ITP Adjuster 1 . 0 : A NewUtility Program to Adjust Charges in the Topology Files Generated by the PRODRG Server

e suitable computation of accurate atomic charges for the GROMACS topology ∗.itp �les of small molecules, generated in the PRODRG server, has been a tricky task nowadays because it does not calculate atomic charges using an ab initio method. Usually additional steps of structure optimization and charges calculation, followed by a tedious manual replacement of atomic charges in the ∗.itp �le, are needed. In order to assist this task, we report here the ITP Adjuster 1.0, a utility program developed to perform the replacement of the PRODRG charges in the ∗.itp �les of small molecules by ab initio charges.

e topology �les preparation for performing EM and MD simulations with GROMACS [1] is very important in investigations of biological processes involving drug interactions with their molecular targets.e force �elds available for biomolecules in the literature [13][14][15][16] are generally based on macromolecules, such as proteins and nucleic acids, making the preparation of consistent and reliable topology �les for small ordinary molecules a challenging task.e PRODRG server [12] is a reliable tool for quickly generating topologies and coordinates of ligands in protein-ligand complexes, using the empirical GROMOS96 force �eld [14], from a variety of different input �les.is tool has been used in several studies and a wide range of knowledge areas [17][18][19][20][21][22][23][24][25].is server works with the concept of charge groups, which is de�ned as a group of bonded atoms that sums an integer total charge.Lemkul et al. [26] investigated the quality of topologies generated by the PRODRG server [12] for small molecules for the GROMOS96 43A1 force �eld [14] and described their functional groups.eir results show that the atomic partial charges are critically incompatible with the GROMOS force �elds.Furthermore, when we use the PRODRG [12] parameters, the behavior of the system is notably different than the one observed using the GROMOS parameters.us, they suggest a more consistent strategy for the calculation of partial atomic charges, avoiding the deviation of behavior in the system.
Several computer programs are available for structure optimization and charge calculations using quantum chemical calculations [27][28][29].e Frisch et al. soware [30] has been reported as the most standard for quantum chemical calculations of small molecules [31][32][33].us, the ITP adjuster 1.0 was built to make the interface of Frisch et al.  [30] with the GROMACS package [1].However, it may also be easily modi�ed to work with different quantum chemical sowares.
�ere are several problems to ad�ust the output �les of PRODRG [12] based on Frisch et al. [30] output �les ( * .out�les): (i) the manual edition of the topology charges is an exhausting, tedious, and time-demanding assignment and, so, very susceptible to mistakes; (ii) PRODRG [12] uses a charge group concept, differently from Frisch et al. [30] and any other quantum chemical sowares; (iii) the numbering of hydrogen atoms in the * .itp�les is incompatible with the output �les from quantum chemical sowares; (iv) the bonds information in the Frisch et al. [30] output �les may have some inconsistencies, such as hydrogen atoms bonded to two different atoms.e ITP Adjuster 1.0 (Figure 1) performs a quick and safe edition of the * .itp�les considering the charge calculations from Frisch et al. [30] using Mulliken or Chelpg (Charges from Electrostatic Potentials) charges.It handles charges of up to six decimal places.At the end of the execution, a message box will inform the total charge of the molecule in the edited * .itp�le, which may be used as a guide to detect inconsistencies between the * .itpand * .out�les.

Considerations for
Using ITP Adjuster.Some requirements for using the ITP Adjuster 1.0 are important to guarantee its proper installation and use, as discussed below.
ITP Adjuster 1.0 needs the Microso.Net Framework 4 or later installed for running properly.e authors recommend the Frisch et al. [30] calculations and PRODRG server [12] topology generation.It is highly desirable to have a * .pdb�le with numbered atom names before submitting it to the PRODRG server [12].It guarantees the correspondence of the atom names between the * .outand the �nal * .itp�les.ITP Adjuster 1.0 also has a functionality named �PDB Adjustment, � which veri�es a * .pdb�le and, if necessary, numbers the atom names.
e hydrogen charges are based on the information of bonds presented in the Frisch et al. [30] output �les, which is necessary to provide an * .itp�le which is compatible with the * .out�le indicated.An usual mistake occurs when the PRODRG server [12] removes hydrogen atoms from oxygen and nitrogen atoms or adds hydrogen atoms that do not exist in the * .out�le.In these cases, the user must use the ADDHYD, DELHYD, PATCH, or a combination of these commands in the PRODRG server [12] in order to correct these inconsistencies before using ITP Adjuster 1.0.Not performing this strategy leads to unreliable �nal * .itp�les and meaningless total charges.

Results and Discussion
3.1.Validating ITP Adjuster 1.0.We validated the ITP Adjuster 1.0 with the three common molecules in biological systems: folic acid, adenosine-5′-triphosphate (ATP), and cystein.Structures of these molecules are shown in Figure 2. First we built their 3D structures in the * .pdbformat using Gauss View 4.0 and Frisch et al. [30] and submitted them to energy minimization with Frisch et al. [30] using the algorithm B3LYP/3-21G with charge chelpg, in order to generate their * .out�les.Besides, the * .itp�les of each molecule were generated at the PRODRG server [12].e * .outand * .itp�les were then loaded into the ITP Adjuster 1.0 to change the charges of the * .itp�les generated by the PRODRG server [12] by using ab initio charges of the * .out�les from Frisch et al. [30].e results are presented in Tables 1, 2, and 3.As it can be seen, the ITP Adjuster 1.0 was able to recognize the atoms in each �le.It properly adjusted the charges without modifying the positions of the atoms.e ITP Adjuster 1.0 was also tested against several other small molecules (data not shown) and, without exception, it was able to adjust charges accordingly.

Conclusion
We reported here the development of the ITP Adjuster 1.0, a utility program built to adjust ab initio charges into * .itp�les generated by the PRODRG server [12].is utility program is already in use in our laboratory and proved to be very accurate in the interface of Frisch et al. [30] and GROMACS [1], providing a friendly user interface and a quick way to generate suitable topology �les to perform EM and MD calculations with the GROMACS package [1].
is utility program is available free of charge by request at lmm@puc-rio.br.

�on��ct of �nterests
All the authors declare that there is no con�ict of interests related to the publishing of this paper on the Journal of Chemistry or any direct �nancial relation with the trademarks mentioned in the paper.

F 2 :
Structures of some molecules tested with ATP adjuster 1.0.