially available 1-O-acetyl-2,3,5-tri-O-benzoyl-ß-d-ribose. The key step involves stereoselective α-O-glycosylation of the corresponding 7-bromo-6-chloro-2',3'-O-isopropylidene-ß-d-tubercidin with 2,3,4,6-tetra-O-benzyl-glucopyranosyl trichloroacetimidate. All spectra are in accordance with the reported data for natural 5'-O-α-d-glucopyranosyl tubercidin. Meanwhile, 5'-O-ß-d-glucopyranosyl tubercidin was also prepared using the same strategy.

Tricyclic Nucleobase Analogs and Their Ribosides as Substrates and Inhibitors of Purine-Nucleoside Phosphorylases III. Aminopurine Derivatives.

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2--D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2--d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9- -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.

Novel Nucleoside Analogues as Effective Antiviral Agents for Zika Virus Infections.

Previously considered a neglected flavivirus, Zika virus has recently emerged as a public health concern due to its ability to spread rapidly and cause severe neurological disorders, such as microcephaly in newborn babies from infected mothers, and Guillain-Barré syndrome in adults. Despite extensive efforts towards the identification of effective therapies, specific antivirals are still not available. As part of ongoing medicinal chemistry studies to identify new antiviral agents, we screened against Zika virus replication in vitro in a targeted internal library of small-molecule agents, comprising both nucleoside and non-nucleoside agents. Among the compounds evaluated, novel aryloxyphosphoramidate prodrugs of the nucleosides 2'-C-methyl-adenosine, 2-CMA, and 7-deaza-2'C-methyl-adenosine, 7-DMA, were found to significantly inhibit the virus-induced cytopathic effect in multiple relevant cell lines. In addition, one of these prodrugs exhibits a synergistic antiviral effect against Zika virus when applied in combination with an indirect antiviral agent, a l-dideoxy bicyclic pyrimidine nucleoside analogue, which potently inhibits vaccinia and measles viruses in vitro by targeting a host pathway. Our findings provide a solid basis for further development of an antiviral therapy for Zika virus infections, possibly exploiting a dual approach combining two different agents, one targeting the viral polymerase (direct-acting antiviral), the second targeting a host-directed autophagy mechanism.

Viral RNA-Dependent RNA Polymerase Inhibitor 7-Deaza-2'-C-Methyladenosine Prevents Death in a Mouse Model of West Nile Virus Infection.

West Nile virus (WNV) is a medically important emerging arbovirus causing serious neuroinfections in humans and against which no approved antiviral therapy is currently available. In this study, we demonstrate that 2'-C-methyl- or 4'-azido-modified nucleosides are highly effective inhibitors of WNV replication, showing nanomolar or low micromolar anti-WNV activity and negligible cytotoxicity in cell culture. One representative of C2'-methylated nucleosides, 7-deaza-2'-C-methyladenosine, significantly protected WNV-infected mice from disease progression and mortality. Twice daily treatment at 25 mg/kg starting at the time of infection resulted in 100% survival of the mice. This compound was highly effective, even if the treatment was initiated 3 days postinfection, at the time of a peak of viremia, which resulted in a 90% survival rate. However, the antiviral effect of 7-deaza-2'-C-methyladenosine was absent or negligible when the treatment was started 8 days postinfection (i.e., at the time of extensive brain infection). The 4'-azido moiety appears to be another important determinant for highly efficient inhibition of WNV replication in vitro However, the strong anti-WNV effect of 4'-azidocytidine and 4'-azido-aracytidine was cell type dependent and observed predominantly in porcine kidney stable (PS) cells. The effect was much less pronounced in Vero cells. Our results indicate that 2'-C-methylated or 4'-azidated nucleosides merit further investigation as potential therapeutic agents for treating WNV infections as well as infections caused by other medically important flaviviruses.

Triazine-Modified 7-Deaza-2'-deoxyadenosines: Better Suited for Bioorthogonal Labeling of DNA by PCR than 2'-Deoxyuridines.

6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labeling of oligonucleotides by Diels-Alder reactions with inverse electron demand. We synthetically attached this functional group to the 7-position of 7-deaza-2'-deoxyadenosine triphosphate and to the 5-position of 2'-deoxyuridine triphosphate. Both modified nucleotide triphosphates were used in comparison for primer extension experiments (PEX) and PCR amplification to finally yield multilabeled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]nonyne-rhodamine conjugate. These experiments show that 6-ethynyl-1,2,4-triazine is much better tolerated by the DNA polymerase when attached to the 7-position of 7-deaza-2'-deoxyadenosine in comparison to the attachment at the 5-position of 2'-deoxyuridine. This became evident both by PAGE analysis of the PCR products and real-time kinetic observation of DNA polymerase activity during primer extension using switchSENSE. Generally, our results imply that bioorthogonal labeling strategies are better suited for 7-deaza-2'-adenosines than conventional and available 2'-deoxyuridines.

Transient use of a systemic adenosine kinase inhibitor attenuates epilepsy development in mice.

OBJECTIVE: Over one-third of all patients with epilepsy are refractory to treatment and there is an urgent need to develop new drugs that can prevent the development and progression of epilepsy. Epileptogenesis is characterized by distinct histopathologic and biochemical changes, which include astrogliosis and increased expression of the adenosine-metabolizing enzyme adenosine kinase (ADK; EC 2.7.1.20). Increased expression of ADK contributes to epileptogenesis and is therefore a target for therapeutic intervention. We tested the prediction that the transient use of an ADK inhibitor administered during the latent phase of epileptogenesis can mitigate the development of epilepsy. METHODS: We used the intrahippocampal kainic acid (KA) mouse model of temporal lobe epilepsy, which is characterized by ipsilateral hippocampal sclerosis with granule cell dispersion and the development of recurrent hippocampal paroxysmal discharges (HPDs). KA-injected mice were treated with the ADK inhibitor 5-iodotubercidin (5-ITU, 1.6 mg/kg, b.i.d., i.p.) during the latent phase of epileptogenesis from day 3-8 after injury; the period when gradual increases in hippocampal ADK expression begin to manifest. HPDs were assessed at 6 and 9 weeks after KA administration followed by epilepsy histopathology including assessment of granule cell dispersion, astrogliosis, and ADK expression. RESULTS: 5-ITU significantly reduced the percent time in seizures by at least 80% in 56% of mice at 6 weeks post-KA. This reduction in seizure activity was maintained in 40% of 5-ITU-treated mice at 9 weeks. 5-ITU also suppressed granule cell dispersion and prevented maladaptive ADK increases in these protected mice. SIGNIFICANCE: Our results show that the transient use of a small-molecule ADK inhibitor, given during the early stages of epileptogenesis, has antiepileptogenic disease-modifying properties, which provides the rationale for further investigation into the development of a novel class of antiepileptogenic ADK inhibitors with increased efficacy for epilepsy prevention.