New Journal of Chemistry

Study of the interaction of 2-hydroxy-6-pentadecyl-benzoic

acid with the active site of Trypanosoma cruzi GAPDH
enzyme by docking and molecular fractionation with
conjugate caps (MFCC) analysis

Journal: New Journal of Chemistry

Manuscript ID NJ-ART-03-2019-001162

Article Type: Paper

Date Submitted by the

05-Mar-2019
Author:

Complete List of Authors: Marinho, Marcia; Universidade Federal do Ceará, Departamento de

Análises Clínicas e Toxicológicas
Santos, Ricardo; Universidade Federal do Ceará, Departamento de
Engenharia de Computação
Bezerra, Eveline; Universidade Federal Rural do Semi-Árido
Ferreira-Costa, Roner; Universidade Federal Rural do Semi-Árido
Siqueira-Figueira, Ciro; Universidade Federal do Ceara
Martins, Alice; Universidade Federal do Ceará, Departamento de Análises
Clínicas e Toxicológicas
Lima-Neto, Pedro; Universidade Federal do Ceará, Departamento de
Química Analítica e Físico-Química
Marinho, Emmanuel; Universidade Estadual do Ceará
Freire, Valder; Universidade Federal do Ceará, Departamento de Física
Albuquerque, E.; Universidade Federal do Rio Grande do Norte, Biofísica
e Farmacologia
Page 1 of 8 New Journal of Chemistry

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3 New Journal of Chemistry
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Professor Mir Wais Hosseini
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6
Editor-in-Chief
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9 Study of the interaction of 2-hydroxy-6-pentadecyl-benzoic acid with the active site
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of Trypanosoma cruzi GAPDH enzyme by docking and molecular fractionation
12 with conjugate caps (MFCC) analysis
13
14 M. M. Marinho, R. P. dos-Santos, E. M. Bezerra, R. F. Costa, C. S. Figueira,
15
16 A. M. C. Martins, P. de Lima-Neto, E. S. Marinho, V. N. Freire and E. L. Albuquerque
17
18 Dear Editor,
19
20
21 Enclosed, please find the original manuscript submitted for publication in New
22
Journal of Chemistry. This work is part of the graduate research of M. M.
23
24 Marinho supported by Brazilian agencies.
25
26 This paper was devoted to study the theoretical interactions between anacardic
27
28 acid (AA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma
29 cruzi (TcGAPDH). The inhibition of TcGAPDH is directly correlated with the life cycle
30
31 of the parasite. Although AA is a natural inhibitor of TcGAPDH enzyme, there are no
32
33 known co-crystallization of TcGAPDH along with AA and mechanistical considerations
34
between them are still unknown. In order to fulfill this gap, theoretical considerations are
35
36 carried out to study the TcGAPDH inhibition by AA in its saturated alkyl chain (AA0).
37
38 Computational studies, such as fragmentation method with conjugated layers (MFCC)
39
40 with molecular docking showed the AA0 molecule occupying neighborhood active sites
41 of the chalepin of TcGAPDH enzyme. The residual aminoacids that interacts with AA0
42
43 are: TYR339, CYS166, ILE13, MET16, HIS194, THR167 and THR 226, being IL13 the
44
45 residue with strongest binding energy throughout van der Waals interactions. Those
46
47
unprecedented way of inhibition is a novelity in this research field.
48 Considering these arguments and hoping for a prompting and favorable reviewing
49
50 process, the authors are convinced that the manuscript includes sufficient scientific
51
52 novelties to recommend its publication in New Journal of Chemistry.
53
With kindest regards,
54
55 The Authors
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57
58
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8 ARTICLE
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11 Study of the interaction of 2-hydroxy-6-pentadecyl-benzoic acid
12
13
with the active site of Trypanosoma cruzi GAPDH enzyme by
14 Received 00th January 20xx,
docking and molecular fractionation with conjugate caps (MFCC)
15 Accepted 00th January 20xx analysis
16
17 DOI: 10.1039/x0xx00000x
M. M. Marinho a, R. P. dos-Santos b, E. M. Bezerra c, R. F. Costa c, C. S. Figueira b, A. M. C. Martins a,
18 www.rsc.org/
19 P. de Lima-Neto d, E. S. Marinho e, V. N. Freire f and E. L. Albuquerque g
20 The mechanism of action of trypanocidal drugs is generally not well understood. The inhibition of glyceraldehyde-3-
21 phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi (TcGAPDH) was proposed as an explanation for the
22 trypanocidal effect of the drugs, because it can change the energy metabolism and ATP production through anaerobic
23 glycolysis in the cytoplasm, important for the parasite's life cycle. Anacardic acid (AA) is an important natural inhibitor of
24 TcGAPDH; however, TcGAPDH has not yet been co-crystallized with AA and details of its interaction mechanism with the
25 enzyme active site is yet to be elucidated. From this perspective, the present work aims at elucidating the possible
26 interaction mechanism of AA with the saturated alkyl chain (AA0) in the TcGAPDH enzyme. To obtain more reliable
27 quantitative information on the interaction of anacardic acid with the active site of the TcGAPDH enzyme, we combined the
28 fragmentation method with conjugated layers (MFCC) with molecular docking. We can observe that the AA0 molecule
29 occupies a region near the active site of the chalepin molecule in the TcGAPDH enzyme and the Ile13 residue has the
30 strongest binding energy of approximately 25 kcal / mol with AA0, through a strong van der Waals interaction.
31
32 The mechanism of action of trypanocidal drugs is generally not
33 Introduction well understood. Many published studies assume that the drug
34 produces free radicals to which Trypanosoma cruzi is particularly
Caused by the parasite protozoan hemoflagellate
35 sensitive 7, but some of the proposed mechanisms of action have
Trypanosoma cruzi, Chagas' disease is commonly transmitted to
36
humans by the insect vector Triatoma infestans 1. This is an endemic been recently challenged 8. The inhibition of glyceraldehyde-3-
37
disease in tropical and subtropical regions of Latin America, Africa phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi
38
(TcGAPDH) on the other hand, was also proposed as an explanation
39 and Asia, affecting millions of people each year 2. Through
40 immigration, the disease has also affected non-endemic regions, for the trypanocidal effect of drugs 9–11. This enzyme is responsible,
41 such as the United States 3,4. Benznidazole and nifurtimox are the in the glycolytic pathway of the parasite, for the conversion of
42 drugs used to treat Chagas' disease, are not effective against certain glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate in the
43 forms of the disease, in addition to having significant side effects 5,6. presence of nicotinamide-adenine-dinucleotide (NAD) + inorganic
44 Therefore, it is important to develop new drugs so these problems phosphate 12,13. Through anaerobic glycolysis in the cytoplasm, its
45 can be minimized or eliminated. inhibition can modify the energetic metabolism and the production
46 of ATP, important for the parasite's life cycle 14,15. In addition to its
47 primordial role in glycolysis in the cytoplasm, GAPDH has also been
48 associated with several non-metabolic events, such as the onset of
49 a. Department of Clinical and Toxicological Analysis, Universidade Federal do Ceará,
60430270 Fortaleza, CE, Brazil. E-mail: marinho.marcia@alu.ufc.br apoptosis, axoplasmic transport, activation of transcription, and
50 b. Computer Engineering / Biotechnology, Universidade Federal do Ceará (UFC),
transport of ER to Golgi 16,17. Thus, this enzyme can be an important
51 62042-280 Sobral, CE, Brazil.
target for the rational planning of new drugs.
52
c. Universidade Federal Rural do Semi-Árido (UFERSA), 59625-900 Mossoró, RN,

Brazil. Various substances extracted from medicinal plants have been

53 d. Department of Analytical Chemistry and Physical-Chemistry, Universidade Federal
tested for TcGAPDH inhibition 18–20. For instance, chalepin (Fig. 1a),
54 do Ceará, 60455-760 Fortaleza, Ceará, CE, Brazil.
e. Faculdade de Filosofia Dom Aureliano Matos, Universidade Estadual do Ceará, a furanocoumarin isolated from species of the Rutaceae family,
55 62930-000 Limoeiro do Norte, CE, Brazil.
showed high inhibitory effect on TcGAPDH 21,22. Another important
56 f. Department of Physics, Universidade Federal do Ceará, 60455-760 Fortaleza,

natural product inhibitor of TcGAPDH is the 2-hydroxy-6-pentadecyl-

57 Ceará, CE, Brazil.
g. Department of Biophysics and Pharmacology, Universidade Federal do Rio
benzoic acid, also known as anacardic acid (AA) 23(Fig. 1b). It is found
58 Grande do Norte, 59072-970 Natal, RN, Brazil.
in the shells of cashew nuts, the Anacardium occidentale, a plant
59
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3 native to the northeastern Brazilian coast and also found in Central MFCC combined with the docking approach can provide more
4 America and in countries such as India, Asia and Africa 24. Several reliable quantitative information on the interaction of drugs with the
5 biological activities of AA have been reported: antimicrobial, active site of macromolecular targets.
6 antifungal, antioxidant, antitumoral, antiparasitic, molluscicidal, Devipriya and Kumaradhas (2012) 43 performed molecular
7 larvicidal, and insecticidal 25–29. The AA is a phenolic lipid, having the
docking and the DFT approach to show changes in the conformation
8 structure of a salicylic acid substituted by an alkyl chain with 15
and charge density of AA molecules before and after interaction with
9 carbons. The alkyl chain may be saturated or unsaturated (monoene,
the active site of the p300 enzyme. Pereira (2007) 44 and Freitas et al.
10 diene or triene) 30. All forms of AA (saturated or unsaturated alkyl
(2009) 31 studied the interaction of AA and analogues with TcGAPDH
11 chain) show inhibitory effect on TcGAPDH; however, the form with
through molecular docking and biochemical assays. However, a more
12 the saturated alkyl chain shows the greatest effects 31. Because of its
13 high inhibition capacity, the AA has been studied as a promising detailed analysis of this interaction (using the MFCC method)
14 trypanocidal drug. TcGAPDH has not yet been co-crystallized with AA together with conformational and charge density changes of the
15 and details of its mechanism of interaction with the enzyme site of ligand molecule (before and after docking) have not been conducted.
16 action are not well elucidated. To better elucidate the possible interaction mechanism of AA with
17 the saturated alkyl chain (AA0) in the TcGAPDH enzyme, molecular
18 OH OH docking and MFCC analyzes of the AA0 molecule were performed at
19 (a) the active site of the enzyme. Results were compared with the
20 O
binding energy (calculated by MFCC) of NAD+ cofactor crystalized
21 with TcGAPDH.
CH3
22
23 Results and discussion
24 CH2
25 H3C The molecular electrostatic potential surface (MESP)
26 (b) The molecular electrostatic potential surface (MESP), allows to
CH3
27 visualize the relative polarity of the compounds 45 and allows us to
28 HO CH3 predict the binding site between biological molecules and their
29 receptors 46,47, as well as being an important tool in the study of new
O
30 CH3 O O
drugs 48,49. Associated with the dipole moment, the electronegativity
31 Fig. 1 – Molecular structures of AA (a) and chalepin (b).
and partial charge of the molecular electrostatic potential 50, can be
32
33 Aiming to potentially fulfill this drawback, computational used to predict the types of intermolecular interaction, the molecule
34 chemistry has been widely used for the planning of biologically active may carry as well as the sites of these interactions. The electrostatic
35 compounds and the understanding of their mechanisms of action 32. potential at a point (x, y, z) is given by the electrostatic potential
36 DFT (Density Functional Theory) is a quantum computational method energy between a hypothetical positively charged (+1) ion located at
37 that allows the determination (with acceptable accuracy) of various (x, y, z) and the molecule. Being the ion attracted by the molecule,
38 drug properties, such as geometric configurations of the lowest then the potential molecule is negative, and the ion repulsion means
39 energy, expected positions of the constituent atoms, electronic the potential molecule is positive. Thus, electron-rich regions usually
40 have negative potentials and electron-poor regions usually have
charge distributions, vibrational frequencies, and so on 33,34. Changes
41 positive potentials, and the molecular electrostatic potential (MEP)
in the geometric configuration and charge distribution of a drug
42 at a point r in the space around a molecule (in atomic units) can be
43 molecule caused by its interaction with the active site of an enzyme
play a key role in its biological activity 35. Molecular docking are expressed as 51:
44
45 computational methods usually based on force fields of classical
𝑍𝐴 𝜌(𝑟 )
46 molecular mechanics that predict the mode of binding of a small- 𝑉(𝑟) = ∑ −∫ 𝑑𝑟 ′
|𝑅⃗𝐴 − 𝑟 | |𝑟 ′ − 𝑟 |
47 molecule ligand to the active site of a macromolecular ligand to form 𝐴
48 a stable complex 36. They are used in the search for new ligands for
49 drug therapy because of their low computational cost when where ZA is the charge on nucleus A, located at RA and ρ(r’) is the
50 compared to methods based on quantum mechanics 37. However, electronic density function for the molecule. V(r) is the resulting
51 the typically used docking methods do not correctly predict the electrostatic net effect produced at the point r by both the electrons
52 binding energy of a ligand-receptor complex 38,39. Moreover, the and the nuclei of the molecule, where the first term represents the
53 contributions due to the potential for electrons and the second term
MFCC (molecular fractionation whith conjugate caps) is a more
54 due to the nuclei. The knowledge of the charge distribution,
powerful approach using quantum mechanics calculations to
55
determinate interaction energies for protein-ligand systems 40,41. In identifying their partial densities, helps in understanding the form of
56
this method, the interaction energy is calculated by summing the interaction between a molecule and another, and thus, one can
57
identify the molecular reactive site 52,53.
58 interactions between the individually capped protein fragments
59 (individual amino acid-based fragments) and the ligands 42. The
60

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3 The electrostatic potential map shown in Fig. 2 illustrates the
4 three-dimensional charge distributions of the molecule, while
5 negative electrostatic potentials (with intensity proportional to the
6 absolute value of the potential energy) are shown in red, positive
7 electrostatic potentials are visualized in blue and green, we perceive
8 areas with potentials close to zero.
9
10
11
12
13
14
15
16
17
18
19 Fig. 3 – TcGAPDH complexes crystalized with (a) nicotinamide-adenine-
20 dinucleotide (NAD), (b) chalepin. Docking between TcGAPDH and anacardic
21 acid (AA0) (c) and binding site of AA0 and chalepin in the TcGAPDH (d).
22 Fig. 2 – Molecular electrostatic potential surface (MESP) of anacardic acid
23 (AA0).
Fig. 4 shows the molecular 2D interaction map for AA0 and TcGAPDH
24 complex. As it can be seen, there are many kinds of interaction, since
25 We observed a partial negative charge density of carboxyl weak interactions by London dispersion forces (pale pink), sigma-pi
26 located in the region, thus indicating a region rich in electrons, and interactions (lilac), pi-pi stacking (darker pink) and hydrogen bonding
27 this fact because the presence of pairs of nonbonding electrons of (bright green) are found. Those interactions are in agreement with
28 oxygen, causing the region to become a nucleophilic site. While in AA0/chapelin docking with TcGAPDH complex. The TcGAPDH-AA0
29 the alkyl (green region) groups, we observe a region as a potential complex (with the AA0 better conformation in the docking
30 near zero (bonds C-C), featuring a hydrophobic region of the simulation) was used in the MFCC calculations.
31
molecule (predominantly nonpolar region).
32
33
34 Molecular docking and MFCC
35 The best-fit conformation in the docking simulation of AA0 is
36 shown in fig. 3, together with the chalepin docking configuration
37 (obtained from crystallographic data of the TcGAPDH complex -
38
chalepin) for comparison. We can observe that the AA0 molecule
39
occupies a region near the active site of the chalepin molecule in the
40
41 TcGAPDH enzyme. The AA0 can interact with a large number of
42 residues due to its long chain. In addition, it consists of a polar head
43 with a benzene ring and a long hydrophobic tail. The polar head
44 favors the formation of hydrogen bonds, while the tail can interact
45 with amino acids that have a hydrophobic character.
46
47
48
49 Fig. 4 – Molecular 2D map for intermolecular interactions between AA0 and
50 TcGAPDH residues.
51
52 Based on the density functional theory (DFT), the interaction
53 energy of the amino acid residues was determined, through
54 computations using the MFCC approach. The amino acids that
55 showed significant interaction energy for stabilizing the AA0
56 molecule were: Ile13; Met16; Ser134,224; Ala135,164,228; Pro136;
57 Cys165; Thr167,197,226; His194; Gly227; Arg249; Asp334; Asn335;
58 Glu336 and Tyr339. The BIRD (Binding site, Interaction energy and
59
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3 Residues Domain) panel (Fig. 4) shows a graphical display with reported and finally, a weak interaction of hydrogen with Asp210 is
4 interaction energies between ligand and the most important amino intermediated by W812 water. Due to the structural differences of
5 acid residues comprising the binding site of the TcGAPDH enzyme. the ligands, the above amino acids may or may not show molecular
6 This panel shows relevant residues interacting with anacardic acid interactions relevant to AA0 stabilization, as it can be observed in the
7 contributing to its energy stabilization at the binding site of the BIRD panel.
8 gGAPDH enzyme. This panel is a new version of BIRD presented by Residue Ile13 has the strongest binding energy, approximately
9 Costa et al in 2012 41 and shows: (i) the interaction energy of each 25 kcal/mol with AA0, through a strong Van der Walls interaction
10
residue with the AA0 molecule using horizontal bars, based on which involving the AA0 tail and the side chain of Ile13, both with
11
can quantitatively estimate the role of each residue in the binding hydrophobic characteristics. Although the hydrophobic interactions
12
13 site, i.e., its efficacy to attract or repel the drug; (ii) the most are admittedly one of the weakest, it is relatively strong because of
14 significant residues for the bond on the left side; (iii) the atoms that the proximity between the chemical groups of both binder and
15 show the shortest distance between the amino acid residue and the residue, which is about 2 Å, as well as the extent of the side chain of
16 ligand; (iv) the left side panel containing the minimum distance in the Ile13 residue, which is approximately 4 Å.
17 angstroms between amino acid ligands; (v) the bars indicating the The second amino acid with the strongest interaction energy is
18 interaction strength between ligand and enzyme residues of Glu336, with a binding energy around 15.0 kcal / mol, due to the
19 TcGAPDH show colors that depend on the side chain of each amino natural formation of an AA0 electrostatic dipole inside the tail-
20 acid score: binding site, with absence of electrons, which favors the interaction
21 with amino acids that have more electronegative atoms or side
22 chains, which is the case of Glu336 that has the COO- group.
23 The third most important residue, Tyr339, has a binding energy
24 with the AA0 of 14.1 kcal/mol due to a sigma-pi interaction, caused
25 by the presence of an aromatic ring rich in electrons in the side chain
26 of that amino acid, as well as positive characteristic of the AA0 tail.
27 We can also observe that the method used to calculate the
28 interaction energy was able to describe a pi-pi interaction between
29 the aromatic rings of AA0 and the side chain of the His194 residue,
30 with an intensity of about 5.0 kcal/mol. The residues Thr167 and
31 Thr226 interact with AA0 through a hydrogen interaction of about
32 12.0 kcal/mol and 13.0 kcal/mol, respectively. The hydrogen of the
33 OH group of Thr167 interacts with the oxygen of the CO group of the
34 polar region of AA0, which are separated by a distance of 1.95 Å,
35 whereas the other hydrogen bond is due to the interaction of the
36 hydroxyl hydrogen of the residue Thr226 and the oxygen of the AA0
37 Fig. 5 – (a) Anacardic acid binding site obtained from molecular docking,
hydroxyl group. Together, Ser165 and Cys166 create a region with a
38 highlighting important residues of TcGAPDH: Tyr339, Ser165, Cys166,
Thr167, Ile13, Thr226 and Glu336. (b) Another view of the anacardic acid positive electrostatic potential, which couples with the region of the
39 binding site obtained from molecular docking, highlighting Pro136, Ala 135, AA0 hydrophobic tail that has electronegative characteristics, thus
40 Thr197 and Met16 residues. (c) Interaction energy, binding site and domain producing a relevant attractive interaction between these amino
41 of residues, showing relevant residues interacting with anacardic acid,
acids of approximately 13.0 kcal/mol.
42 contributing to its energy stabilization at the binding site of the TcGAPDH
43 enzyme. Amino acid residues have colors that depend on the side chain of
each: Nonpolar, Aliphatic; Aromatic; Polar, Uncharged; Sulfur-Containing;
44 Charged Negative (Acidic) and Charged Positive (Basic). Experimental
45 Geometry optimization
46 We can see in the attached BIRD (Fig. 5) that twenty-two
47 Initially, the geometry optimization of the AA three-dimensional
residues contribute to the stabilization of AA0: Arg12, Ile13, Arg15,
48 structure (with the pentadecyl chain) obtained from the ZINC
Met16, Asp38, Glu109, Pro136, Ala164, Ser165, Cys166, Thr167, database (ZINC code: 8234355) 55 was performed using DFT (B3LYP)
49
His194, Thr197, Thr226, Ala228, Arg249, Asp254, Asp334, Asn335, calculations. The Gaussian 03 program (Gaussian, Inc.) was used,
50
Glu336, Tyr339, and Arg342, whereas only one Ala135 amino acid adopting the B3LYP hybrid functional exchange-correlation and the
51
52 residue has positive (repulsive) interaction energy. Pavão et al. 54 6-311+G (d,p) basis set to expand the electronic states. Structural
53 reported that the residues Thr167, Cys166, Arg249, and Asp210 of convergence of isolated neutral AA0 (gas phase) was reached when
54 the gGAPDH enzyme have important interactions with Chalepin. the following thresholds were met: maximum force per atom < 1.5 x
55 According to Pavão et al. 54, residue Thr167 interacts with the oxygen 10−5 Ha Å−1 and RMS force < 1.0 x 10−5 Ha Å−1, self-consistent field
56 of the CO group of Chalepin through the W739. The Cys166 residue energy variation < 10-7 Ha, and maximum atomic displacement < 6 x
57 shows a characteristic hydrophobic interaction with the 1,1- 10−5 Å. The lowest energy configuration (optimized) was represented
58 as AA0. With the AA0 optimization data, it was possible to plot the
dimethylallyl group; a hydrogen interaction with the Arg249 is also
59 molecular electrostatic potential surface (MESP).
60

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3 Molecular docking the self-consistent field convergence threshold to 10-6 Ha. The
4 Molecular docking simulation was performed using automated binding energy of chain A of TcGAPDH-NAD+ cofactor complexes was
5 coupling with the AutoDock Vina program (Molecular Graphics Lab) also calculated (by MFCC) to be compared with the TcGAPDH-AA0
6 56. The crystallographic structure of TcGAPDH complexed with complex.
7
chalepin (TcGAPDH-chalepin) was obtained from RCSB Protein Data
8 Conclusions
Bank (code: 1K3T). Pavão et al (2002) elucidated such a complex [55].
9
The ligand used was the optimized AA0 and the receptor was the In conclusion, we obtained more reliable quantitative
10
11 chain C of the TcGAPDH enzyme after the removal of the chalepin of information on the interaction of anacardic acid with the active site
12 the complex. A box region with 820 nm3 centered on and of the enzyme TcGAPDH by combining MFCC with molecular docking.
13 encompassing the active site was defined as the receptor search We verified that the AA0 molecule occupies a region near the active
14 volume. The best-fit conformation in the docking simulation of AA0 site of the chalepin molecule in the TcGAPDH enzyme and that
15 the binding is the binding site used for the construction of the residue Ile13 has the strongest binding energy, approximately 25
16 TcGAPDH-AA0 complex. To compare the position of the ligand at the kcal/mol with AA0, through a strong Van der Waals interaction.
17 binding site, the Chain A of the TcGAPDH-NAD+ cofactor (PDB code:
18 1QXS) 10 complexes were used. Molecular graphics and docking were Conflicts of interest
19 performed using the UCSF Chimera 1.8 package 57. There are no conflicts to declare.
20
21
Molecular fractionation with conjugate caps (MFCC) Acknowledgements
22
This research received financial support from Brazilian National
23 To estimate the total energy of interaction, all 359 amino acid
Council (CNPq) projects 151182/2013-2 and 447592/2014-9.
24 residues of the A chain of the enzyme of the TcGAPDH-AA0 complex
This study was financed in part by the Coordenação de
25 were taken into account, seeking to disregard important Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES)
26 interactions. For each amino acid residue, sets of four amino acids – Finance Code 001. Alice-Maria Costa-Martins, Eudenilson Lins-
27 were used as caps, two on each side, in order to increase accuracy Albuquerque, Pedro Lima-Neto, and Valder Nogueira-Freire are
28
when measuring the interaction energy. Interaction energies senior researchers from Brazilian National Council (CNPq) and
29
between the AA0 molecule and the amino acid residues of the acknowledge the financial support received during the
30
TcGAPDH enzyme were estimated by using the MFCC strategy 43. The development of this work from the Brazilian Research Agency
31
binding energy (interaction) between the inhibitor molecule AA0 and (CNPq).
32
33 the amino acid residue Ri, is given by E(AA-Ri):
34 E(AA-Ri) = E(AA-Ci-1RiCi+1) - E(Ci-1RiCi+1) - E(AA-Ci-1Ci+1) + E(Ci-
References
35 1Ci+1)

36 where the Ci (cap) is obtained by attaching a carboxyl or amine group 1 A. Rassi, A. Rassi and J. A. Marin-Neto, The Lancet, 2010,
37 375, 1388–1402.
to the dangling bond of the residue Ri. On the right side of the
38 2 G. A. Schmunis, Memorias Do Instituto Oswaldo Cruz, 2007,
equation, E(AA-Ci-1RiCi+1), is the total energy of the system formed by
39 102, 75–85.
the AA molecule and the capped residue; the E(Ci-1RiCi+1) term is the
40 3 Y. Jackson, L. Gétaz, H. Wolff, M. Holst, A. Mauris, A.
total energy of the blocked residue alone; E (AA-Ci-1Ci + 1) is the total Tardin, J. Sztajzel, V. Besse, L. Loutan, J. M. Gaspoz, J.
41
energy of the system formed by the AA and the covers alone. To Jannin, P. A. Vinas, A. Luquetti and F. Chappuis, PLoS
42
conclude, E (Ci-1Ci + 1) is the total energy of the system formed only Neglected Tropical Diseases, 2010, 4, 1–7.
43
44 by the molecular caps. For the First-Principles Calculations, the 4 C. Bern, S. Kjos, M. J. Yabsley and S. P. Montgomery,
45 methodology seen in early works 42,58 was used; in the other words, Clinical Microbiology Reviews, 2011, 24, 655–681.
46 the positions of the non-hydrogen atoms were kept fixed while the 5 F. Nagajyothi, F. S. Machado, B. A. Burleigh, L. A. Jelicks, E.
47 positions of the hydrogen atom and AA0 were in principle optimized Scherer, S. Mukherjee, M. P. Lisanti, L. M. Weiss, N. J. Garg
48 using the CHARMm force field 59, which has parameters specific for and H. B. Tanowitz, 2013, 14, 634–643.
49 amino acids. Then, simulations were performed within the formalism 6 J. R. Coura and S. L. de Castro, Memorias do Instituto
50 Oswaldo Cruz, 2002, 97, 3–24.
of the Density Functional Theory, using local density approximation
51 7 C. A. Morillo, J. A. Marin-Neto, A. Avezum, S. Sosa-Estani,
for the correlation-change functional with a dispersion correction
52 A. Rassi, F. Rosas, E. Villena, R. Quiroz, R. Bonilla, C. Britto,
scheme (DFT/LDA/OBS) were carried out using the Dmol3 code 60,61.
53 F. Guhl, E. Velazquez, L. Bonilla, B. Meeks, P. Rao-Melacini,
In order to expand the Kohn-Sham electron orbitals, a set of J. Pogue, A. Mattos, J. Lazdins, A. Rassi, S. J. Connolly, S.
54
numerical bases and double polarization (DNP) was selected, Yusuf and BENEFIT Investigators, The New England journal
55
considering all electrons, explicitly and with restricted spin. The DNP of medicine, 2015, 373, 1295–306.
56
57 basis set has an accuracy equivalent to the 6-311+G(3df,2pd) 8 S. Sosa-Estani, R. Viotti and E. Segura Leonor, Memórias do
58 Gaussian basis set 60–62, with negligible basis set superposition error Instituto Oswaldo Cruz, 2009, 104, 167.
59 (BSSE). The orbital cutoff radius for this basis set was set to 3.7 Å and 9 B. M. Bakker, H. V. Westerhoff, F. R. Opperdoes and P. A.
60

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1 ARTICLE NJC
2
3 M. Michels, Molecular and Biochemical Parasitology, 2000, 28 M. Hemshekhar, M. Sebastin Santhosh, K. Kemparaju and
4 106, 1–10. K. S. Girish, Basic and Clinical Pharmacology and
5 10 S. Ladame, M. S. Castilho, C. H. T. P. Silva, C. Denier, V. Toxicology, 2012, 110, 122–132.
6 Hannaert, J. Périé, G. Oliva and M. Willson, European 29 J. Tan, B. Chen, L. He, Y. Tang, Z. Jiang, G. Yin, J. Wang and
7 Journal of Biochemistry, 2003, 270, 4574–4586. X. Jiang, Chinese journal of cancer research = Chung-kuo
8 11 I. M. Prokopczyk, J. F. R. Ribeiro, G. R. Sartori, R. Sesti- yen cheng yen chiu, 2012, 24, 275–83.
9 Costa, J. S. Silva, R. F. Freitas, A. Leitão and C. A. Montanari, 30 J. H. P. Tyman, Journal of the Chemistry Society Perkin,
10 Future Medicinal Chemistry, 2013, 6, 17–33. 1973, 1, 1639–1647.
11 12 J. I. . Harris and M. Waters, ed. P. . Boyer, Academic Press, 31 R. F. Freitas, I. M. Prokopczyk, A. Zottis, G. Oliva, A. D.
12 New York, 3 ed., 1976, pp. 1–49. Andricopulo, M. T. S. Trevisan, W. Vilegas, M. G. V. Silva
13 13 V. L. Zinsser, E. M. Hoey, A. Trudgett and D. J. Timson, and C. A. Montanari, Bioorganic and Medicinal Chemistry,
14 Biochimica et Biophysica Acta - Proteins and Proteomics, 2009, 17, 2476–2482.
15 2014, 1844, 744–749. 32 S. G. Dahl and I. Sylte, Basic & clinical pharmacology &
16 14 M. W. Nowicki, L. B. Tulloch, L. Worralll, I. W. McNae, V. toxicology, 2005, 96, 151–5.
17 Hannaert, P. A. M. Michels, L. A. Fothergill-Gilmore, M. D. 33 T. Ziegler and J. Autschbach, Chemical Reviews, 2005, 105,
18 Walkinshaw and N. J. Turner, Bioorganic and Medicinal 2695–2722.
19 Chemistry, 2008, 16, 5050–5061. 34 E. M. Bezerra, M. Z. S. Flores, E. W. S. Caetano, V. N. Freire,
20 15 S. Yang, C. Pannecouque, D. Daelemans, X. D. Ma, Y. Liu, F. V. Lemos, B. S. Cavada and J. L. de L. Filho, Journal of
21 E. Chen and E. De Clercq, European Journal of Medicinal Physics: Condensed Matter, 2006, 18, 8325–8336.
22 Chemistry, 2013, 65, 134–143. 35 B. Devipriya and P. Kumaradhas, Journal of Theoretical
23 16 A. Tarze, A. Deniaud, M. Le Bras, E. Maillier, D. Molle, N. Biology, 2013, 335, 119–129.
24 Larochette, N. Zamzami, G. Jan, G. Kroemer and C. 36 N. Brooijmans and I. D. Kuntz, Annual Review of Biophysics
25 Brenner, Oncogene, 2007, 26, 2606–2620. and Biomolecular Structure, 2003, 32, 335–373.
26 17 C. Nicholls, H. Li and J. P. Liu, Clinical and Experimental 37 S. F. Sousa, P. A. Fernandes and M. J. Ramos, Proteins:
27 Pharmacology and Physiology, 2012, 39, 674–679. Structure, Function, and Bioinformatics, ,
28 18 I. R. A. Menezes, J. C. D. Lopes, C. A. Montanari, G. Oliva, F. DOI:10.1002/prot.21082.
29 Pavão, M. S. Castilho, P. C. Vieira and M. T. Pupo, Journal of 38 D. B. Kitchen, H. Decornez, J. R. Furr and J. Bajorath, Nature
30 Computer-Aided Molecular Design, , Reviews Drug Discovery, 2004, 3, 935–949.
31 DOI:10.1023/A:1026171723068. 39 S. G. P. Sooderhjelm and U. Ryde, ed. ed. H. Gohlke, Wiley-
32 19 S. Hanau, E. Rinaldi, F. Dallocchio, I. H. Gilbert, C. VCH Verlag GmbH & Co, 2012, pp. 121–144.
33 Dardonville, M. J. Adams, S. Gover and M. P. Barrett, 40 G. Zanatta, I. L. Barroso-Neto and V. B.- Junior, Journal of
34 Current medicinal chemistry, 2004, 11, 2639–50. Proteomics & Bioinformatics, 2012, 05, 155–162.
35 20 A. A. De Marchi, M. S. Castilho, P. G. B. Nascimento, F. C. 41 R. F. Da Costa, V. N. Freire, E. M. Bezerra, B. S. Cavada, E.
36 Archanjo, G. Del Ponte, G. Oliva and M. T. Pupo, Bioorganic W. S. Caetano, J. L. De Lima Filho and E. L. Albuquerque,
37 and Medicinal Chemistry, 2004, 12, 4823–4833. Physical Chemistry Chemical Physics, 2012, 14, 1389–1398.
38 21 P. C. Vieira, J. Mafezoli, M. T. Pupo, J. B. Fernandes, M. F. 42 D. W. Zhang and J. Z. H. Zhang, Journal of Chemical Physics,
39 D. G. F. da Silva, S. de Albuquerque, G. Oliva and F. Pavão, 2003, 119, 3599–3605.
40 Pure and Applied Chemistry, 2001, 73, 617–622. 43 B. Devipriya and P. Kumaradhas, Journal of Molecular
41 22 G. H. Schmelzer and ; Ameenah Gurib-Fakim, Plant Graphics and Modelling, 2012, 34, 57–66.
42 Resources of Tropical Africa 11(1) : Medicinal plants 1, 44 B. Cnpq, .
43 2008, vol. 11. 45 A. E. Reed and F. Weinhold, The Journal of Chemical
44 23 J. M. Pereira, R. P. Severino, P. C. Vieira, J. B. Fernandes, M. Physics, 1985, 83, 1736–1740.
45 F. G. F. da Silva, A. Zottis, A. D. Andricopulo, G. Oliva and A. 46 E. J. Yearley, E. A. Zhurova, V. V. Zhurov and A. Alan
46 G. Corrêa, Bioorganic and Medicinal Chemistry, 2008, 16, Pinkerton, Journal of Molecular Structure, 2008, 890, 240–
47 8889–8895. 248.
48 24 R. P. Santos, A. A. X. Santiago, C. A. A. Gadelha, J. B. 47 P. Politzer and J. S. Murray, Theoretical Chemistry
49 Cajazeiras, B. S. Cavada, J. L. Martins, T. M. Oliveira, G. A. Accounts, 2002, 108, 134–142.
50 Bezerra, R. P. Santos and V. N. Freire, Journal of Food 48 D. E. Hibbs, J. Overgaard, J. A. Platts, M. P. Waller and M. B.
51 Engineering, 2007, 79, 1432–1437. Hursthouse, The Journal of Physical Chemistry B, 2004, 108,
52 25 N. Mendes and A. Oliveira, Rev. Soc. bras. Med. …, , 3663–3672.
53 DOI:10.1590/S0037-86821990000400007. 49 P. Politzer, J. S. Murray and Z. Peralta-inga, 2001, 684, 676–
54 26 I. Kubo, N. Masuoka, T. J. Ha and K. Tsujimoto, Food 684.
55 Chemistry, 2006, 99, 555–562. 50 C. E. Ophardt, Virtual ChemBook,
56 27 M. S. C. Oliveira, S. M. de Morais, D. V. Magalhães, W. P. http://chemistry.elmhurst.edu/vchembook/index.html,
57 Batista, Í. G. P. Vieira, A. A. Craveiro, J. E. S. A. de Manezes, (accessed 25 January 2018).
58 A. F. U. Carvalho and G. P. G. de Lima, Acta Tropica, 2011, 51 N. Prabavathi, A. Nilufer and V. Krishnakumar,
59 117, 165–170. Spectrochimica Acta - Part A: Molecular and Biomolecular
60

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Please do not adjust margins

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1 NJC ARTICLE
2
3 Spectroscopy, 2013, 114, 101–113.
4 52 J. Seminario, Ed., Recent Developments and Applications of
5 Modern Density Functional Theory, Amsterdam, The
6 Netherland, 1st edn., 1996.
7 53 G. E. Herring, R. H. Petrucci, W. S. Harwood and J. Madura,
8 General Chemistry: Principles & Modern Applications,
9 Pearson Education Inc, New Jersey, ninth., 2007.
10 54 F. Pavão, M. S. Castilho, M. T. Pupo, R. L. A. Dias, A. G.
11 Correa, J. B. Fernandes, M. F. G. F. Silva, J. Mafezoli, P. C.
12 Vieira and G. Oliva, FEBS Letters, 2002, 520, 13–17.
13 55 J. J. Irwin, T. Sterling, M. M. Mysinger, E. S. Bolstad and R.
14 G. Coleman, Journal of Chemical Information and
15 Modeling, 2012, 52, 1757–1768.
16 56 O. Trott and A. Olson, Journal of Computational Chemistry,
17 2010, 31, 455–461.
18 57 E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D.
19 M. Greenblatt, E. C. Meng and T. E. Ferrin, Journal of
20 Computational Chemistry, 2004, 25, 1605–1612.
21 58 I. L. Barroso-Neto, J. P. C. Marques, R. F. Da Costa, E. W. S.
22 Caetano, B. S. Cavada, C. Gottfried and V. N. Freire, Journal
23 of Physical Chemistry B, 2012, 116, 3270–3279.
24 59 F. A. Momany and R. Rone, Journal of Computational
25 Chemistry, 1992, 13, 888–900.
26 60 B. Delley, The Journal of Chemical Physics, 1990, 92, 508–
27 517.
28 61 B. Delley, The Journal of Chemical Physics, 2000, 113,
29 7756–7764.
30 62 Y. Inada and H. Orita, Journal of Computational Chemistry,
31 2008, 225–232.
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
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