IntroductionHuman telomerase is a ribonucleoprotein complex composed of the protein telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC1) that acts as a template. TERT has emerged as a crucial drug target in cancer research due to its reactivation being essential for the unlimited proliferation potential of human cancer cells2,3,4. Numerous TERT-specific compounds designed to inhibit its reverse transcriptase activity have been identified and developed as anti-cancer drugs aimed at limiting tumor growth5,6. Conversely, TERT represents a therapeutic target for cell regeneration, as shortened telomeres are associated with various chronic diseases7. Moreover, TERT activity is relevant for anti-aging and regenerative therapies since it extends telomeric ends by adding hexameric repeats (TTAGGG)8,9. While TERT supplies the enzymatic activity critical for DNA synthesis, TERC serves as the template for forming these telomeric DNA repeats10.The TERT/TERC ribonucleoprotein complex contains two key binding sites: an ATP-binding site and a magnesium-binding site, both of which are essential for its activity11,12. The ATP-binding site shares structural homology with the ATP-binding site of HIV reverse transcriptase, which has enabled the repurposing of several antiviral reverse transcriptase inhibitors for cancer treatment13,14. However, there are currently no TERT-specific drugs approved by the FDA. The development of TERT inhibitors has become a pivotal focus in anticancer therapy due to their potential to restrict the immortality of cancer cells. Small molecule inhibitors, such as BIBR1532, have been shown to directly interact with TERT, thereby inhibiting its activity15. In addition, the natural substance epigallocatechin-3-gallate (EGCG) has shown significant inhibitory effects on TERT function16. These inhibitors often function by obstructing the binding sites critical for the enzyme’s function, such as the ATP-binding site and the RNA interaction domain. Targeted approaches have also been explored, such as using antisense oligonucleotides to downregulate TERT expression. For example, GRN163L (Imetelstat), a synthetic oligonucleotide, binds to the RNA template region of TERC, effectively blocking the elongation of telomeres17. This inhibition of TERT activity leads to progressive telomere shortening that induces senescence and apoptosis in cancer cells. GRN163L is subject to several past and active clinical trials. One innovative strategy is the use of immunotherapy to target TERT18. Vaccines that elicit an immune response against TERT-expressing cells have shown promise in preclinical studies, offering a potential route to specifically target cancer cells while sparing normal cells. Additionally, combination therapies that involve TERT inhibitors and conventional chemotherapy or radiotherapy are being studied to enhance the overall efficacy of cancer treatments19,20. These varied approaches underscore the significance of TERT as a multifaceted target in cancer therapy. However, challenges such as the need for improved specificity and reduced off-target effects remain. Continued research and development in this domain hold the potential to yield highly effective therapeutic strategies against cancer and other telomere-related diseases. To facilitate the evaluation of larger compound libraries, simpler assay systems are required in addition to the frequently used but complex telomeric repeat amplification protocol (TRAP) assay21. In this study, we investigate the development of an effective nonradioactive chip-based assay for screening potential TERT inhibitors22,23,24. TERT was obtained either as recombinant protein synthesized in E. coli and purified via Immobilized Metal Affinity Chromatography (IMAC) or derived from cell culture lysates to test compound binding.We have developed a microarray-based TERT binding assay that exploits the ability of the TERT complex to bind single-stranded 5’-TTAGGG-3' repeats. It is hypothesized that substances affecting this binding can modulate TERT activity25,26. The microarray-based assay revealed that epigallocatechin-3-gallate (EGCG), a known TERT activity inhibitor, demonstrated dose-responsive inhibitory effects. This targeted assay format was employed to test various compounds, including novel cytochalasin derivatives that were generated through mutational and semisynthetic approaches. These derivatives were purified from Pyricularia grisea strains and assessed for binding to TERT27,28,29. Cytochalasins are a diverse group of specialised metabolites. Their major cellular target is the polymerisation of actin. However, some non-actin targets are known such as the GLUT1 transporter30. Their potential effects on non-actin targets such as TERT may suggest a new avenue for therapeutic intervention31.ResultsPrior to the development of a binding assay for TERT inhibitors, we investigated the molecular interaction of the known TERT inhibitor, epigallocatechin-3-gallate (EGCG), with TERT through in silico docking experiments. Previous studies have demonstrated the inhibitory activity of EGCG on TERT25,26,32,33. The architecture of human TERT, coupled with the RNA sequence TERC, the DNA primer 5’-TTAGGGTTAGGGTTAGGG-3', one ATP molecule, and ten magnesium ions, was modeled using AlphaFold334,35. The modeled TERT structure shows the DNA primer threaded through a pore into a protein cavity, where it is bound in contact with the complementary RNA sequence, while ATP, along with two magnesium ions, is bound at the end of the primer sequence (Fig. 1A–D, TERT1.pdb; Figure S1). Structural alignment with the crystal structure of the insect telomerase from Tribolium castaneum (PDB:6USO)—a structural homolog to human TERT—revealed high domain homology (Fig. 1C) providing support for the model structure. The modeled TERT structure without TERC and DNA primer was used for docking experiments with EGCG using the software Diff-Dock-L36. The program tested 100 positions for binding at TERT and identified different binding position with a calculated affinity35,36,37,38,39,40. Positions with a binding affinity lower than zero are marked in red (Figure S2A) and mapped to the TERT structure (Figure S2B). Predictions indicate that EGCG could bind with the ATP’s adenine ring or the magnesium ions and phosphate backbone. EGCG predicted a binding affinity of − 3.6 kcal/mol at the nucleotide-binding site (Figure S2C, yellow) and − 2.8 kcal/mol near to a magnesium binding site (white, Figure S2C,41).Fig. 1Complex TERT architecture generated by AlphaFold3. (A) Visualization of TERT electrostatic surface charges of the protein architecture with a calculated electronegativity between -5 and + 521. (B) Presentation of the complex structure of TERT including TERC-RNA sequence in wheat and the nucleotides as tubes whereas the DNA primer given in magenta sticks and magnesium dots in green. (C) Inside the TERT cavity at the end of the bound DNA-primer showing three dG´s (magenta sticks), bound ATP with two magnesium ions and complementary paired RNA (green sticks) from TERC sequence. (D) Presentation as a cartoon of the alignment of the TERT complex and crystal structure from insect telomerase (pdb: 6USO).Full size imageTo validate the in-silico findings with a binding assay, we used several cell lines that express TERT; and human TERT obtained through recombinant expression in E. coli for the development of a highly miniaturized microarray-based nonradioactive TERT assay24. TERT was synthesized using pET28aTERT in E. coli and purified via Ni-IMAC (Figure S3). The TRAP assay confirmed the presence of TERT in NIH3T3 and HeLa cells, but not in HUVEC cells due to high passages (Fig. 2, Figure S4). For the microarray-based TERT binding assay, Hsp90α, purified TERT, and cell lysates were spotted onto nitrocellulose microarray slides in columns of ten dots as described earlier for other target-oriented tests on microarrays (Fig. 3). The results were evaluated using the same experimental system as reported in previous studies42,43,44,45,46,47. Hsp90α served as a control for nonspecific primer binding. The microarrays were incubated with a primer cocktail containing TERC, 5'-Cy5-TTAGGGTTAGGGTTAGGG-3' (Cy5-primer), Cy5-ATP, and dNTPs to enable pairing for enhanced binding (Fig. 3A). Only Cy5-ATP incubation showed ATP binding by human Hsp90α (Fig. 3B), whereas the addition of the primer cocktail led to fluorescence emission at all cell lysates and purified TERT (Fig. 3C). The advantage of this highly miniaturized TERT assay is the low material consumption, as between 800 and 1600 pL are used for a single spot at a Hsp90 concentration of 3 mg/mL. This would allow thousands of tests to be conducted from a single 50 µL cell lysate, for example. Another advantage is that the microarrays with Hsp90 are storable with printed proteins for at least one month. The microarray-based assay was utilized to test EGCG (Fig. 4A,B), a known TERT inhibitor25, by performing a dose-dependent titration using cell lysates or purified recombinant TERT at concentrations ranging from 0.01 to 100 µM (Figure S5A with the complete microarray). The main fluorescence signal originated from Cy5-primer, stabilized by the presence of TERC, Cy5-ATP, and dNTPs (Fig. 4B, Figure S5A with the complete microarray). For better visualization a section of three pads is shown in Fig. 4B. The binding of the fluorescence label was reduced as a function of EGCG concentration in a dose-responsive manner for purified TERT and cell lysates with an EC50 between 1.26 and 1.76 µM (Fig. 4C). The thermophoresis assay also estimated TERT’s affinity for EGCG, identifying a dose-responsive affinity with a Kd of ~ 26 µM (Fig. 4D). The observed difference in microarray-based TERT and thermophoresis assays may result from different accessibilities of the primer cocktail, in the microarray-based assay the proteins become fixed to the nitrocellulose surface. The TRAP assay further validated the inhibitory activity of EGCG, demonstrating dose-dependent inhibition within a range of 1 µM to 15 µM (Fig. 4E,F, Figure S6). This result corroborates the microarray assay’s screening capability for novel TERT-binding compounds.Fig. 2TRAP for detection of telomerase activity. Different concentration of recombinant hTERT isolated from E. coli and purified with Ni-IMAC, HeLa, NIH3T3 and HUVEC at passage 3 (P3) and 9 (P9) cell lysates is shown. Δ indicates corresponding heat inactivated cell lysate.Full size imageFig. 3Development of a microarray based binding assay monitoring TERT activity. (A) Cell lysates-containing TERT or recombinant expressed become spotted on nitrocellulose pad of a microarray using spotting technology (1). Stabilization of the nucleo-complex at TERT after incubation of microarray slide in chambers with TERC and Cy5-primer (2). Monitoring of bound fluorescence after washing and drying of the microarray slide (3). Lower panel shows the identified fluorescence spots of the microarray. Standard incubation chamber is shown left with inset for separation of pads. (B) ATP-binding assay using 100 nM ATP-Cy5 or (C) as cocktail containing 10 nM Cy5-primer100 nM dNTP, 0.1 µg TERC-RNA and 10 nM Cy5-ATP diluted in buffer containing 20 mM Hepes, 50 mM KCL, 5 mM MgCl2, 0.01% Tween 20, 0.1 mg/mL BSA, 1 mM DTT on spotted proteins (human Hsp90a or recombinant TERT) or cell lysates. The proteins or cell lysates were spotted in columns of ten dots.Full size imageFig. 4Evaluation of the binding affinity of epigallocatechin-3-gallate (EGCG) for TERT. (A) Structure of EGCG. (B) Microarray based binding assay monitoring TERT binding activity. The proteins or cell lysates were spotted in columns of ten dots. (left pads) EGCG series in presence of 100 nM Cy5-ATP; (right pads) EGCG series in presence of 10 nM primer cocktail. All binding experiments performed in binding buffer. The complete microarray is shown in Figure S5A, whereas in B is shown a section of six pads and in presence of 10 µM, 50 µM or 100 µM EGCG, respectively. (C) Histogram of fluorescence intensities (Φ) as a function EGCG concentration at proteins or cell lysates. The mean of the fluorescence intensities and standard deviation obtained from the microarray was used to estimate relative fluorescence as a function of different concentrations of EGCG from 0.01 to 100 µM. (D) Microscale thermophoresis (MST) experiment performed with TERT. Affinity of substances was performed with unlabelled TERT. Unlabelled TERT protein (30 µg) was incubated with primer cocktail and substance concentration as indicated overnight at 4° C. The ΔF data were normalized and fitted in the Kd model. (inset) Relative fluorescence as a function of time at different EGCG concentration. (E) Dose-dependent inhibition of cell lysate with high TERT activity in a TRAP assay by EGCG, observed across concentrations ranging from 1 to 15 µM. ∆ indicates heat inactivated cell lysate. (F) Quantification of respective TRAP samples. n = 3; *p