Biochemical mechanism and biological effects of the inhibition of silent information regulator 1 (SIRT1) by EX-527 (SEN0014196 or selisistat)

Abstract The human sirtuin silent information regulator 1 (SIRT1) is a NAD+-dependent deacetylase enzyme. It deacetylates many protein substrates, including histones and transcription factors, thereby controlling many physiological and pathological processes. Several synthetic inhibitors and activators of SIRT1 have been developed, and some therapeutic applications have been explored. The indole EX-527 and its derivatives are among the most potent and selective SIRT1 inhibitors. EX-527 has been often used as a pharmacological tool to explore the effect of SIRT1 inhibition in various cell types. Its therapeutic potential has, therefore, been evaluated in animal models for several pathologies, including cancer. It has also been tested in phase II clinical trial for the treatment of Huntington’s disease (HD). In this review, we will provide an overview of the literature on EX-527, including its mechanism of inhibition and biological studies.


Introduction
Human silent information regulator 1 (SIRT1) belongs to the sirtuin family of enzymes, which constitute class III of the histone deacetylase family (HDAC). It is the most studied of the seven human sirtuins known to date. It is a NAD þ -dependent deacetylase, which deacetylates many protein substrates, including histones and transcription factors 1 . SIRT1 has been linked to type 2 diabetes 2 , cancer 3 , Alzheimer disease 4 , and more generally diseases of ageing 5,6 . In particular, the contradictory roles of human SIRT1 in cancer have been reviewed and are still a subject of debate 7,8 . To study these biological activities, the modulation of SIRT1 expression and activity by bioengineering (mutations, overexpression, siRNA, or knockout for example) has been largely employed 7,9,10 .
In addition to these genetic manipulation studies, pharmacological modulation of SIRT1 has been the subject of intense research. SIRT1 modulators in general and their roles in cancer in particular have been often reviewed, usually giving an overview of several inhibitors and activators, but limited information on each one [11][12][13][14] . We present here an overview of the literature data on the SIRT1 selective and potent inhibitor EX-527 (SEN0014196 or selisistat) since its first disclosure in 2005 15 . Key data are reported, regarding its mechanism of inhibition and inhibitory potency in vitro, its effect on various cell types (used alone or in combination with other molecules), biological studies in animal models, and results of a clinical trial. This review primarily describes studies in which EX-527 is the main compound of interest, but we also included selected studies using EX-527 as a control and/or pharmacological tool to explore SIRT1 related pathways. To complete this overview, we also included some examples in which the inhibitor EX-527 was used to counteract the effects of other molecules, such as SIRT1 activators.

2.
In vitro assays of EX-527 on isolated enzymes and mechanism of inhibition 2.1. Discovery, properties, IC 50 values, and structure/activity relationship studies EX-527 was identified in 2005 by high throughput screening of libraries of compounds on the enzyme SIRT1 (Figure 1) 15 . It has now been the subject of more than 200 articles.
A typical synthesis of this family of compounds is depicted in Scheme 1. These compounds were obtained by a Bischler indole synthesis. In the first step, a b-keto ester was brominated on a to the ketone, affording a bromo keto ester, which was heated in the second step with an aniline, affording the tetrahydrocarbazole ester. The ester was then converted to the primary amide under pressure. In case enantiomerically pure material was needed, separation by chiral column chromatography was achieved 15 .
EX-527 is a potent and selective SIRT1 inhibitor, with IC 50 values as low as 38 nM, depending on assay conditions 16 . In the first report, it was shown to be more selective for SIRT1 than for SIRT2 or SIRT3 (200-500-fold) 15 . EX-527 does not inhibit class I/II HDAC activity at concentrations up to 100 mM. EX-527 is racemic, the active isomer (designated EX-243) being (S), whereas the other (R) isomer (designated EX-242) is inactive. IC 50 values for sirtuin inhibition by EX-527 have been measured in several studies, using a variety of assay methods and peptide substrates (Table 1). They range from 0.038 to 3 mM, usually between 0.1 and 1 mM. They depend mostly on the nature and concentration of the peptide substrates and on NAD þ concentration, because of the uncompetitive inhibition mechanism of EX-527 (see below). Very stringent structure/activity relationships were described in the original article 15 and were later explained in light of the crystal structure published in 2013 (see below) 31 . Compound 35 (Figure 1) is an analogue of EX-527, very potent inhibitor of SIRT1: the IC 50 of the (S) isomer is 60 nM, and the IC 50 of the racemic mixture is 124 nM. It is selective for SIRT1, with an IC 50 for SIRT2 of 2.77 mM 15 .
EX-527 was also identified independently in 2006 from another high throughput screening. The screened compound was in fact the N-((dimethylamino)methylene)acetamide derivative (a dimethylformamide adduct), which was rapidly hydrolysed in aqueous solution to form EX-527 and dimethylformamide (Scheme 2) 32 . EX-527 is also able to block the protein-protein interaction taking place between deleted in breast cancer 1 (DBC1) and SIRT1 33 . DBC1 is an endogenous protein shown to interact with SIRT1 and to inhibit its catalytic activity 34,35 . The regulation of this interaction is complex. For example, DBC1 itself is a substrate of SIRT1, and deacetylated DBC1 does not bind to SIRT1 33 . However, the team of Sinclair showed that EX-527 blocks the interaction via an acetylation-independent mechanism in vitro. They also demonstrated, using a luciferase complementation assay, that the inhibitor is able to block the SIRT1-DBC1 interaction in cells with an IC 50 of approximately 1 mM 33 .
In addition to sirtuins, EX-527 and racemic 35 (rac-35) have been tested in vitro on other isolated enzyme and receptor targets. Overall, they displayed very little to no activity. They did not inhibit class I and II HDACs and NAD þ glycohydrolase at 100 mM 15 . PARP are enzymes using the NAD þ as cosubstrate for ADP-ribosyl transfer, producing nicotinamide, like sirtuins. Therefore, inhibitors targeting the nicotinamide binding pocket like EX-527 could have an inhibitory effect on PARP enzymes. No inhibition was observed on PARP1 and PARP10 29,36 . On cardiac potassium channels (hERG/ I Kr ), EX-527 had an IC 50 of 43 mM, with 0% inhibition at 10 mM 37 , and rac-35 displayed only 10% inhibition at 10 mM 15 . Cytochrome P450 are key enzymes involved in metabolism of drugs. They are largely evaluated in screening panels of new biologically active molecules, to identify P450 substrates or inhibitors. On cytochromes P450 (3A4, 2D6, 2C9, 2C19, 1A2, 2C8, and 2E1), both molecules had weak or no inhibitory potency at 1 mM, the highest values being 23% inhibition for 2C19 and 1A2 with rac-35. IC 50 values determined for EX-527 were higher than 100 mM for all cytochromes P450 except 2C9 (62.4 mM), 2C19 (72.2 mM), and A2 (8.7 mM) 15,37 .

Mechanism of inhibition and crystal structures
A simplified mechanism of deacetylation of a substrate catalysed by sirtuins is represented in Figure 2(A) 38 . The acetylated substrate makes a nucleophilic substitution on the C1 0 of the NAD þ cofactor, releasing nicotinamide. The 1 0 -O-alkylimidate intermediate formed reacts intramolecularly to generate a bicyclic intermediate. This intermediate is subsequently hydrolysed to form the deacetylated product and the 2 0 -O-AcADPr coproduct.
The mechanism of SIRT1 inhibition by EX-527 is represented in Figure 2(B), adapted from Gertz et al. 25 . Mechanistic studies on SIRT1, SIRT3, and Sir2Tm (sirtuin from Thermotoga maritima) demonstrated in all three cases that the inhibition by EX-527 was noncompetitive with substrate and uncompetitive with NAD þ . Therefore, the inhibition potency depends on the NAD þ concentration. Binding parameters are summarised in Table 2. K d values for EX-527 measured for the apo enzymes and in the presence of NAD þ confirmed the uncompetitive nature of the inhibition. Indeed, EX-527 does not bind to the apo enzyme, but binds with low micromolar affinity in the presence of NAD þ .
Another interesting aspect of these mechanistic studies concerns the specificity of EX-527 for sirtuin isoforms. The authors propose that the difference between EX-527-sensitive enzymes (like SIRT1 and Sir2Tm) and less sensitive ones (like SIRT2 and SIRT3) comes from differences in their kinetics of catalysis, and not from differences in the binding pockets, which are very similar 25 . Indeed, they suggest that binding of EX-527 either after or before the rate-limiting step leads to differences in inhibition potency.
In all these structures, the inhibitors occupy the nicotinamide binding pocket (the so-called C-pocket) of the sirtuin, and one of the following molecules is also co-crystallised, forming a ternary complex: NAD þ , the coproduct 2 0 -O-AcADPr, or ADPr ( Figure 3). This observation is in agreement with the uncompetitive nature of the inhibition with the cofactor NAD þ , which is required for efficient inhibition, as mentioned above. The inhibitors are deeply buried in the C-pocket and make hydrogen bonds contacts and hydrophobic interactions with the enzyme, which explain the stringent structure/activity relationships observed 15 .
Moreover, the mechanistic studies showed that sirtuin inhibition with EX-527 allows the formation of one molecule of product per molecule of enzyme, indicating that the inhibitor binds most efficiently after bicyclic intermediate formation and allows coproduct formation 25 . The authors proposed that EX-243 inhibits sirtuins mostly by binding in the presence of the coproduct 2 0 -O-AcADPr. Finally, from the comparison of crystals structures with and without the inhibitor, it appears that a flexible cofactor-binding loop moves towards the inhibitor and the coproduct during inhibition, resulting in a "closed" conformation preventing product release 25 .

Cellular assays of EX-527
EX-527 has been tested on several cell lines, either as the main molecule of interest for potential therapeutic applications, or as a control experiment for comparison with other sirtuin modulators (inhibitors or activators). Often, it has been used as a pharmacological tool to demonstrate the involvement of SIRT1 in a biological response. An overview of literature data is summarised in Table 3. f Radioactive nicotinamide release assay using a peptide substrate derived from the sequence of p53 (K382): Ac-RHKK(Ac)-AMC. g Radioactive nicotinamide release assay using a peptide substrate derived from the sequence of p53 (K330): Ac-QPKK(Ac)-AMC. h Microfluidic mobility shift assay using a labelled peptide substrate derived from the sequence of p53 (K382): fluorescein-SKKGQSTSRHKK(Ac)LMFKTEGPDS. i NAD þ bioluminescence assay using a peptide substrate derived from the sequence of p53 (K382): HLKSKKGQSTSRHKK(Ac)LMFK. j Enzyme-coupled system detecting nicotinamide formation, using a peptide substrate derived from the sequence of histone H3 (K14) named AcH3: KSTGGK(Ac)APRKQ. k Charcoal-binding assay using [ 3 H]AcH3. l Fluorimetric assay using a peptide substrate derived from the sequence of p53 (K330): Ac-QPKK(Ac)-AMC. m Mass spectrometry assay using the peptide substrate derived from the sequence of p53 (K382): Ac-RHKK(Ac)W-NH 2 . n Enzyme-coupled system detecting nicotinamide formation, using a peptide substrate derived from the sequence of p53 (K382): RHKK(Ac)LMFK. o Enzyme-coupled system detecting nicotinamide formation, using a peptide substrate derived from the sequence of acetyl-CoA synthetase 2 (ACS2, K642): TRSGK(Ac)VMRRL. p Sir2Tm: sirtuin from Thermotoga maritima. q Enzyme-coupled system detecting nicotinamide formation, using a peptide substrate derived from the sequence of carbamoyl phosphate synthetase 1 (CPS1, K527): FKRGVLK(Ac)EYGVKV. r Fluorimetric assay using a peptide substrate derived from the sequence of histone H3 (K56): Ac-RYQK(Ac)-AMC. s Luminescence assay using a peptide substrate derived from the sequence of p53 (K330): Z-QPK(Me) 2 K(Ac)-aminoluciferin. t Fluorometric assay using the substrate Cbz-K(Ac)-AMC. u Fluorometric assay kits, undisclosed substrates. On tumour cell lines, several reports demonstrated the ability of EX-527 to increase p53 acetylation from 1 to 25 mM concentrations, when used either alone or in combination with cytotoxic molecules 16,23,44,46,51,56,63 . EX-527 was shown to improve the efficiency of cytotoxic agents on cancer cells, with several chemotherapeutic and genotoxic agents 40,42,60 . However, in few cases, EX-527 administered alone increased cell proliferation of cancer cell lines 49,71 . The conclusion of one of these studies on the role of SIRT1 in cancer cells is a simple summary of these apparently contradictory results: In summary, our results suggest that both activators and inhibitors of SirT1 have therapeutic potential as anti-tumor agents. A simple scenario is that SirT1 activators may impart cancer prevention effects by enhancing the growth-inhibitory effect of SirT1 in benign tumors. Its effect on advanced stage tumors may be heterogeneous, depending on whether a tumor has evolved to rely on SirT1 for survival. However, when tumors are being treated with chemotherapy, SirT1 inhibitors may be useful for enhancing apoptotic response 40 .
Ten years after this report, the list of EX-527 studies has grown to reinforce this view (Table 3). For example, a decrease in cell survival and migration and an increase in apoptosis was recently observed on hepatocellular carcinoma (HCC: HepG2 and Huh7) cell lines with EX-527 alone 63 . Moreover, the same study demonstrated that EX-527 induced the downregulation of ABC transporters P-gp and MRP3 in HepG2 cells, suggesting an additional potential application of this SIRT1 inhibitor in combination with conventional therapeutic drugs to overcome multi-drug resistance (MDR) during HCC therapy 63 . Indeed, one of the most potent effect was obtained when EX-527 was used in combination with Hsp-90 inhibitors on CSCs (cancer stem-like cells) or MDR variants, with a potent increase in cytotoxicity of the Hsp-90 inhibitor with only 10 nM EX-527 52,53 . Moreover, EX-527 at 1 mM decreased colony formation of ovarian carcinoma cells, with or without overexpression of SIRT1 72 . At 600 nM, it suppressed cell migration and inhibited the occurrence of epithelial-mesenchymal transition  25 . E: enzyme. Note that former studies of SIRT1 inhibition by substrate analogues suggested (i) a random addition of substrates (therefore, Ac-Pep could be added first to the enzyme, not represented here for simplification) and (ii) a departure of the peptide product from the enzyme in the last step (which would disagree here with the existence of the crystallised complex E/2 0 -O-AcADPr/EX-243) 39 . Table 2. Binding parameters of EX-527 with sirtuins. (EMT) in chemotherapy resistant oesophageal cancer cells 71 . Overall, several factors are important to consider to understand the effect of EX-527 on cancer cells: (i) the type of cell line and the cancer stage, from benign to advanced, (ii) the presence of other agents, conventional chemotherapy, or additional HDAC inhibitors for example, and (iii) the dose, because at higher doses (ex. 40 mM or above), EX-527 may significantly inhibit SIRT2 and may have other targets. For potential anti-cancer therapeutic applications, aiming for a specific SIRT1 inhibition at low concentrations of EX-527 (ex. 1 mM or below) in combination with cytotoxic agents may be the most promising strategy.
On non-cancer cell lines, fewer studies were published than on cancer-cell lines. For example on HUVEC, EX-527 was shown to protect from H 2 O 2 damage 49 , but to abolish the protective effect of resveratrol under high-glucose conditions 67 . Several articles described effects on cells involved in the immune system, macrophages, and T cells. Beneficial effects on autoimmune diseases and graft rejection problems can be envisioned from these cell assays, for example through reduction of effector T cell proliferation and differentiation 57,69 , and increase in the number and suppressive function of T regulatory cells Tregs (see Chapter undefined for in vivo results) 64 .
Many of the studies evaluating the role of EX-527 in cells summarised in this review incorporated control experiments with SIRT1 knockdown, mostly with anti-SIRT1 siRNA. These studies, in which the same effects were obtained with anti-SIRT1 siRNA or with its pharmacological inhibition with EX-527, make a strong case for the use of EX-527 as a pharmacological tool to study SIRT1 activity. However, the fact that EX-527 only targets SIRT1 must be tempered. Indeed, in vitro studies show that the extent of its specificity, in particular towards SIRT2, depends on the assay types (nature of the substrate and concentration of NAD þ for example) and may not be so high under certain conditions (Table  1). Consequently, its specificity inside cells or in vivo is even less predictable and quantifiable. Therefore, the results of studies concluding that SIRT1 is involved in the observed effect must be taken with caution, if they are solely based on the effect of EX-527 as a pharmacological control. SIRT2 and other unknown potential protein targets may be involved.

In vivo assays of EX-527
EX-527 has been tested in several organisms, mostly mice and rats, but also in the nematode C. elegans, in Drosophila melanogaster (D. melanogaster) and in humans in exploratory clinical trials (Tables 4 and 5).
Pharmacokinetic data were obtained in mice and human, both in female and male. Selected parameters are given in Table 4. In R6/2 mice model of Huntington's disease (HD) with 10-20 mg/kg dosing, average plasma concentrations over 24 h were in the low micromolar range (1.5-3.2 mM) 47 . In healthy male human volunteers with 150-300 mg doses, average plasma concentrations over 24 h were also in the low micromolar range (1.6-3.9 mM) 37 . However, a higher than proportional concentration (11.8 mM) was observed with 600 mg dosing, suggesting that one or more clearance mechanisms are approaching saturation at this dose. For multiple oral doses (for ex. 300 mg daily for 7 d for male), the data suggested that the pharmacokinetic steady-state was reached within 4 d, with an exposure higher than predicted from singledose data.
The fraction of unchanged EX-527 excreted in the urine was very low for all doses in male subjects (<0.02% up to 24 h postdose). The compound was transformed in vivo by hydroxylation and oxidative deamination followed by glucuronic acid conjugation, across all species studied (mouse, rat, dog, and human) 37 .
Pharmacogenomics studies suggested that EX-527 treatment in human was associated with a specific transcriptional signature in blood cells, with genes involved in mechanisms of signal transduction and transmembrane transport, as well as metabolic and redox processes 37 .   The conclusion of the safety study in healthy volunteers indicated that EX-527 was safe and well tolerated by female and male subjects after single doses up to 600 mg and multiple doses up to 300 00/d for 7 d. Moreover, no meaningful cardiovascular effects were observed in beagle dogs up to 100 mg/kg 37

.
In vivo, numerous studies have been carried out to explore the effect of EX-527 under physiological or pathological conditions (see Table 5 for representative examples). Although most cellbased assays used cancer cells, in vivo, EX-527 was assayed in a more diverse set of pathologies, and only in a small number of cancer models on mice xenograft. Overall, it appeared very well tolerated when administered alone, in agreement with the phase I clinical trial described above 37 .
Apparent detrimental effects of EX-527 often consisted in inhibition of beneficial effects induced by additional compounds. For example, mice and rats suffering from ischaemia, sepsis, or chronic obstructive pulmonary disease were treated with several natural products including melatonin 76,[87][88][89] , diallyl trisulphide 90 , and punicalagin 86 . Other examples include the effects of ghrelin 82,84 , hydrogen-rich saline 83 , carbon monoxide 62 , the SIRT1 activators resveratrol 67,79,91 and scopolin 92 , and the PARP inhibitor 3aminobenzamide 36 . In all these cases, EX-527 was used as a pharmacological tool to demonstrate that SIRT1 activation was involved in the beneficial effects of the compounds under study. When used alone, a detrimental effect of EX-527 on pancreatic tumour xenograft was observed in one study, which gave surprising results 77 . Indeed, EX-527 increased the cytotoxic effect of gemcitabine in vitro in PANC-1 cells, in agreement with another study 50 , but it activated the tumour xenograft of the same cells in vivo 77 . The activity of EX-527 on other cell types in the tumour microenvironment is a possible explanation for this discrepancy. We note that in this xenograft study, the addition of EX-527 at 10 mg/kg with gemcitabine apparently did not have any effect, but the tumour growth in the control experiments with gemcitabine alone was already very limited.
Beneficial effects were observed in several pathologies. In cancer, EX-527 decreased the tumour growth of xenografted mice with endometrial and lung cancer cells 60,55 . In immunity-related diseases, a first report in 2011 indicated that, when used in combination with rapamycin, it prolonged heart allograft survival in mice 74 . The involvement of Tregs through increased expression of Foxp3 was proposed. Other studies confirmed these beneficial effects of EX-527 on Tregs through increased Foxp3 expression and acetylation, and the possible involvement of another SIRT1 substrate, NF-jB 69,75,93 . In a mouse model of multiple sclerosis, an immune disorder, it strongly suppressed the number of paralysed mice, through an effect of Th17 effector cells 57 .
In a phase II clinical trial involving HD patients, EX-527 was found to be safe and well-tolerated 73 . However, no clinical benefit was observed after the two weeks treatment. For this slowly progressive neurodegenerative disease, longer treatment durations of 2 years may be required to observe clinical benefits. In addition, and maybe for the same reason, no effects on the levels of soluble mutated huntingtin (mHtt) in healthy peripheral blood mononuclear cells (PBMCs) were observed.

Conclusion
EX-527 has been tested on many cell lines, alone or in combination with other molecules, resulting in a variety of cellular effects. Moreover, it displayed several biological effects in vivo in various pathological conditions. These results are in agreement with the fact that its specific target SIRT1 is a key regulator of cell fate,     through its deacetylation action on a large number of protein substrates. The expression and the activity of SIRT1 can be either upor down-regulated, depending on the cellular state in the physiological or pathological conditions under study. The administration of EX-527 appears to be beneficial in cases where the activity of SIRT1 is upregulated. Perhaps the most promising in vivo results have been obtained on mice and rats in autoimmune diseases and allograft tolerance, with a significant increase in survival.
Although the results of a phase II clinical trial in HD did not provide the expected beneficial effects, the safety of EX-527 was demonstrated with patients in phase I clinical trials. Therefore, further preclinical and clinical studies in other pathologies appear attractive. In this way, the SIRT1 Antagonism For Endometrial Receptivity (SAFER) clinical trial with EX-527 (Selisistat) will enrol around 30 women with unexplained failure after embryo transfer with euploid embryos. This phase II trial will start on 1 January 2021, and finish on 31 December 2022. The drug will be administered daily for 5 d, beginning with the start of progesterone therapy, and ending 24 h before embryo transfer. Pregnancy rates and pregnancy outcome will be monitored (trial number NCT04184323).
New derivatives of EX-527 with greater activity and selectivity for SIRT1, as well as improved pharmacokinetic and pharmacodynamic properties, may lead to results that are even more promising, and reach further advanced clinical trials.