Jaime Rubio-Martinez

The Methylerythritol Phosphate (MEP) Pathway for Isoprenoid Biosynthesis as a Target for the Development of New Drugs Against Tuberculosis

Tuberculosis remains a major infectious disease to humans. It accounts for approximately 8-9 million new cases worldwide and an estimated 1.6 million deaths annually. Effective treatments for tuberculosis consist of a combination of several drugs administered over long periods of time. Since Mycobacterium tuberculosis often acquires multiple drug resistant mechanisms, development of new drugs with innovative actions is urgently required. The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway, in charge of the essential biosynthesis of isoprenoids, represents a promising and selective target for developing new drugs against tuberculosis. To date, only fosmidomycin, a molecule that targets the second enzyme of the MEP pathway, has reached clinical trials but recent advances elucidating the structure and kinetics of the MEP enzymes are likely to change this scenario. This review describes the structure, mechanism of action and inhibitors of the seven enzymes of the MEP pathway, with special attention to the reported studies in M. tuberculosis.

Comparative evaluation of MMPBSA and XSCORE to compute binding free energy in XIAP-peptide complexes

Evaluation of binding free energy in receptor-ligand complexes is one of the most important challenges in theoretical drug design. Free energy is directly correlated to the thermodynamic affinity constant, and, as a first step in druglikeness, a lead compound must have this constant in the range of micro- to nanomolar activity. Many efforts have been made to calculate it by rigorous computational approaches, such as free energy perturbation or linear response approximation. However, these methods are still computationally expensive. We focus our work on XIAP, an antiapoptotic protein whose inhibition can lead to new drugs against cancer disease. We report here a comparative evaluation of two completely different methodologies to estimate binding free energy, MMPBSA (a force field based function) and XSCORE (an empirical scoring function), in seven XIAP-peptide complexes using a representative set of structures generated by previous molecular dynamics simulations. Both methods are able to predict the experimental binding free energy with acceptable errors, but if one needs to identify slight differences upon binding, MMPBSA performs better, although XSCORE is not a bad choice taking into account the low computational cost of this method.

Protein–protein recognition as a first step towards the inhibition of XIAP and Survivin anti-apoptotic proteins

Apoptosis, also called programmed cell death, is a conserved mechanism inherent to all cells that sentences them to death when they receive the appropriate external stimuli. Inhibitor of apoptosis proteins (IAPs) are a family of regulatory proteins that suppress such cell death. XIAP is the most commonly studied member of the IAP family. It binds to and inhibits Caspases, an important family of apoptotic proteases. In addition, XIAP over‐expression has been detected in numerous types of cancer. Smac/DIABLO, a mitochondrial protein that binds to IAPs and promotes Caspase activation, has the opposite action to XIAP and can be considered a key protein in the regulation of IAPs. Survivin, the smallest IAP protein, has received a lot of attention due to its specific expression in many cancer cell lines. It has been shown to interact with Smac/DIABLO, even though the structure of this complex has not yet been reported.

Rational design of new class of BH3-mimetics as inhibitors of the Bcl-xL protein.

The Bcl-2 family of proteins plays an important role in the intrinsic pathway of cell apoptosis. Overexpression of pro-survival members of this family of proteins is often associated with the development of many types of cancer and confers resistance against conventional therapeutic treatments. Accordingly, antagonism of its protective function has emerged as an encouraging anticancer strategy. In the present work, we use a pharmacophore for describing interaction between the BH3 domain of different pro-apoptotic members and the pro-survival protein Bcl-x(L) in order to identify new lead compounds. In the strategy followed in the present work, the pharmacophore was derived from molecular dynamics studies of different Bcl-x(L)/BH3 complexes. This pharmacophore was later used as query for 3D database screening. Hits obtained from the search were computationally assessed, and a subset proposed for in vitro testing. Two of the 15 compounds assayed were found able to disrupt the Bcl-x(L)/Bak(BH3) complex with IC(50) values in the lower micromolar range. Finally, docking studies were performed to explore the binding mode of these compounds to Bcl-x(L) for further modifications.

Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle.

Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G1 phase. Aberrant expression of CDK4 and CDK6 is a hallmark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM (the calcein acetoxymethyl-ester) is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G1 phase. The metabolic effects of calcein AM on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phosphate pathway was significantly altered. To elucidate whether these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.

Structural analysis of the inhibition of Cdk4 and Cdk6 by p16INK4a through molecular dynamics simulations

Abstract Cyclin-dependent kinases 4, 6 and 2 (Cdk4/6/2), are proteins that lead progression through the G1-S transition, a step strictly regulated in the process of cell proliferation. The p16INK4a tumor suppressor, whose expression is inhibited in a high number of cancers, binds to Cdk4/6 and inhibits phosphorylation of the retinoblastoma protein, forcing cells to remain in the G1 phase and therefore, arresting cell division. Accordingly, the design of small compounds mimicking the inhibition of p16INK4a appears to be a promising way to treat cancer. In order to get some insight into the key interactions governing recognition between different cyclin-dependent kinases and the p16INK4a tumor suppressor, the present work reports the results of molecular dynamics simulations of both, the Cdk6-p16INK4a complex and the Cdk4-p16INK4a complex, respectively at 300 K. Most of the key interactions observed, were already anticipated in the analysis of the crystal structure of Cdk6-p16INK4a. However, a few different features found out from the analysis of these calculations provide a better understanding of the role of the T-loop conformation, a fragment of Cdks, and the way the ATP binding-site is distorted upon binding of p16INK4a.

Molecular determinants of Bim(BH3) peptide binding to pro-survival proteins.

Proteins of the Bcl-2 family regulate apoptosis through the formation of heterodimers between antiapoptotic or pro-survival proteins and proapoptotic or pro-death proteins. Overexpression of antiapoptotic proteins not only contributes to the progression of many cancers, but also confers resistance to the chemo- and radiotherapeutic treatments. It has been demonstrated that peptides containing the BH3 domain of proapoptotic Bcl-2 family members are able to bind and inhibit antiapoptotic proteins. For this reason, the design of small molecules mimicking the BH3 domain of proapoptotic proteins has emerged as a promising therapeutic strategy for cancer treatment during the last years. However, BH3 domains exhibit different affinities for binding to antiapoptotic proteins; whereas Bim(BH3) and Puma(BH3) are able to bind all antiapoptotic proteins, others like Bad(BH3) and Bmf(BH3) show preference for some proteins over others. Consequently, the ability of a BH3-mimetic to kill tumor cells will depend on the BH3 peptide used as template and thus will have a selective or pan-inhibition effect. Recently, it has been suggested that this last approach could be interesting. Therefore, the present work is aimed to elucidate how the nonselective peptide Bim(BH3) is able to bind to all of the Bcl-2 family antiapoptotic proteins. To unravel the molecular determinants of this pan-inhibition, we used the MM-PB/GBSA approaches to calculate the binding free energy of the different complexes studied and to determine which residues of the peptide have the largest contribution to complex formation. Results obtained in the present work show that the binding of Bim(BH3) to pro-survival proteins is mainly hydrophobic and that specific interactions are fully distributed along the peptide sequence.

Homology modeling of human transketolase: description of critical sites useful for drug design and study of the cofactor binding mode.

Transketolase, the most critical enzyme of the non-oxidative branch of the pentose phosphate pathway, has been reported as a new target protein for cancer research. However, since the crystal structure of human Transketolase is unknown, no structure-based methods can be used to identify new inhibitors. We performed homology modeling of human Transketolase using the crystal structure of yeast as a template, and then refined the model through molecular dynamics simulations. Based on the resulting structure we propose five critical sites containing arginines (Arg 101, Arg 318, Arg 395, Arg 401 and Arg 474) that contribute to dimer stability or catalytic activity. In addition, an interaction analysis of its cofactor (thiamine pyrophosphate) and a binding site description were carried out, suggesting the substrate channel already identified in yeast Transketolase. A binding free energy calculation of its cofactor was performed to establish the main driving forces of binding. In summary, we describe a reliable model of human Transketolase that can be used in structure-based drug design and in the search for new Transketolase inhibitors that disrupt dimer stability and cover the critical sites found.

Mimicking direct protein-protein and solvent-mediated interactions in the CDP-methylerythritol kinase homodimer: a pharmacophore-directed virtual screening approach

The 2C-methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate, which are the precursors of isoprenoids, is present in plants, in the malaria parasite Plasmodium falciparum and in most eubacteria, including pathogenic agents. However, the MEP pathway is absent from fungi and animals, which have exclusively the mevalonic acid pathway. Given the characteristics of the MEP pathway, its enzymes represent potential targets for the generation of selective antibacterial, antimalarial and herbicidal molecules. We have focussed on the enzyme 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase (CMK), which catalyses the fourth reaction step of the MEP pathway. A molecular dynamics simulation was carried out on the CMK dimer complex, and protein–protein interactions analysed, considering also water-mediated interactions between monomers. In order to find small molecules that bind to CMK and disrupt dimer formation, interactions observed in the dynamics trajectory were used to model a pharmacophore used in database searches. Using an intensity-fading matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry approach, one compound was found to interact with CMK. The data presented here indicate that a virtual screening approach can be used to identify candidate molecules that disrupt the CMK–CMK complex. This strategy can contribute to speeding up the discovery of new antimalarial, antibacterial, and herbicidal compounds.

Conformationally Restricted Hydantoin-Based Peptidomimetics as Inhibitors of Caspase-3 with Basic Groups Allowed at the S3 Enzyme Subsite

By using a combination of molecular modeling, combinatorial chemistry, and biological essays, novel scaffold molecules for the inhibition of caspase‐3 have been developed. These compounds have an overall attenuated negative charge and show similar IC50 values for both recombinant and human endogenous caspase‐3. This might provide the basis for a novel strategy for the discovery of potent and more druglike inhibitors of caspase‐3.