Ethenesulfonyl fluoride derivatives as telomerase inhibitors: structure-based design, SAR, and anticancer evaluation in vitro

Abstract Based on our previous docking model, in order to carry out more rational drug design, totally 82 vinyl sulfonyl fluorides, including some 2-(hetero)arylethenesulfonyl fluorides and 1,3-dienylsulfonyl fluorides derivatives as potential human telomerase inhibitors were designed and synthesised. The in vitro anticancer activity assay showed that compound 57 (1E,3E)-4-(4-((E)-2-(fluorosulfonyl)vinyl)phenyl)buta-1,3-diene-1-sulfonyl fluoride exhibited high activity against A375 and MDA-MB-231 cell lines with IC50 1.58 and 3.22 µM, but it manifested obvious un-toxic effect against GES-1 and L-02 with IC50 with IC50 values less than 2.00 mM. By the modified TRAP assay, some compounds including 57 exhibited potent inhibitory activities against telomerase with IC50 values of 0.71–0.97 µM.


Introduction
Telomerase plays an important role in chromosomal integrity. About 80-90% of various cancer cells have detectable telomerase activity, so, it is considered as a potential anticancer target [1][2][3][4] . In the past decade, based on searching for telomerase inhibitors, different approaches have been designed containing G-quadruplex stabilising inhibitors 5,6 , 2 0 -O-MeRNA oligonucleotides and peptide nucleic acids targeting telomerase RNA 7 , ligands targeting telomeric DNA 8 . But, no compounds that inhibit telomerase activity have reached clinical trials so far 9 .
Among the whole telomerase, human telomerase reverse transcriptase (TERT) is a key component of telomerase. The expression of TERT is a rate-limiting factor for telomerase activity; also, most human somatic cells do not show detectable telomerase activity due to the lack of TERT. Therefore, TERT is an important target for the drug discovery [10][11][12][13] . Some hTERT inhibitors with good anticancer activity, including BIBR1532 14 and isothiazolone derivatives have been discovered [15][16][17][18][19] . However, most of them exhibited potential toxicities against somatic cells, there are no inhibitors targeting hTERT have been approved so far. Therefore, the design of potent hTERT inhibitors with high selectivity against cancer cells and somatic cells is a very important and immediate need.
As we know, chemical environment of the warhead plays a key role in inhibitor design since it enables the tuning of its reactivity and thereby increasing its selectivity and stability 20,21 . Among them, vinyl sulfone, which is a dominant electrophilic trap containing unsaturated sulfone Michael acceptor, which could inhibit both the proteasome and cysteine proteases and high selectivity through manipulation of the peptidic portion 22 . Based on the structure of protein TERT using a LigandFit module, the novel docking model was found. The results showed that LYS 189, GLN 308, and ASP 254 were the key residues for telomerase activity. Focusing on these residues and the volume of the active site of hTERT, the fragment of ethenesulfonyl fluoride could be well aligned with the active site. We, therefore, designed some of the drug-like scaffolds which incorporate the moiety of ethenesulfonyl fluoride derivatives and tested their inhibition activity of telomerase ( Figure 1).

Cell proliferation assays
The antiproliferative activities evaluation was conducted by using a modified procedure as described in the literature 23 . Briefly, target tumour cells were grown to log phase in RPMI 1640 medium supplemented with 10% fetal bovine serum. After diluting to 3 Â 10 4 cells mL À1 with the complete medium, 100 mL of the obtained cell suspension was added to each well of 96-well culture plates. The subsequent incubation was performed at 37 C, 5% CO 2 atmosphere for 24 h before subjecting to antiproliferation assessment. The tested samples at pre-set concentrations were added to six wells with Doxorubicin (AMD) co-assayed as a positive reference. After 48 h of exposure period, 25 mL of PBS containing 2.5 mgÁmL À1 of MTT was added to each well. After 4 h, the medium was replaced by 150 mL DMSO to dissolve the purple formazan crystals produced. The absorbance at 570 nm of each well was measured using an ELISA plate reader. The data represented the mean of three experiments in triplicate and were expressed as means ± SD using Student's test. The IC 50 value was defined as the concentration at which 50% of the cells could survive.

Telomerase activity assays
Thirty-six compounds were tested in a search for inhibitors of telomerase activity using the TRAP-PCR-ELISA assay. In detail, the A375 cells were firstly maintained in RPMI 1640 buffer (Hyclone, Miami, FL), supplemented with 10% fetal bovineserum (GIBCO, New York, NY), streptomycin (0.1 mg/mL) and penicillin (100 IU/mL) at 37 C in a humidified atmosphere containing 5% CO 2 . After trypsinisation, 5 Â 10 4 cultured cells in logarithmic growth were seeded into T25 flasks (Corning, New York, NY) and cultured to allow to adherence. The cells were then incubated with Staurosporine (Santa Cruz, Santa Cruz,CA) and the drugs with a series of concentration as 60, 20, 6.67, 2.22, 0.75, 0.25 and 0.082 g/mL, respectively. After 24 h treatment, the cells were harvested by cell scraper orderly followed by washing once with PBS. The cells were lysed in 150 mL RIPA cell lysis buffer (Santa Cruz, Santa Cruz, CA), and incubated on ice for 30 min. The cellular supernatants were obtained via centrifugation at 12,000g for 20 min at 4 C and stored at -80 C. The TRAP-PCR-ELISA assay was performed using a telomerase detection kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. In brief, 2 mL of cell extracts were mixed with 48 mL TRAP reaction mixtures. TRAP primers and Taq polymerase are incubated at 25 C for 30 min. PCR was then initiated at 94 C, 120 s for predenaturation and performed using 35 cycles each consisting of 94 C for 30 s, 50 C for 30 s, 72 C for 90 s. Then 20 mL of PCR products were hybridised to a digoxigenin (DIG)-labelled telomeric repeat specific detection probe. And the PCR products were immobilised via the biotin-labelled primer to a streptavidin-coated microtiter plate subsequently. The immobilised DNA fragments were detected with a peroxidase-conjugated anti-DIG antibody and visualised following the addition of the stop regent. The microtitre plate was assessed on TECAN Infinite M200 microplate reader (Mannedorf, Switzerland) at a wavelength of 490 nm, and the final value were presented as mean ± SD 23 .

Molecular modelling
The Discovery Studio 2017 was used (Accelrys Software Inc., San Diego, CA). Crystal structure of telomerase TERT (PDB: 3DU6) was used as a template. The active site was defined and sphere of 5 Å was generated around the active site pocket, with the active site pocket of BSAI model using C-DOCKER. The structure of protein, substrate were subjected to energy minimisation using CHARMm forcefield as implemented in DS 2017.

Statistical analysis
All results are expressed as Mean ± SE. Statistical significance was determined either by the Student's t-test for comparison between means or one-way analysis of variance with a post-hoc Dunnett's test.

Chemistry
In our previous study, 82 structurally diverse vinyl sulfonyl fluorides, including some 2-(hetero)arylethenesulfonyl fluorides and 1,3-dienylsulfonyl fluorides, were synthesised on a decent scale using a general method. Our results of molecular construction revealed that Heck-type reaction had a broad scope for aryl iodides comprising various types of ethenesulfonyl fluoride derivatives. Under mild reaction conditions used simple operations, catalytic amounts of Pd(OAc) 2   Based on MDA-MB-231, the further structure-activity relationship was summarised as follows. First, about 2-phenylethenesulfonyl fluoride series, compounds with non-substituted or parasubstituted of the benzene ring showed better activity (compounds 1, 5, 6, 11, 12, 14 and 15); For the meta-substituted compounds, strong electron withdrawing group, such as F, CF 3 , NO 2 reflected great contribution to the activity (compounds 18, 20 and 22). Two substitutions containing Cl were beneficial to the activity (compounds 69 and 71). For the R-ethenesulfonyl fluoride, when R is heterocyclic or benzo heterocyclic, it was not beneficial to the activity (compounds 44, 45, 46, 58 and 59). Second, for the series of diene-1-sulfonyl fluoride, most of these compounds had certain activity (compounds 51-56), among them, compound 2-(fluorosulfonyl)vinyl)phenyl)buta-1,3-diene-1sulfonyl fluoride showed the best activity with IC 50 value of 3.22 mM.

Anticancer activity
It was obvious from the data that most compounds showed good activity against A375 cell (compounds 12, 18, 31, 46, 57 and 70, their IC 50s value is around 3.0 mM), among them, compound 46 possessed the highest activity with the IC 50 value of 1.25 mM, which is comparable with the positive control AMD. For the phenylethenesulfonyl fluoride series, most of the para-substituted derivatives showed good activity except for -CN and -COOEt substitutions. But, in addition to F substitution, most of the meta-substituted derivatives displayed weak activity. We then investigated the SAR profiles of the multi-substituted group of ethenesulfonyl fluoride, compared to phenylethenesulfonyl fluoride moiety, the kind of diene-1-sulfonyl fluoride moiety could not significantly improve the activity.

Inhibition assay of human normal cell
In order to determine the selective cancer cell toxicity of some title compounds. We subsequently conducted a proliferative inhibition assay with human normal liver cell (L-02) and gastric mucosa cell (GES-1). As shown in Table 2, all compounds manifested obvious un-toxic effect on GES-1 and L-02 with IC 50 from 1.33 to 2.80 mM. The data indicated that the title compounds have good selectivity against tumour cells over somatic cells (Tables 1 and 2).

Telomerase activity
Screening results of cell activity showed that most compounds had good activity against A375 cell, due to high expression of telomerase in melanoma cells A375, so, to confirm if the title compounds performed anticancer activity via telomerase inhibition, some selected compounds were evaluated using an extraction from A375 cells. The results are summarised in Table 3. Among them, compounds 5, 31, 36, 57, 46 and 70 showed potent inhibitory activities against telomerase with IC 50 values less than 1.5 mM, better than positive control staurosporine with IC 50 value 8.67 mM. One of them, compound 5 showed the most potent activity against telomerase with IC 50 value of 0.71 mM, which is 12 times as much as the positive control staurosporine.
From the structure-activity relationships presented in Table 3, it can be concluded that most compounds had high telomerase activity. Overall, the compounds with low activity against cancer cell expressed the same weak telomerase activity (compounds 25 and 42). There was also a good correlation between the antiproliferative activity and the IC 50s of telomerase inhibition activity (compounds 31, 47, 57 and 70).
In order to further analyse the SAR, the 82 compounds were divided into two types, seriers one is R-ethenesulfonyl fluoride skeleton and series two is R 1 -diene-1-sulfonyl fluoride skeleton. In general, the activity of series one is generally higher than the activity of series two (compounds 5, 15, 18, 31, 36, 46, 47, 58, 67 with 48 and 55). Further SAR analysis was focused on different substitutes of R. For the series one skeleton, when R was a different substitute phenyl、thick ring and heterocyclic ring showed significant effect on the activity against telomerase (com- pounds 5, 31, 46, 47 and 58). For series two skeleton, the nucleus of different rings had little effect on the activity of telomerase (compounds 48 and 55). The result of enzyme inhibition test showed that title compounds had moderate activity against telomerase. For this reason, it is not certain that TERT is the only protein target, responsible for the biological activity.

Conclusions
Inspired by the dual warhead approach, based on our previous TERT model of rational drug design, in this study, 82 sulfonyl fluoride derivatives were designed, synthesised and biologically    evaluated as potential telomerase inhibitors. Total 82 compounds were evaluated for their antiproliferative activities against A375, MDA-MB-231, SMMC-7721, SGC-7901 and MGC-803 cell lines. The structure-activity relationships were discussed in depth. The results showed most compounds had good activity against A375 cell with lower toxicity against normal cells in vitro. Moreover, compounds 5, 31 and 57 displayed high inhibitory activity against telomerase with IC 50 ¼ 0.71, 0.81 and 0.97 mM, respectively. These results are of help in rational design of more efficient telomerase TERT inhibitors in the future.