Antimalarial Activity of Potential Inhibitors of Plasmodium falciparum Lactate Dehydrogenase Enzyme Selected by Docking Studies

The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH) has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH) all exhibit ∼90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH) and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole) were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2). The IC50 values for each drug in both tests were similar, were lowest for posaconazole (<5 µM) and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use

Antimalarial Activity and Mechanisms of Action of Two Novel 4-Aminoquinolines against Chloroquine-Resistant Parasites

Chloroquine (CQ) is a cost effective antimalarial drug with a relatively good safety profile (or therapeutic index). However, CQ is no longer used alone to treat patients with Plasmodium falciparum due to the emergence and spread of CQ-resistant strains, also reported for P. vivax. Despite CQ resistance, novel drug candidates based on the structure of CQ continue to be considered, as in the present work. One CQ analog was synthesized as monoquinoline (MAQ) and compared with a previously synthesized bisquinoline (BAQ), both tested against P. falciparum in vitro and against P. berghei in mice, then evaluated in vitro for their cytotoxicity and ability to inhibit hemozoin formation. Their interactions with residues present in the NADH binding site of P falciparum lactate dehydrogenase were evaluated using docking analysis software. Both compounds were active in the nanomolar range evaluated through the HRPII and hypoxanthine tests. MAQ and BAQ derivatives were not toxic, and both compounds significantly inhibited hemozoin formation, in a dose-dependent manner. MAQ had a higher selectivity index than BAQ and both compounds were weak PfLDH inhibitors, a result previously reported also for CQ. Taken together, the two CQ analogues represent promising molecules which seem to act in a crucial point for the parasite, inhibiting hemozoin formation

Epigenetic mechanisms in silico: understanding demethylation and rational design of bromodomain inhibitors

Histone octamer proteins are crucial for DNA packaging and storage in the confined space of the cell nucleus. Epigenetic post-translational modifications to specific histone monomers facilitate changes in chromatin flexibility that are necessary for access by transcription machinery, and therefore have control over gene expression. Targeting enzymes that regulate these powerful modifications has been established as a promising strategy in the treatments of some diseases, such as cancer, male infertility and adult obesity. The work presented in this thesis aims to shed light on the mode of action of epigenetic proteins, and in so doing, aid in the design of more potent and selective small molecules that target them (e.g. Jumonji C (JmjC) and bromodomain containing-proteins). We have used a diverse array of computational techniques such as quantum mechanics (QM), classical Molecular Dynamics (MD) simulations, and hybrid Newtonian molecular mechanics/quantum mechanics (QM/MM) approaches. Following an introduction to these computational chemistry techniques (Chapter 1) and a description of the methodology used in this thesis (Chapter 2), we then present our studies on: (a) improving Bromodomaincontaining proteins inhibition (Chapters 3, 4) and (b) providing novel insights into the lysine demethylation catalysed by JmjC proteins (Chapters 5, 6). In Chapter 3, we have investigated a series of dihydroquinoxalinone (DHQ) derivatives, analogues of (R)-2 (Figure A.1), previously proven inactivators of the epigenetic molecular target CREBBP bromodomain, resulting in the first potent inhibitors of a bromodomain outside the Bromomodomain and extra-terminal (BET) family. Through QM and MD calculations, I established the significance of cation-π interactions for the binding of CREBBP inhibitors. In Chapter 4, we then developed a novel electrostatic model for the quantification of cation-π interactions in biological environments, inspired by a good fit between experimental binding affinity data of a series of fifteen 5-isoxazolylbenzimidazole derivatives and electrostatic potential (ESP) surfaces. This model has been prospectively applied to newly synthesised DHQ derivatives. Our approach could be used for the development of force fields and docking scoring functions with increased reliabilities to reproduce cation-π interactions. Together with the understanding of DHQ binding in CREBBP, I have investigated one of the most common but poorly understood epigenetic processes: histone lysine demethylation by a JmjC protein, JMJD2A (Figure A.2). In Chapter 5, in the first QM/MM studies reported for this system, we describe the importance of the protein environment on the binding of molecular O2, while in Chapter 6 we analyse the PES of the elementary steps associated with enzymatic demethylation: cofactor/ O2 activation, C-H abstraction and hydroxyl-rebound. Insights into this reaction mechanism and energetic contributions have been used to the role of specific JMJD2A residues in this process, which can be used to accelerate the design of effective drug molecules.</p

Epigenetic mechanisms in silico: understanding demethylation and rational design of bromodomain inhibitors

Histone octamer proteins are crucial for DNA packaging and storage in the confined space of the cell nucleus. Epigenetic post-translational modifications to specific histone monomers facilitate changes in chromatin flexibility that are necessary for access by transcription machinery, and therefore have control over gene expression. Targeting enzymes that regulate these powerful modifications has been established as a promising strategy in the treatments of some diseases, such as cancer, male infertility and adult obesity. The work presented in this thesis aims to shed light on the mode of action of epigenetic proteins, and in so doing, aid in the design of more potent and selective small molecules that target them (e.g. Jumonji C (JmjC) and bromodomain containing-proteins). We have used a diverse array of computational techniques such as quantum mechanics (QM), classical Molecular Dynamics (MD) simulations, and hybrid Newtonian molecular mechanics/quantum mechanics (QM/MM) approaches. Following an introduction to these computational chemistry techniques (Chapter 1) and a description of the methodology used in this thesis (Chapter 2), we then present our studies on: (a) improving Bromodomaincontaining proteins inhibition (Chapters 3, 4) and (b) providing novel insights into the lysine demethylation catalysed by JmjC proteins (Chapters 5, 6). In Chapter 3, we have investigated a series of dihydroquinoxalinone (DHQ) derivatives, analogues of (R)-2 (Figure A.1), previously proven inactivators of the epigenetic molecular target CREBBP bromodomain, resulting in the first potent inhibitors of a bromodomain outside the Bromomodomain and extra-terminal (BET) family. Through QM and MD calculations, I established the significance of cation-&pi; interactions for the binding of CREBBP inhibitors. In Chapter 4, we then developed a novel electrostatic model for the quantification of cation-&pi; interactions in biological environments, inspired by a good fit between experimental binding affinity data of a series of fifteen 5-isoxazolylbenzimidazole derivatives and electrostatic potential (ESP) surfaces. This model has been prospectively applied to newly synthesised DHQ derivatives. Our approach could be used for the development of force fields and docking scoring functions with increased reliabilities to reproduce cation-&pi; interactions. Together with the understanding of DHQ binding in CREBBP, I have investigated one of the most common but poorly understood epigenetic processes: histone lysine demethylation by a JmjC protein, JMJD2A (Figure A.2). In Chapter 5, in the first QM/MM studies reported for this system, we describe the importance of the protein environment on the binding of molecular O2, while in Chapter 6 we analyse the PES of the elementary steps associated with enzymatic demethylation: cofactor/ O2 activation, C-H abstraction and hydroxyl-rebound. Insights into this reaction mechanism and energetic contributions have been used to the role of specific JMJD2A residues in this process, which can be used to accelerate the design of effective drug molecules.</p

ITP Adjuster 1.0: A New Utility Program to Adjust Charges in the Topology Files Generated by the PRODRG Server

The suitable computation of accurate atomic charges for the GROMACS topology *.itp files 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 file, 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 files of small molecules by ab initio charges

Antimalarial activity of BAQ and MAQ in mice infected with <i>P. berghei</i> after treatment with daily doses of the compounds during three consecutive days.

*<p>Reductions ≤30% were considered as inactive, 30–50% as partially active and ≥50% as active drugs.</p

Inhibition of hemozoin formation by the CQ analogs BAQ and MAQ (mean ± SD from triplicates), in two different experiments.

<p>Statistical differences as compared to drug-free controls are indicated in each graph by an asterisk (p<0.05). The p and r values represent the statistical correlation analyses.</p

Compounds docked in dimeric hematin.

<p>(A) Protonated CQ, (B) MAQ-1, (C) MAQ-2, (D) MAQ-3, (E) BAQ-1, (F) BAQ-2.</p