In silico analysis of V48A dihydropteroate synthase mutation to dapsone on Mycobacterium leprae from Papua

Background: Leprosy is a disease caused by Mycobacterium leprae. Drug resistance is one of the challenges in leprosy elimination especially in Papua. The presence of mutations in folP1 gene that encode dihydropteroate synthase (DHPS) was considered as the exclusive basis for molecular detection of dapsone resistance in leprosy. The objective of this study was to detect mutations in the folP1 gene of Mycobacterium leprae from Papua, Indonesia and to analyze the effect of these mutations on dapsone using the in-silico method. Methods: Identification of mutations in the folp1 M. leprae gene is carried out through the Basic Local Alignment Search Tool (BLAST) process in the gene bank. The analysis of the effects of mutations using the Have (y)Our Protein Explained (HOPE) server. Bindings pocket prediction is done using the Computed Atlas of Surface Topography of proteins (CASTp). Homology modeling 3D structure of DHPS using the Iterative Threading ASSEmbly Refinement (I-TASSER) server. Docking analysis was performed using AutoDock Vina which is integrated with the Python Prescription (PyRx) application. Results: The sequencing results showed a variation in the folP1 M. leprae gene, namely a change from thymine (T) to cytosine (C) in nucleotide 143. The mutated residue (V48A) is in a domain that is essential for the activity of the protein and in contact with residues in another domain. It is possible that this interaction is important for the correct function of the protein. V48A mutants did not significantly affect the stability of DHPS M. leprae. Conclusion: Based on molecular docking analysis, this mutation does not affect binding affinity dapsone against M. leprae dihydropteroate synthase. These results indicate that the V48A mutant is likely to remain susceptible to dapsone. Thus, it is necessary to do an in vivo test to confirm the effect of the V48A mutation. (Health Science Journal of Indonesia 2020;11(2):70-6)

Leprosy is a skin infection, membrane and prefers nerve disease. 1 Leprae is a neglected disease that still occurs in about 120 countries with more than 200,000 new cases reported annually. The regional proportions of all new cases in 2019 were: 71.3% (143 787) in South-East Asian Region (SEAR), 14.9% (29 936) in Americas Region (AMR), 9.9% (20 205) in African Region (AFR), 2.1% (4211) in Eastern Mediterranean Region (EMR), 1.9% (4004) in WPR and 42 in European Region (EUR). 2 This disease is still a significant disease in Jayapura City, Papua, furthermore, the burden disease of leprosy in Jayapura is considered as high. 3 Based on the Disability Number, Papua was the highest number in Indonesia in 2013 (26,88), followed by Aceh (18,62), and West Papua (17,72). The data from General Directorate of Disease Control and Health Environment in 2013 reveals that Case Detection Rate (CDR) of Papua was 35,64 in 100.000 people and declined to 30,43/100.000 on 2014. 4,5 Since 1995, WHO has supplied Multi Drug Therapy (MDT) to all country with leprosy burden. The MDT consists of three antibiotics, contained rifampicin, clofazimine and dapsone for Multibacillary (MB) and rifampicin and dapsone combination for Paucibacillary (PB). 6 Unfortunately, MDT program in Papua and West Papua faces challenges such as the low awareness of patients, geographical barriers to access the health facility and other people's paradigm about drugs. DHPS is an enzyme that plays a role in the biosynthesis of folate in bacteria including M. leprae, which targets dapsone by inhibiting p-aminobenzoic acid (PABA). 7 The presence of point mutations in folP1 gene that encodes dihydropteroate synthase (DHPS) was considered as the exclusive basis for molecular detection of dapsone resistance in leprosy. 8 Dapsone-resistant M. leprae isolates have shown mutation at codon 53 or 55 in the folP1 gene. 9,10,11 The most frequently detected mutation associated with dapsone resistance in M. leprae is CCCCTC in codon 55 of folP1 resulting in the substitution of leucine for a proline residue (Pro55Leu) in the DHPS. 12 Predictively, the mutation will decrease the effectiveness of dapsone therapy. The effect of drug resistance due to the point mutation on the amino acid residues of the targeted protein can be studied by bioinformatics simulation (in-silico). This method is relatively accurate, rapid and cost-effective compared to in vitro and in vivo method. Therefore, computational studies can be performed to study drug resistance. 13,14 Many of the molecular docking is successful in predicting the binding form of the ligand in the receptor binding sides. 15 The objectives of this study were to detect mutations in the folP1 gene of M. leprae from Papua Island, Indonesia and to analyze the effect of these mutations on dapsone using the in silico method. Homology of 3D dihydropteroate synthase structure was analyzed by using I-TASSER 16 (https://zhanglab. ccmb.med.umich.edu/I-TASSER/). Variants in T53A, V48A, and P55L were constructed using the fold-X 17 based on the wildtype structure (P0C0X1).

e. Preparation of drug molecules
The 3D structure of dapsone (CID:2955) was downloaded from Pubchem database, available at https://pubchem.ncbi.nlm.nih.gov/. Dapsone molecule was downloaded in SDF format. Molecule energy was reduced and converted pdbqt using Open Babel software.

f. Molecular docking
Docking analysis was performed by using AutoDock Vina 18 which is integrated in PyRx application. 19 Docking results were visualized using PyMol 1.8.6 and Discovery Studio 2017.

RESULTS
From 100 clinical samples, 53 PCR positive result samples were obtained and qualified to be proceed to sequencing. The BLAST result in NCBI shows that variation was identified in M. leprae Papua strain compared with TN strain in folP1 gene. The type of mutation is missense where Thymine (T) was replaced by Cytosine (C) on nucleotide 143 ( Figure  1). This mutation gave rise to the replacement of amino acid Valine become Alanine on DHPS ( Figure  2b). The wild type and mutant amino acids differ in size. Alanine is smaller than Valine. The mutation will cause an empty space in the core of the protein.
Among the 53 samples that were successfully amplified, there were six samples that show the mutation.
Mutation in the 143 nucleotides folP1 gene of M. leprae was identified as a single peak (Figure 2b) and in a double peak (Figure 2c). This mutation caused a change from the Thymine (Figure 2a) to Cytosine. In some samples this mutation was found in a mixed allele (Figure 2c). As a comparison, we used the P55L, T53A variants which were confirmed as mutations that cause resistance to dapsone. 10,11

. Comparison of electropherogram between wild type and mutant (a), Thymine mutation becomes Cytosine with single pic (b) and mix between mutant and wildtype (c).
V48A T53A P55L

Figure 3. Overview of the protein in ribbon-presentation. The protein is colored grey, the side chain of the mutated residue is colored magenta and shown as small balls.
Protein structure analysis of DHPS conformation on mutant of M. leprae at V48A, P55L, T53A, showed that each mutation influences the protein function ( Figure 3). The mutation on V48A is located within a domain, annotated in UniProt as Pterin-binding. The mutation introduces an amino acid with different properties, which can disturb this domain and abolish its function 20 (Figure 3a). The wildtype residue on 55 DHPS is a proline. Prolines are known to be very rigid and therefore induce a special backbone conformation which might be required at this position. The mutation P55R/L can disturb this unique conformation 20 (Figure 3b dan 3c).
The wild-type residue on 53 DHPS is a threonine. The mutation T53A introduces an amino acid with different properties, which can disturb this domain and abolish its function 20 (Figure 3).
The wide and the volume of binding pocket DHPS were predicted using CASTp servers. 21 Binding pocket from DHPS is in the red area ( Figure 4). The V48A mutant showed the identical area and volume as wildtype while the binding pocket of the T53A and P55L mutants showed the increasing area and volume (Table 1). WT   The wildtype DHPS (P0C0X1) modeling was performed using I-TASSER, while the mutant DH PS protein structure was derived using fold-X by emphasizing the mutations in the amino acids 48, 53 and 55. The model used in this study was the model with the highest confidence level based on the C-score. 16,22 Mutations in all three amino acids did not significantly alter the stability of the energy of the molecules ( Table  2). Molecular docking between dapsone and DHPS showed the ability of dapsone to interact with wild type DHPS and mutant DHPS. Binding affinity between dapsone was higher on different sides of wildtype, specifically on variants T53A and P55L, whereas the binding affinity between dapsone and wild type and V48A of DHPS were equal ( Table 2). Interaction between dapsone and DHPS consisted of hydrogen bond, unfavorable donor-donor, Pi-cation, Pi-sulfur, and Pi-Pi T-shaped ( Figure 5).

DISCUSSIONS
We found mutations in the folP1 gene of M. leprae from Papua in nucleotide no 143 where there has been a change of amino acids from Thymine (T) to Cytosine (C) (Figure 1). This mutation caused changes in the amino acid Valine to Alanine in the DHPS enzyme ( Figure 2b). These mutations exist in the region of Drug Resistance-Determining Regions (DRDR) of the folP1 gene. The folP1 gene is the gene that encodes the formation of the DHPS enzyme. This enzyme is the target of dapsone in the treatment of leprosy. 9 WHO has recommended this area for surveillance of drug resistance using PCR-direct sequencing. 12 This mutation was found in a single allele (Figure 2a) as well as multiple alleles (Figure 2a). Confirmation of mixed alleles in the folP1, gyrA, and rpoB genes of M. leprae has been reported previously. 23,24 Mutation V48A has been detected by Nakata et al in their clinical samples. 25 In this study, we analyze V48A mutant using in silico method. Former studies show mutations in the folP1 gene were missense mutations located at codon 53 (Thr53Ile, Thr53Arg and Thr53Ala) or codon 55 (Pro55Arg, Pro55Leu). 11 Nakata et al, found all mutations that cause amino acid substitutions at codon 55 resulted in dapsone resistance and mutations at codon 53 also gave rise to dapsone resistance except for the T53S substitution, which resulted in less resistance to dapsone than the wildtype sequence. 25 Mutation on V48A is located within a domain, annotated in UniProt as Pterin-binding which can disturb this domain and abolish its function. 20 The wild-type residue is very conserved, but a few other residue types have been observed at this position too. The mutant residue was not among the other residue types observed at this position in other, homologous proteins. However, residues that have some properties in common with your mutated residue were observed. This means that in some rare cases mutation V48A might occur without damaging the protein. The mutant residue is located near a highly conserved position important for the activity of the protein and in contact with residues in another domain. It is possible that this interaction is important for the correct function of the protein (Figure 3). This mutation does not affect the volume binding pocket (Table 1) and protein stability ( Table  2). However, the mutation may affect this interaction and as such affect protein function. 20 On P55L mutant, the mutated residue is located in a domain that is important for the activity of the protein and in contact with residues in another domain. The mutant increased the volume binding pocket of DHPS (Table 1). It is possible that this interaction is important for the correct function of the protein.
The mutation can affect this interaction and as such affect protein function. 20 The mutated residue of T53A mutant is located in a domain that is important for the activity of the protein and in contact with another domain that is also important for the activity. The interaction between these domains could be disturbed by the mutation, which might affect the function of the protein. 20 On wildtype, the binding position of dapsone mutants V48A, T53A and P55L present in the binding pocket.
The binding affinity between dapsone in T53A DHPS was stronger than wildtype, but preferably bond to a site that was different from wildtype. Dapsone linked with the T53A mutant through four hydrogen bonds on the residues Q51, E54, D86 and R253 ( Figure 5). Dapsone is bound to the mutant P55L with three hydrogen bonds, R54, G181, and R253. Binding affinity formed was lower than that of wildtype, but also on sites that were different from wildtypes (Table 1). This indicated that the variants of P55L and T53A caused Dapsone to experience orientation changes in binding to DHPS. So that dapsone activity in inhibiting the performance of DHPS in both variants had decreased, or resistance to dapsone. T53A mutations, P55L in the folP1 gene have also been confirmed to cause resistance to dapsone. 10,11 Chaitanya also found the greatest decrease in free energy bindings was present in the T53I and T55V mutants. 8 The energy and changes in the bonding patterns revealed the structural and mechanistic effects of these mutations on inducing dapsone resistance in leprosy. 8 The binding affinity of V48A mutant was similar to wildtype ( Figure 5). Dapson was bound with V48A through three covalent bonds of hidrogen namely 2 hidrogen bonds on residual E51 and 1 on D177 residual. This interaction was similar to the dapsone interaction on the wildtype (Gambar 5). This shows that the V48A mutant might still possess the ability to interact with dapsone. The testing of the effect of mutant 448A based on MIC values has been conducted before and it is known that the V48A mutation effect might give rise to low-level resistance to dapsone in M. leprae based on MIC value. 25 Thus it is necessary to do an in vivo test to confirm the effect of the V48A mutation.
In conclusion, based on molecular docking analysis, this mutation does not affect binding affinity dapsone against M. leprae dihydropteroate synthase. These results indicate that the V48A mutant is likely to remain susceptible to dapsone. Thus it is necessary to do an in vivo test to confirm the effect of the V48A mutation. 26