Lys05

Binding of hydroxychloroquine and chloroquine dimers to palmitoyl-protein thioesterase 1 (PPT1) and its glycosylated forms: a computational approach
Gérard Vergoten & Christian Bailly
To cite this article: Gérard Vergoten & Christian Bailly (2021): Binding of hydroxychloroquine and chloroquine dimers to palmitoyl-protein thioesterase 1 (PPT1) and its glycosylated
forms: a computational approach, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2021.1908167
To link to this article: https://doi.org/10.1080/07391102.2021.1908167

 
Published online: 20 Apr 2021.

 

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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS https://doi.org/10.1080/07391102.2021.1908167

 

Binding of hydroxychloroquine and chloroquine dimers to palmitoyl-protein thioesterase 1 (PPT1) and its glycosylated forms: a computational approach

Gtierard Vergotena and Christian Baillyb
aInserm, INFINITE – U1286, Institut de Chimie Pharmaceutique Albert Lespagnol (ICPAL), Faculteti de Pharmacie, University of Lille, Lille, France; bOncoWitan, Lille (Wasquehal), France
Communicated by Ramaswamy H. Sarma

 

ABSTRACT
The lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1) removes thioester-linked fatty acid groups from membrane-bound proteins to facilitate their proteolysis. A lack of PPT1 (due to gene mutations) causes the progressive death of cortical neurons and is responsible for infantile neural ceroid lipofuscinosis (INCL), a severe neurodegenerative disorder in children. Conversely, PPT1 is often over-expressed in cancer, and considered as a valid target to control tumor growth. Potent and select- ive inhibitors of PPT1 have been designed, in particular 4-amino-7-chloro-quinoline derivatives such as hydroxychloroquine (HCQ) and the dimeric analogues Lys05 and DC661. We have modeled the inter- action of these three compounds with the enzyme, taking advantage of the PPT1 crystallographic structure. The molecules can fit into the palmitate site of the protein, with the dimeric compounds forming more stable complexes than the monomer. But the molecular modeling suggests that the most favorable binding sites are located outside the active site. Two sites centered on residues Met112 and Gln144 were identified, offering suitable cavities for drug binding. According to the calcu- lated empirical energies of interaction (DE), the dimer DC661 forms the most stable complex at site Met112 of palmitate-bound PPT1. N-glycosylated forms of PPT1 were elaborated. Paucimannosidic gly- cans (M2FA and M3F) and a bulkier tetra-antennary complex glycan were introduced at asparagine residues N197, N212 and N232. These N-glycans do not impede drug binding, thus suggesting that all glycoforms of PPT1 can be targeted with these compounds.

Abbreviations: HCQ: hydroxychloroquine; PPT1: palmitoyl-protein thioesterase 1
ARTICLE HISTORY Received 16 January 2021 Accepted 19 March 2021

KEYWORDS
PPT1; hydroxychloroquine; palmitoylated proteins; drug-protein binding; molecular modeling

 
1.Introduction
The enzyme palmitoyl-protein thioesterase 1 (PPT1) is impli- cated is diverse human pathologies, notably in a severe pedi- atric neurodegenerative disease called infantile neuronal ceroid lipofuscinosis (INCL, or infantile Batten disease), in neu- rodegeneration and some cancer (Koster & Yoshii, 2019; Sapir et al., 2019). INCL is a neurodegenerative lysosomal storage disease caused by inactivating mutations in the CLN1 gene. To date, about 75 mutations in the CLN1 gene (encoding PPT1), including deletion/duplications, have been reported (Sheth et al., 2018). As a result of loss-of-function mutations, the enzyme can no longer catalyze the deacylation of S-palmitoy- lated proteins to facilitate their degradation and clearance by lysosomal hydrolases. The deficiency causes the selective death of cortical and retinal neurons. The enzyme normally protects against apoptosis mediated by different stimuli and cytotoxic drugs (Cho & Dawson, 2000). PPT1-defisicient cells are highly susceptible to reactive oxygen species-induced cell death (Balouch et al., 2021). Small molecules, such as N-(tert- butyl)hydroxylamine, functionally mimicking PPT1 can limit neurological deterioration and may have therapeutic implica- tions (Sarkar et al., 2013, 2020). On the opposite, inhibitors of

 

PPT1 are searched to treat cancer, because inhibition of PPT1 increases the susceptibility of tumor cells to apoptotic cell death whereas overexpression of PPT1 led to increased resist- ance to cell death (Cho et al., 2000, 2001).
PPT1 depalmitoylates proteins in the lysosome prior to their degradation and functions as a key regulator of the autophagy pathway. It is one of the major proteins upregu- lated by the transcription factor EB (TFEB) which is a master regulator of lysosome and autophagosome biogenesis (Koster & Yoshii, 2019). In cancer cells, the enzyme modulates apoptosis induced by cytotoxic agents and TNFa (Dawson et al., 2002; Tardy et al., 2009). PPT1 is frequently over- expressed in cancer cells notably in colorectal carcinoma (Tsukamoto et al., 2006) and neuroblastoma to protect against apoptotic death (Cho & Dawson, 2000). The enzyme is now considered as a valid target for cancer therapy and selective inhibitors are actively searched.
A few inhibitors have been discovered recently, in particu- lar the dimeric chloroquine derivative DC661 identified as a potent inhibitor of PPT1 in cells, with remarkable anticancer properties (Rebecca et al., 2019). Previously, the related quinacrine dimeric compound DQ661 was found to bind dir- ectly to PPT1, to induce accumulation of palmitoylated
CONTACT Christian Bailly [email protected] OncoWitan, Lille (Wasquehal) 59290, France
ti 2021 Informa UK Limited, trading as Taylor & Francis Group
proteins in cells, and to perturb mTOR signaling and autoph- agy. The compound showed marked antitumor activities in vitro (Nicastri et al., 2018). DQ661 markedly reduced tumor progression in a melanoma xenograft model and improved survival in mice models of colon and pancreatic cancer (Rebecca et al., 2017). DQ661 and DC661 bear the same spermidine linker between the two aromatic units. They both derive from the compound Lys05 (DC221) with a shorter tria- mine linker between the two 7-chloroquinoline heterocycles. Lys05 is a prototypic autophagy inhibitor, ten-fold more potent than the monomeric compound hydroxychloroquine (HCQ) (Figure 1(a)). This water-soluble compound accumu- lates within and deacidifies lysosomes, leading to impaired autophagy and tumor growth (Amaravadi & Winkler, 2012; McAfee et al., 2012). It is also a convenient tool to study inhibition of lysosomal functions in cells (Bagri et al., 2020). The elongation of the linker chain between the two aromatic units (Lys05 ! DC221) further reinforces the anticancer activity of the dimeric molecules (Rebecca et al., 2017, 2019). PPT1 represents a common target for HCQ, Lys05 and DC661 (Rebecca et al., 2019) but the position of the drug binding site within the enzyme has not been studied.
The crystal structure of PPT1 with and without bound palmitate has been elucidated (Bellizzi et al., 2000). In the present molecular modeling analysis, we took advantage of this tridimensional structure to investigate the interaction between the enzyme and the chloroquine monomeric and dimeric derivatives HCQ, Lys05 and DC661. Models of the drug-enzyme complexes were elaborated, and potential drug binding sites were identified and analyzed. In addition, we investigated drug binding to glycosylated forms of the enzyme, to determine the possible effect of different N-gly- cans on the drug-enzyme interaction. N-glycosylation is essential for PPT1’s activity and intracellular transport (Lyly et al., 2007). It is thus important to evaluate the possible effect of N-glycans, including paucimannosidic and branched glycans, on drug binding.

2.Materials and methods
2.1.In silico molecular docking procedure
Two tridimensional structures of the PPT1 protein were retrieved from the Protein Data Bank (www.rcsb.org) under the PDB codes 1EI9 (Crystal structure of palmitoyl protein thi- oesterase 1) and 1EH5 (Crystal structure of palmitoyl protein thioesterase 1 complexed with palmitate) (Amaravadi &
Winkler, 2012). Docking experiments were performed with the GOLD software (GOLD 5.3 release, Cambridge Crystallographic Data Centre, Cambridge, UK). Before starting the docking procedure, the structure of the ligands has been optimized using a classical Monte Carlo conformational searching procedure as described in the BOSS software (Jorgensen & Tirado-Rives, 2005). Molecular docking was per- formed according to published procedures (Dalal et al., 2021; Dhankhar, Dalal, Kotra, et al., 2020; Dhankhar, Dalal, Singh, et al., 2020; Kumari et al., 2021; Shakya et al., 2019).
We essentially used 1EH5 which refers to a palmitate- bound PPT1 structure (removing the palmitate ligand when

necessary). With this structure, based on shape complemen- tarity criteria, three possible binding sites for HCQ or Lys05 or DC661 have been defined around amino acid residues Gln144 and Met112, and at the active site (palmitate site). A fourth weaker site was identified around position Ala203. Shape complementarity and geometry considerations are in favor of a docking grid centered in the volume defined by these amino acids. In each case, within the binding site, side chains of specific amino acids have been considered as fully flexible. At the palmitate site, the flexible amino acids are Met41, Phe114, Ser115, Gln142, Leu167, Leu180, Gln182, Tyr185, Trp186 and His289. At the Gln144 site, flexible amino acids are His143, Gln144, His187, Glu192, Arg196, Glu208, Phe244, Tyr247, Ser261 and Ile256. At the Met112 site, flex- ible amino acids are Tyr109, Met112, Phe114, Leu121, Ile137, Leu283, Phe297, Ile301, Phe304 and Leu305. The ligand is always defined as flexible during the docking procedure. Up to 100 poses that are energetically reasonable were kept while searching for the correct binding mode of the ligand. The decision to keep a trial pose is based on ranked poses, using the PLP fitness scoring function (which is the default in GOLD version 5.3 used here (Jones et al., 1997)). In add- ition, an empirical potential energy of interaction DE for the ranked complexes is evaluated using the simple expression DE(interaction) ¼ E(complex) – (E(protein) þ E(ligand)). For that purpose, the Spectroscopic Empirical Potential Energy function SPASIBA and the corresponding parameters were used (Lagant et al., 2004; Vergoten et al., 2003). Free energies of hydration (DG) were estimated using the MM/GBSA model in Monte Carlo simulations within the BOSS software (Jorgensen et al., 2004). Molecular graphics and analysis were performed using Discovery Studio Visualizer, Biovia 2020 (Dassault Systtiemes BIOVIA Discovery Studio Visualizer 2020, San Diego, Dassault Systtiemes, 2020).

3.Results
3.1.PPT1 enzyme-palmitate complex
We used the crystal structure of the enzyme PPT1 covalently bound to the substrate palmitate (Bellizzi et al., 2000) to model the interaction of the chloroquine derivatives with the enzyme. Two structures are available, one without the palmi- tate ligand (PDB access code 1EI9) and one with the palmi- tate molecule covalently attached to Ser115 (PDB access code 1EH5). We used this latter structure for the docking measurements. The presence of the palmitate ligand does not markedly change the 3 D structure of the enzyme (Figure 1(b)). The fatty acid fits into a hydrophobic groove of the enzyme to establish contacts with the catalytic triad com- posed of residues Ser115, His289, and Asp233, critical for the enzyme activity (Bellizzi et al., 2000). We modeled the palmi- tate-PPT1 bonding interaction to determine the empirical
energy of interaction (DE ¼ ti 37.71 kcal/mol) and the free energy of hydration (DG ¼ ti 19.20 kcal/mol) for the non- covalent (bonding, not binding) interaction (palmitate mol-
ecule removed from structure 1EH5). Values of DE ¼ ti 45.00 kcal/mol and DG ¼ ti24.10 kcal/mol were obtained

 

 

 

 

 

 

 

 

 

 

 

 
Figure 1. (a) Structure of the chloroquine derivatives. (b) Molecular models of PPT1 free (cyan, PDB: 1EI9) or bound to palmitate (green, PDB: 1EH5). The structure corresponds to residues 28–306 of the enzyme, after cleavage of the 27-residue signal peptide (Bellizzi et al., 2000). The bound palmitate molecule does not mark- edly impact the enzyme structure.

 

 

 

 

 

 

 

 

Figure 2. Molecular models of HCQ (a), Lys05 (b) and DC661 (c) bound to the palmitate site of PPT1. In each case, the drug is located in a central cavity of the pro- tein (in green). There is an extended groove to accommodate the monomeric or dimeric ligand. (d) Overlay of the different compounds bound to the palmitate site, with the protein a-helices in red and b-sheets in blue.
with structure 1EI9 (for palmitate prior to bonding with Ser115 residue).
3.2.Binding of the chloroquine derivatives to PPT1
Models of compounds DC661, Lys05 and HCQ bound to PPT1 were constructed starting from the X-ray structure of the enzyme (PDB code 1EH5). We performed a molecular docking to identify preferred drug binding sites. On the one hand, the molecules were docked into the active site of the enzyme, after removal of palmitate. On the other hand, bind- ing positions outside the active site were screened (while maintaining the palmitate bound).
We found that the three drugs can form stable complexes with PPT1 upon binding to the hydrophobic palmitate cavity. Molecular models are presented in Figure 2 and the calcu- lated energies of interaction are given in Table 1. With the
three drugs, the calculated DE values are superior to that cal- culated with palmitate not covalently bond. The calculations indicate that the two dimeric compounds Lys05 and DC661 form more stable PPT1 complexes than the monomeric drug HCQ, in agreement with the published experimental data (Rebecca et al., 2019). The drug dimerization leads to a very significant gain of binding energy of about 38%. There is a long groove in the protein suitable to accommodate an extended ligand like Lys05 or DC661, or even possibly a lon- ger ligand (Figure 3(a)). In all cases, the drug can establish multiple interactions with the protein, including H-bond interactions, hydrophobic and van der Waals contacts. The contact map for DC661 is shown in Figure 3(b), with up to 26 different interactions contributing to the stability of the drug-protein complex (23 interactions for Lys05 and 20 for HCQ, not shown).
The protein groove used by DC661 is not fully occupied, as represented in Figure 3(a). Apparently, there is room for a
longer ligand. We computer-designed two longer molecules compounds C7 and C8, with a -(CH2)7-N(CH3)-(CH2)7- and a
-(CH2)8-N(CH3)-(CH2)8- linker respectively, in place of the C6 linker of DC661 and evaluated their binding to the PPT1 active site. No improvement was found with compound C7
(DE ¼ ti63.0 kcal/mol; DG ¼ ti33.1 kcal/mol) compared to DC661. In contrast, a better fit was observed with the C8 compound which can occupy almost the full length of the groove (data not shown), leading to more stable complexes
with the enzyme (DE ¼ ti75.8 kcal/mol; DG ¼ ti38.2 kcal/
mol). It could be useful to synthesize and test such longer analogues of DC661 (certainly with a more water-sol- uble linker).
But in fact, the palmitate cavity does not seem to offer the optimal binding site for the three studied compounds. We could identify two other binding locations, centered on

amino acid residues Gln144 and Met112 of PPT1, which apparently provide better binding sites for the studied mole- cules. If we refer to the calculated empirical energy of inter- action (DE values in Table 1), the best site would be Met112 for DC661 and Gln144 for Lys05. The optimal site may be Met112, considering the calculated free energy of hydration (DG values) which is for favorable at site Met112 compared to Gln144 for the three ligands (Table 1). For the two sites, the compounds rank in the order DC661 > Lys05 > HCQ (more DE negative values), again in agreement with the bet- ter inhibition of PPT1 in cells, previously reported (Rebecca et al., 2019). Models of HCQ and Lys05 bound to site Gln144 on PPT1 are presented in Figure 4, to illustrate the possible coexistence of palmitate bound to the active site of the enzyme and the chloroquine derivative bound at a vicinal site. There is no overlap between the two sites. Similarly, at site Met112 it is possible to correctly position the chloro- quine derivative without impacting the insertion of the

Table 1. Calculated potential energy of interaction (DE) and free energy of hydration (DG) for the interaction of hydroxychloroquine (HCQ) and its two dimeric analogues (Lys05 and DC661) with the PPT1 enzyme, at the indicated sites (kcal/mol).
Palmitate site Site Gln144 Site Met112
Compound DE DG DE DG DE DG
HCQ –46.90 –23.10 –53.96 –17.60 –57.51 –27.50
Lys05 –66.90 –33.60 –67.65 –22.35 –59.60 –26.30
DC661 –68.85 –38.51 –73.51 –23.20 –77.80 –35.10
palmitate substrate into the enzyme active site. A model of DC661 bound to the Met112 site with palmitate occupying the active site of PPT1 is presented in Figure 5. In this case, the DC661-PPT1 complex is stabilized by a diversity of molecular interactions, including van der Waals contacts, alkyl interactions and a key H-bond between the NH group of the drug amino-quinoline and residue Ile301 of the enzyme (not shown).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
Figure 3. (a) Close-up view of DC661 bound into the palmitate site of PPT1, within the extended groove delimited by a yellow dashed line. (b) Binding map con- tacts for DC661 bound to PPT1 at the palmitate site.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
Figure 4. (a) Molecular models of HCQ (top) and Lys05 (bottom) bound to site Gln144 of palmitate-bound PPT1. The protein model shows a-helices in red and b-sheets in blue. Drug binding to the Gln144 site does not impede palmitate binding to the proximal active site. (b) Binding map contacts for HCQ (top) and Lys05 (bottom) bound to PPT1 at the Gln144 site.

 
3.3.Drug binding to glycosylated PPT1
In cells, PPT1 can be N-glycosylated at asparagine residues 197, 212 and 232 (hereafter designated N197, N212 and N232). N-glycosylation of N197 and N232, but not N212, is essential for the activity and intracellular transport of the enzyme (Lyly et al., 2007). For this reason, we determined the potential impact of PPT1 N-glycans on drug binding. Different models of PPT1 N-glycosylated at these three sites were constructed using two distinct types of N-glycans: a paucimannosidic type (M2FA and M3F) glycan and a more bulky, branched tetra-antennary glycan, as represented in Figure 6. In the crystal structure (PDB code 1EH5), the pro- tein has no glycan but bears a N-acetyl glucosamine (GlcNAc) residue at positions N197 and N212, and a GlcNAc- GlcNAc disaccharide at N232. Those sugar residues were replaced with the indicated N-glycans.
With both the paucimannosidic and branched glycans, it can be seen immediately that the N-glycans have no major impact on palmitate binding. The active site remains fully accessible, not shielded by the N-glycans, even with the extended tetra-antennary glycan. The presence of N-glycans has no impact on the access of palmitate (Figure 6) or the other molecules to the active site. Figure 7 shows a model of DC661 bound simultaneously to the three sites (palmitate, Met112 and Gln144) of the PPT1 enzyme bearing three tetra- antennary N-glycans (at N197, N212 and N232). No major obstacle to drug binding was observed. Similarly, the binding of HCQ to those sites is not impacted by the presence of N- glycans (not shown). In both cases, the drug binding site remains fully accessible to the small molecule.
At this point, it is worth to mention a potential weaker site, centered on residue Ala203, which could also accommo- date the drug. We did not prioritize this site because the

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5. (a) Molecular models of DC661 bound to site Met112 of palmitate-bound PPT1. (b) A close view of the drug-protein interface, with the H-bond donor/
acceptor sites indicated. (c) Close-up view of the binding of DC661 to the Met112 site of PPT1 with the palmitate ligand bound to the vicinal active site.

 

 

calculated energies are less favorable compared to sites Met112 and Gln144 (binding to site Ala203: DE ¼ ti47.9,
ti55.0 and ti75.2 for HCQ, Lys05 and DC661, respectively). However, this site would be the only one possibly impacted by a N-glycosylation because it is located between the glyco- sylated positions N197 and N212 (data not shown). In this case only, the N-glycosylation with a bulky tetra-antennary N-glycan could restrict the drug access to the site. But it is a weaker site, thus not prioritized.

4.Discussion
PPT1 removes palmitate from palmitoylated proteins. It is the only lysosomal enzyme that cleaves thioester bonds from palmitoylated proteins, liberating these proteins from mem- branes to facilitate proteolysis. PPT1 inhibitor are actively searched for the treatment of cancer and other diseases. Different small molecule inhibitors have been identified and dimeric compounds like DQ661 (quinacrine dimer) and DC661 (chloroquine dimer) stand as highly potent inhibitors. The bio- logical activity of DQ661 is mediated through inhibition of PPT1- dependent depalmitoylation of palmitoylated proteins (Rebecca
et al., 2017, 2019). There are other chemical archetypes of PPT1 inhibitors, such as the anticancer drug didemnin (Crews et al., 1996; Meng et al., 1998; Potts et al., 2015), but the chloroquine dimers look very promising as anticancer agents. The combination of DC661 (or HCQ) with a monoclonal antibody targeting the PD- 1 immune checkpoint has revealed a remarkable efficacy in a model of melanoma. The small molecule targeting PPT1 enhances the antitumor efficacy of the anti-PD-1 mAb in melanoma, pro- moting the secretion of interferon-b in macrophages, to promote T cell-mediated cytotoxicity (Sharma et al., 2020). It is thus import- ant to comprehend how molecules likes HCQ and DC661 bind to PPT1 and interfere with its functioning. Our molecular modeling analysis can be useful in that respect, although we are well aware of the limitations of an in silico approach. It is a theoretical ana- lysis, which will require an experimental validation, but it provides new ideas and research perspectives.
Our molecular modeling analysis conveys several import- ant information. Firstly, it indicates that the chloroquine- based compounds, monomer or dimers, can form stable complexes with PPT1, binding either to the palmitate site or, more likely, to a proximal site distinct from the palmitate active site. Lys05 and DC661 appear to form stable com- plexes with PPT1 upon binding to the sites designated

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
Figure 6. Molecular models of N-glycosylated palmitate-bound PPT1. Protein models bearing three N-glycans at position N197, N212 and N232 were elaborated, using either a paucimannosidic N-glycan (M2A as shown, or M3A not shown) or a complex branched tetra-antennary N-glycan. In both cases, the N-glycans do not hinder palmitate binding.

 

 

 

 

 

 

 

 

 

Figure 7. Models of DC661 bound to the different sites of N-glycosylated palmitate-bound PPT1. The protein bears three branched tetra-antennary N-glycans (N197, N212 and N232). The three drug binding sites are indicated.

 

Met112 and Gln144. The Met112 site offers a suitable cavity for the binding of DC661 to PPT1. In this case, there is no competition for drug binding between the palmitate bound to the active site and DC661 interacting with the vicinal Met112 site. This hypothesis is interesting because it echoes the mechanism of inhibition of PPT1 by the cyclic depsipep- tide didemnin B. Indeed, this marine compound potently inhibits PPT1 (Ki ¼ 92 nM) but the inhibition is
uncompetitive; PPT1 binding of didemnin family members requires the presence of the substrate (Meng et al., 1998). A similar situation may occur with the chloroquine-based com- pounds, upon binding to site Met112 or Gln144, proximal to the active center. If this is the case, it will be necessary to determine how the compound can inhibit the enzyme activ- ity without occupying the active center, via indirect effects. Secondly, the study provides a rational basis to explain the
superior inhibitory potency of dimeric DC661 versus mono- meric HCQ. There is apparently a long groove at the surface of the enzyme that allows the extended dimer to occupy a long area and to establish multiples contacts with the pro- tein. The dimeric molecules like Lys05 and DC661 can sit into the groove. It is likely that slightly longer molecules can fit equally well because the groove does not seem to be fully occupied when DC661 is bound.
Thirdly, the presence of N-glycans does not impede drug binding. This information suggests that the designed inhibi- tors will be able to target all glycoforms of PPT1, whatever the nature of the N-glycan. Recently, we have modeled two glycosylated proteins using bi-antennary N-glycans, the PD- L1 immune checkpoint protein that is frequently expressed at the surface of cancer cells (Bailly & Vergoten, 2020) and the secreted pro-inflammatory protein HMGB1 (Vergoten &
Bailly, 2020). Here for the first time, we modeled two other types of N-glycans, paucimannosidic glycans (M2A/M3A) and a complex tetra-antennary glycan. To our knowledge, this has never been done before. Apparently these glycans do not hinder PPT1 recognition by HCQ, Lys05 or DC661. The molecular models elaborated will be useful to study other types of interactions. N-glycans are increasingly considered as drug targets for small and large bioactive molecules (Bravo et al., 2020; Shimomura et al., 2018; Wang et al., 2019; Zhou, 2018).

CRediT authorship contribution statement
Gtierard Vergoten: Visualization; Software; Computations; Molecular modeling. Christian Bailly: Conceptualization; Visualization; Writing – original draft; Writing – review
& editing.

Disclosure statement
The authors declare no conflict of interest associated with this publica- tion and there has been no significant financial support for this work that could have influenced its outcome.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

ORCID
Christian Bailly http://orcid.org/0000-0002-2973-9357

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