The inhibition of adenylate kinase by 2 , 4-thiazolidinedione evaluated by protein-ligand docking

Due to its crucial role in nucleotide metabolism, adenylate kinase deserves a special attention in screening of potential inhibitors. Herein, we report the assessment of the relative orientation of the ligand 2,4thiazolidinedione to adenylate kinase crystallized in closed conformation. Protein-ligand docking was performed to estimate the binding energy and inhibition constant of 2,4-thiazolidinedione to the adenylate kinases’ active sites from different organisms. Our results revealed the best orientation of 2,4thiazolidinedione is with Gram-positive and acid fast bacteria adenylate kinase – Ki = 0.76±0.1 mM and binding energy -4.26±0.08 kcal/mol. Human adenylate kinases display unfavourable interactions, the binding affinity fluctuating among Ki=0.84 mM and 8.8 mM (3.88±3.51); the energy binding -3.56±0.57. From the three human adenylate kinases analysed, only isoenzyme 2 shows a binding conformation similar to its counterpart from E. coli. Adenylate kinase this small enzyme needed for survival of every organisms interacts differently with 2,4-thiazolidinedione, this selectivity being the most important evidence of the present study.


Introduction
Bacterial resistance to antibiotics is an evolving problem of medical practice [1,2].Discovery of new antibacterial agents and/or reevaluating of different targets are impetuously needed [3], [4].These kinds of studies are usually constructed by comparison of well characterized pathogens.Escherichia coli (E.coli) is an opportunistic Gram-negative pathogen, widely isolated from nosocomial infections [5].Streptococcus pneumoniae (S. pneumoniae) -a Gram-positive cocci -is an extracellular organism responsible for pneumoniae, otitis media, meningitis and even sepsis in very young children and adults above 65 years [6,7].Tuberculosis treatment remains one of the most challenging problems for clinicians; multidrugresistant Mycobacterium tuberculosis (M.tuberculosis) strains considerably diminish treatment options [8].
Adenylate kinase (AK) -ATP: AMP phosphotransferase; EC 2.7.3.4 -belong to the nucleotide kinase super family which catalyzes conversion between adenylate nucleotide by the following reaction: Mg 2+ ATP + AMP ↔ Mg 2+ ADP + ADP.AK is widely studied due to its implication in nucleotide metabolism and to its ubiquities -it is described in Archaea, prokaryotes and eukaryotes.Some AKs are very well characterized, many of them have the crystal structures solved and biochemical parameters established.AK is a flexible protein, which can adopt different conformations during catalytic process [9,10].
AK is, due to its universality and its flexibility, an attractive target for screening of new inhibitors.Thiazolidinediones (TZDs) derivates -synthetic agonists for the peroxisome proliferator-activating receptorgamma receptor -were demonstrated as potential inhibitors of S. pneumoniae out-growth [11].As we are interested in inhibition of AK by thiazolidine derivates [12], the present study is focused on 2,4thiazolidinedione interaction with the active site of bacterial and human AKs.

Data Collection and Design
The sequences and annotations for the proteins examined were collected from NCBI Protein Database (http://www.ncbi.nlm.nih.gov/protein).AKs X-ray 3D structures were downloaded from the Protein Data Bank (http://www.rcsb.org/pdb).AKs co-crystallized with the substrates or substrates analogs like the inhibitor P1,P5-di(adenosine-5')pentaphosphate (Ap5A) (C 20 H 29 N 10 O 22 P 5 ), were stored for further procedures.The structure of the ligand 2,4-thiazolidinedione (C 3 H 3 NO 2 S) was retrieved from the Open Chemistry Database -National Center for Biotechnology Information PubChem Compound Database; CID=5437, https://pubchem.ncbi.nlm.nih.gov/compound/5437.This open database provided chemicals as SDF files.
AKs included in the present study were presented in the Table 1.Firstly, from the 3D structure, substrate or substrate analogs (Ap5A) and all solvent molecules were removed and a closed conformation was obtained, which allows an algorithm based on a rigid protein structure.In our case, this docking algorithm was sufficient to obtain reliable data.The Lamarckian Genetic Algorithm (LGA), with a maximum of 2,500,000 energy evaluations, was choose as searching parameter [16].
Statistical evaluations: Data were expressed as mean ± SD.
Inclusion criteria: AKs from any microorganism or from human origin with crystal structure determined by X-ray crystallography co-crystallized with substrate or substrate analogs, so a closed conformation is provided.
Exclusion criteria: AKs with no crystal structure recorded in protein databases or 3D structure in open conformation.

Protein-ligand docking
AKs from different sources were subjected to docking with 2,4-thiazolidinedione.It is already established that AK is a natural flexible protein, binding and releasing of its substrates are followed by motions of the AMP-binding domain and LID domain.As a result of sophisticated conformational transitions a so-called closed conformation results during catalytic process [23,24].Starting from these premises, rigid protein structures were considered for further docking algorithm.For each AK, ten conformations were obtained as a result of docking process.The conformation with the smallest binding energy (∆G) and the smallest inhibition constant (K i ) was considered the best.As it is shown in the Table 2, regarding AKs from Gram-positive and acid fast species, ∆G values are almost the same with a mean of -4.26±0.08 kcal/mol, and K i are clustered around the mean 0.76±0.1 mM.On the other hand, the human counterpart AKs exhibit more diverse values.Human AK isoenzyme 2 (2C9Y) -mitochondrial origin -exhibits values similar to enzymes of bacterial sources.
Active sites residues which interact with the ligand 2,4-thiazolidinedione are shown in the Table 3.The most striking observation is related to AKs of human origin, as it is illustrated in the Figures 1 and 2. The AK5 (2BWJ) -expressed exclusively in the brain [25] -is clearly a poor target, none of the residues from the active site being involved in 2,4-thiazolidinedione binding.Further, in the case of AK2 (2C9Y), the ligand forms standard hydrogen bonds with two nonpolar residues from AMP-binding region (Gly100 and Phe101) and with Lys28 from ATP-binding domain.Besides, from the AKs examined in the present study, AK2 and one AK from E. coli 3HPQ are the only ones which bind 2,4-thiazolidinedione with P-loop domain residues (Lys28 and Lys13, respectively).Notably, the same oxygen atom (O2) (Scheme 1) of the ligand forms hydrogen bond with lysine but different atoms are involved -NZ regarding 2C9Y and CE for 3HPQ (Figures 3  and 4).On the contrary, apart from the residues involved in AMP-binding, AK1 shows only a glycine from NMP-binding domain which binds the ligand.This might be a reasonable explanation of almost twofold value K i of AK1 (1.99 mM) compared with AK2 (0.84 mM).
A unique detail is observed in the case of Gram-positive and acid fast AKs -a favorable interaction between Pi-sulfur of the ligand and the nonpolar residue Phe35 from NMP-binding domain (Figures 5 and  6) might play an important role in stabilization of AK-2,4-thiazolidinedione complex [26].Multiple sequence alignment exhibits a conservative replacement of Phe35 with leucine on other AKs.Moreover, in A. aeolicus' AK, van der Waals' forces between Leu35 and the only sulfur atom of the ligand are noticed.The interactions of 2,4-thiazolidinedione with AKs of Gram-negative bacteria A. aeolicus -a hyperthermophilic species -and two structures from E. coli are shown in the Figures 3 and 4. In the latter cases, even though the aminoacid sequences and the structures are identical, different ligand orientations were observed.One explanation could reside on the fact that the two enzymes being co-crystallized with different substrate analogs (Table 1).It is well known that AK is a flexible enzyme, the LID domain and NMP-binding domain being highly dynamic.Therefore, the transition between open and closed states is characterized by important conformational fluctuations (dynamics).Studies performed with AK mutated in the residues which do not alter its structure, demonstrated that the binding interactions of different ligands affect the dynamics of AK, without accompanying structural changes of the enzyme [23,27,28].

Moreover, glutamine, a polar residue strictly conserved in AKs -the last residue of the consensus sequence motif [LIVMFYWCA] -[LIVMFYW] (2)-D-G-[FY]-P-R-X(3) -[NQ]
-promotes hydrogen bonds with O2 / O3 in A. aeolicus, E. coli (1ANK), AK1, M. tuberculosis, and Gram-positive AKs.On these six AKs, sulfur atom contributes to protein-ligand complex stabilization also.Contrary to AKs of Gram-positive and acid fast bacteria where Pi-sulfur interactions were observed, hydrogen bonds were noticed for the rest.Regarding 1ANK and AK1, the valine residue Val59, respectively Val67, strictly conserved in AKs, participates in hydrogen bonding with sulfur atom.

4W5J
1ZIP 2CDN In Gram-negative microorganisms, the ligand 2,4-thiazolidinedione binds different residues.Looking at E. coli's AKs, we noticed that Gly85, another residue strictly conserved from the consensus sequence motif, promotes a hydrogen bonding with the sulfur in the instance of 3HPQ, but failed to establish a valid interaction in 1ANK.On the contrary, in human AK2, this residue (Gly100) interacts with the O2 atom from the 2,4-thiazolidinedione structure.
As it is demonstrated in the Figures 5 and 6 and in multiple sequence alignment (Figure 7), AKs from Gram-positive and acid fast microorganisms, bind almost in the same manner the residues involved in AMPbinding (Thr31, Gln93, and Phe35), a notable difference is for AK from B. stearothermophilus In this latter instance, the smallest binding affinity is noticed -K i = 0.64 mM.One can speculate other Gram-positive pathogens AKs' have the same inhibition pattern.
AKs of human origin display unfavorable interactions, the binding affinity fluctuating among 0.84 mM and 8.8 mM.Mitochondrial isoenzyme 2 of AK behaves in the same manner as one E. coli's AK -3HPQ.As demonstrated in the Figure 7, they bind the same residues and have comparable binding affinity, K i = 0.84 mM and 0.86 mM (Table 2).On the contrary, even though AK2 has energy binding and K i comparable to Gram-positive microorganisms, 2,4-thiazolidinedione binds AMP-binding pocket and ATP-binding domain simultaneously.Considering these different binding patterns of human AKs the selectivity of 2,4thiazolidinedione is an advantage in the context of antimicrobial treatment.

Multiple sequence alignment
Primary structure offers useful details; comparisons of even small identities could be exploited for finding interesting patterns.It would be of interest to speculate that enzymes with similar primary structure, behave the same with otherwise well characterized enzyme.Sometimes, observing minute details on primary structure has a major impact in decipher intriguing mechanism of a particular protein.
The ligand chooses for the present work, 2,4-thiazolidinedione, has proven to bind important residues involved in AMP-binding.The main limitation of the present work is the availability of AKs' crystal structures deposited in the public databases.Even though some databases have some weaknesses -some of them being redundantthey remain a powerful tool in fundamental research.Unfortunately, kinetic experiments were not performed, which was another limit of the present study.But the biochemical characterization of a particular enzyme, not always deciphers all capabilities of the protein of interest.
To put these findings in perspective, two approaches could be taken in consideration.Either the screening of library protein molecules [28] to find receptors to the highest affinity to 2,4-thiazolidinedione, or design novel TZDs derivates with better binding affinity.Inevitably, inhibitors used for bacterial infections could interact with human counterpart's target.Although these evidences are not strong enough to persuade us that 2,4-thiazolidinedione is selective inhibitors for bacterial AKs, a novel perspective of AK potentiality is revealed.Results obtained in this study lead us to the conclusion that refining the structure of 2,4-thazolidinedione could be a viable idea in developing potential inhibitors for vital enzymes like AK.

Conclusions
This study demonstrated an effort to reveal interactions of AK of bacterial / human origin with 2,4thiazolidinedione.The most noteworthy evidences were achieved referring to Gram-positive and acid fast bacteria's AKs; 2,4-thiazolidinedione interacts with the same residues belonging to NMP-binding region.Further, the complex comprising AKs of S. pneumoniae and M. tuberculosis exhibit the same ligand-protein pattern.Human AKs interactions with 2,4-thiazolidinedione are divergent; a single notable connection being observed in case of mitochondrial isoenzyme 2 and its counterpart from E. coli.AK, a small enzyme needed for survival, could be an attractive target for TZDs derivates, at least in case of M. tuberculosis and Gram-positive microorganisms.

Figure 7 .
Figure 7. Multiple sequence alignment.In gray background is marked ATP-binding domain -the Walker A motif GXXXXGKGT/S; in blue background are shown leucine and valine residues, very well conserved in AKs family; in green background is highlighted the consensus sequence [LIVMFYWCA] -[LIVMFYW] (2)-D-G-[FY]-P-R-X(3)-[NQ], were aspartic acid and arginine, strictly conserved in AKs family, are bolded; in red are colored the residues which interact with 2,4-thiazolidinedione

Table 1
The main characteristics of the AKs used in protein-ligand docking

Table 2 .
Energy binding and inhibition constant (Ki) of the best conformation of the complex AK-2, 4-thiazolidinedione

Table 3 .
Residues of substrate binding domains, according to Protein Database (www.rcsb.org/pdb/home/home.do)details Code color for interactions with 2,4-thiazolidinedione: in green are shown conventional hydrogen bonds; blue -van der Waals' forces, brown -carbon hydrogen bonds; orange -Pi-sulfur, red -unfavorable acceptor-