Developing Inhibitors Targenting Enhanced Intracellular Survival (Eis) Protein of Mycobacterium tuberculosis (Mtb)

Developing Inhibitors Targenting Enhanced Intracellular Survival (Eis) Protein of Mycobacterium tuberculosis (Mtb)

• Author
• Midiane Correia Gomes
• Co-authors
• Vitoria de Melo Batista , Igor José dos Santos Nascimento , Thiago Mendonça de Aquino , Maria Eugênia Gouveia de Freitas , Francisco Jaime Bezerra Mendonça-Júnior , Valnês S. Rodrigues Junior , Edeildo Ferreira da Silva Júnior
• Abstract

•  

  Developing Inhibitors Targenting Enhanced Intracellular Survival (Eis) Protein of Mycobacterium tuberculosis (Mtb)

  Midiane Correia Gomes1, Vitoria de Melo Batista2, Igor José dos Santos Nascimento3, Thiago Mendonça de Aquino1, Maria Eugênia G. de Freitas4, Francisco Jaime Bezerra Mendonça-Júnior5, Valnês S. Rodrigues-Junior4,5, Edeildo Ferreira da Silva-Júnior1.

  1Post-Graduation Program in Chemistry and Biotechnology, Institute of Chemistry and Biotechnology, Federal University of Alagoas, CEP: 57072-970, Maceió-AL, Brazil.

  2Institute of Pharmaceutical Sciences, Federal University of Alagoas, CEP: 57072-970, Maceió-AL, Brazil.

  3Post-Graduation Program of Pharmaceutical Sciences, Pharmacy Department, State University of Paraíba, Campina Grande, Brazil

  4National Institute of Science and Technology in Tuberculosis (INCT-TB), Brazil

  5Post-Graduation Program in Natural Products and Bioactive Synthetics, Federal University of Paraíba, João Pessoa, Brazil

   

  INTRODUCTION: Tuberculosis (TB) is a infectious disease caused by Mycobacterium tuberculosis (Mtb) that mainly affects the lungs, but can also affect other organs and systems [1,2]. TB is transmitted through the air when an infected person coughs, sneezes or talks, releasing small droplets containing the bacteria into the air. Before the COVID-19 outbreak, tuberculosis (TB) was one of the leading causes of death worldwide, with more than one million deaths recorded annually [3]. The pandemic has had a significant impact on TB control initiatives, leading to an increase in cases, a decrease in diagnostic tests and reduced access to medicines. The current therapeutic protocol for tuberculosis (TB) includes a combination of drugs, including isoniazid (INH), rifampicin (RIF), pyrazinamide (PYZ) and ethambutol (EMB) [4]. Drug-resistant TB, which includes both multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), poses a significant threat because these strains are resistant to conventional treatments [5]. According to the literature, compounds derived from pyrimidines, thienopyrimidines and indoles have shown promise against TB [6-9]. Therefore, based on the findings in the literature, our research group decided to synthesise a small library of compounds, including thienopyrimidines and their derivatives, quinazolines, quinazolinimines and indole derivatives, with the aim of evaluating their efficacy as anti-tuberculosis agents against the H37Ra strain of MTb. Among the synthesised compounds, LQM495 was the most promising and was subjected to detailed in silico analysis and the Enhanced Intracellular Survival (Eis) protein of MTb was identified as a potential target. Therefore, the in silico studies indicate that LQM495 has potential for the development of new inhibitors targeting the Eis protein of MTb H37Ra.

  AIMS

  The study aimed to discover a potential inhibitory substance against the Eis protein of MTb via compound synthesis, biological assessment, and in silico analyses. The goal was to contribute important insights towards the development of fresh therapeutic remedies for tuberculosis.

   METHODS

  The production of the compounds comprised multiple stages, beginning with the Gewald reaction. This initial phase involved the combination of ethyl cyanoacetate, sulfur, morpholine, and cyclohexanone to produce 2-amino-thiophene intermediates through cyclization. These intermediates were subsequently merged with distinct isocyanates to yield thiourea derivatives, which were then cyclized with NaOH to produce thienopyrimidines. The thienopyrimidines were subsequently reacted with substituted 2-bromoacetophenones to yield the final S-substituted thienopyrimidine compounds, and the same synthesis procedure was employed to produce the quinazoline analogues. In addition, in silico studies were conducted using a consensus reverse docking (CRD) strategy with both Vina and ChemPLP GOLD searching algorithms to investigate the interactions between compound LQM495 and 98 possible targets within the H37Ra MTb (PDB id: 6VUR). Following the study, Eis was identified as a feasible target. To gain insights into the reactivity of the compound based on its electronic properties, the investigation was advanced with 400 ns molecular dynamics (MD) simulations and Density Functional Theory (DFT) calculations using B3LYP/D3 to optimize the LQM495-Eis complex geometry.

   

   RESULTS AND DISCUSSION

  Among the tested compounds, LQM495 exhibited the most potential, displaying a minimum inhibitory concentration (MIC) of 50 µM, whereas the remaining compounds demonstrated limited or no impact. Co-administration of LQM495 with MTb infection-targeted pharmaceuticals did not significantly affect MIC values, indicating a distinctive mechanism of action and the involvement of alternative targets. In light of this, further research was conducted through in silico methods to determine potential biological targets of LQM495. The Eis protein of MTb H3Ra (PDB id: 6VUR) was identified as a promising pharmacological target for LQM495. Both the Vina and ChemPLP scores yielded significant binding affinities, with values of -11.1 kcal/mol and 103.94, respectively. This protein can acylate numerous aminoglycoside antibiotics, preventing their binding to the mycobacterium's ribosome. When the MTb parasite promoter undergoes mutations, the overexpression of this protein leads to drug resistance. A benzothieno[2,3d]pyrimidine analogue named SGT366 was cocrystallized at the Eis MTb protein binding site [9], which has raised significant interest. This significant development suggests that our active compound, LQM495, structurally similar to SGT366, may function as an effective inhibitor for this potential target. Furthermore, the LQM495-Eis complex underwent further investigation regarding its interactions and stability under physiological conditions through molecular dynamics (MD) studies. These simulations yielded crucial insights into the LQM495-Eis complex, highlighting its impressive structural dynamics and stability. Across the 400 nanosecond simulations, the complex maintained an equilibrium, indicating a high degree of structural integrity. The analysis of the Ramachandran plots demonstrated that both SGT366 and LQM495 maintained conformations that were compatible with the Eis MTb protein binding site. However, there was a slight structural flexibility observed in LQM495 compared to SGT366. The RMSD (Root Mean Square Deviation) analyses exhibited that LQM495-Eis complex achieved a higher level of stability than the SGT366-Eis complex under similar physiological conditions. The Root Mean Square Fluctuation (RMSF) analysis revealed specific residues that underwent significant fluctuations, particularly in the regions of alpha helices and loops. These findings suggest that the binding of LQM495 requires adaptation. The study also investigated the chemical interactions between LQM495 and the Eis MTb protein, identifying a strong affinity between LQM495 and residues like Phe24, Trp36, and Glu401 through hydrophobic, water bridge, and hydrogen bond interactions. Water bridges were the predominant interaction type detected for specific residues, including Asp26, Glu199 and Glu203. Results from the MD simulations highlighted the outstanding structural and conformational stability of LQM495 when bound to the Eis MTb protein, therefore exhibiting potential for further research into inhibitor development for this target. The examination of the conjugated electronic systems and frontier molecular orbitals (FMOs) of the LQM495-Eis complex has yielded vital insights into its energetic properties and reactivity. Consequently, the LQM495-Eis complex was subjected to DFT calculations [10] that provided all necessary energetic parameters. The B3LYP functional, Grimme's D3 dispersion correction method, and the def2-SVP basis set were utilised for these calculations. The study investigated the LQM495 binding site on the Eis MTb site and determined the values for EHOMO, ELUMO and EHOMO-LUMO gaps. The analysis of the EHOMO-LUMO gaps demonstrated a stable complex between LQM495 and the Eis MTb binding site, indicating an effective interaction. The distribution of electronic densities in the orbitals was revealed through HOMO-LUMO maps. For LQM495, the HOMO was primarily located on the benzopyrimidine ring, whilst the LUMO was dispersed over almost the entire molecule. The HOMO for residue Phe24 was found to be prevalent at the Eis MTb binding site, whereas the LUMO was noted to be on residues Ala20 and Glu199. An investigation of the FMOs revealed pronounced complementarity between the HOMO of LQM495 and the LUMO of the Eis MTb binding site. After evaluating the binding energy, the interaction's stability with the Eis MTb binding site was confirmed. The binding energy was calculated as "Ebinding energy" and determined to be -9.91 kcal/mol. This suggests a high binding affinity and stability of LQM495 to the Eis MTb binding site. These analyses provided detailed information on the energetic properties and interaction of LQM495 with the Eis MTb binding site, indicating its potential as an effective inhibitor for this therapeutic target.

  CONCLUSION

  Therefore, the worldwide concern over the global health problem presented by TB, particularly in light of the emergence of MDR strains of MTb, persists. This study is a notable step forward in the quest for new therapeutic approaches to combat this drug-resistant pathogen, providing valuable insights. The compound LQM495 demonstrated potential as an inhibitor of the Eis protein, a resistant enzyme of the MTb-causing bacterium. During a systematic screening, LQM495 demonstrated remarkable effectiveness in inhibiting the H37Ra strain of MTb, along with other compounds that displayed moderate activity. Computational simulations yielded information on the stability and binding mechanism of the LQM495-Eis complex, which enhanced our comprehension of the molecule's electronic properties and reactivity. The structural resemblance between LQM495 and a ligand found on the Eis protein highlights its potential as an anti-tuberculosis medication. These findings signify a significant progression in the quest for novel therapeutic approaches to combat TB, particularly in light of the surge of drug-resistant strains.

   

  ACKNOWLEDGMENT

  This research is supported by CAPES, CNPq, FAPEAL and UFAL

   

  REFERENCES

   

  1.       Belyaeva, I. V.; Kosova, A.N.; Vasiliev, A.G. Tuberculosis and Autoimmunity. Pathophysiology, 2022, 29, 298–318.

   

  2.       Mei, Y.-M.; Zhang, W.-Y.; Sun, J.-Y.; Jiang, H.-Q.; Shi, Y.; Xiong, J.-S.; Wang, L.; Chen, Y.-Q.; Long, S.-Y.; Pan, C.; Luo, T.; Wang, H.-S. Genomic Characteristics of Mycobacterium Tuberculosis Isolates of Cutaneous Tuberculosis. Front. Microbiol., 2023, 14.

   

  3.       Dartois, V.A.; Rubin, E.J. Anti-Tuberculosis Treatment Strategies and Drug Development: Challenges and Priorities. Nat. Rev. Microbiol., 2022, 20, 685–701.

   

  4.       Loiseau, C.; Windels, E.M.; Gygli, S.M.; Jugheli, L.; Maghradze, N.; Brites, D.; Ross, A.; Goig, G.; Reinhard, M.; Borrell, S.; Trauner, A.; Dötsch, A.; Aspindzelashvili, R.; Denes, R.; Reither, K.; Beisel, C.; Tukvadze, N.; Avaliani, Z.; Stadler, T.; Gagneux, S. The Relative Transmission Fitness of Multidrug-Resistant Mycobacterium Tuberculosis in a Drug Resistance Hotspot. Nat. Commun., 2023, 14, 1988.

   

  5.       Dean, A.S.; Zignol, M.; Cabibbe, A.M.; Falzon, D.; Glaziou, P.; Cirillo, D.M.; Köser, C.U.; Gonzalez-Angulo, L.Y.; Tosas-Auget, O.; Ismail, N.; Tahseen, S.; Ama, M.C.G.; Skrahina, A.; Alikhanova, N.; Kamal, S.M.M.; Floyd, K. Prevalence and Genetic Profiles of Isoniazid Resistance in Tuberculosis Patients: A Multicountry Analysis of Cross-Sectional Data. PLOS Med., 2020, 17, e1003008.

   

  6.       Finger, V.; Kufa, M.; Soukup, O.; Castagnolo, D.; Roh, J.; Korabecny, J. Pyrimidine Derivatives with Antitubercular Activity. Eur. J. Med. Chem., 2023, 246, 114946.

   

  7.       Lagardère, P.; Fersing, C.; Masurier, N.; Lisowski, V. Thienopyrimidine: A Promising Scaffold to Access Anti-Infective Agents. Pharmaceuticals, 2021, 15, 35.

   

  8.       Chiarelli, L.R.; Salina, E.G.; Mori, G.; Azhikina, T.; Riabova, O.; Lepioshkin, A.; Grigorov, A.; Forbak, M.; Madacki, J.; Orena, B.S.; Manfredi, M.; Gosetti, F.; Buzzi, A.; Degiacomi, G.; Sammartino, J.C.; Marengo, E.; Korduláková, J.; Riccardi, G.; Mikušová, K.; Makarov, V.; Pasca, M.R. New Insights into the Mechanism of Action of the Thienopyrimidine Antitubercular Prodrug TP053. ACS Infect. Dis., 2020, 6, 313–323.

   

  9.       Punetha, A.; Ngo, H.X.; Holbrook, S.Y.L.; Green, K.D.; Willby, M.J.; Bonnett, S.A.; Krieger, K.; Dennis, E.K.; Posey, J.E.; Parish, T.; Tsodikov, O. V.; Garneau-Tsodikova, S. Structure-Guided Optimization of Inhibitors of Acetyltransferase Eis from Mycobacterium Tuberculosis. ACS Chem. Biol., 2020, 15, 1581–1594.

   

  10.    Mucs, D.; Bryce, R.A. The Application of Quantum Mechanics in Structure-Based Drug Design. Expert Opin. Drug Discov., 2013, 8, 263–276.

   

   

• Keywords
• Quinazolinimines, Thienopyrimidine, Indole, Mycobacterium tuberculosis, Eis MTb.
• Modality
• Pôster
• Subject Area
• Drug Design and Discovery, Synthesis and Natural Products