Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca2+ binding, altering transport Ca2+ dependence without actually affecting the equilibrium binding affinity of the Ca2+-coordinating sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca2+ affinity of SERCA by interrupting allosteric activation of the pump by ATP.

Missense variants in phospholamban and cardiac myosin binding protein identified in patients with a family history and clinical diagnosis of dilated cardiomyopathy.

As the genetic landscape of cardiomyopathies continues to expand, the identification of missense variants in disease-associated genes frequently leads to a classification of variant of uncertain significance (VUS). For the proper reclassification of such variants, functional characterization is an important contributor to the proper assessment of pathogenic potential. Several missense variants in the calcium transport regulatory protein phospholamban have been associated with dilated cardiomyopathy. However, >40 missense variants in this transmembrane peptide are currently known and most remain classified as VUS with little clinical information. Similarly, missense variants in cardiac myosin binding protein have been associated with hypertrophic cardiomyopathy. However, hundreds of variants are known and many have low penetrance and are often found in control populations. Herein, we focused on novel missense variants in phospholamban, an Ala15-Thr variant found in a 4-year-old female and a Pro21-Thr variant found in a 60-year-old female, both with a family history and clinical diagnosis of dilated cardiomyopathy. The patients also harbored a Val896-Met variant in cardiac myosin binding protein. The phospholamban variants caused defects in the function, phosphorylation, and dephosphorylation of this calcium transport regulatory peptide, and we classified these variants as potentially pathogenic. The variant in cardiac myosin binding protein alters the structure of the protein. While this variant has been classified as benign, it has the potential to be a low-risk susceptibility variant because of the structural change in cardiac myosin binding protein. Our studies provide new biochemical evidence for missense variants previously classified as benign or VUS.

Mechanisms for cardiac calcium pump activation by its substrate and a synthetic allosteric modulator using fluorescence lifetime imaging.

The discovery of allosteric modulators is an emerging paradigm in drug discovery, and signal transduction is a subtle and dynamic process that is challenging to characterize. We developed a time-correlated single photon-counting imaging approach to investigate the structural mechanisms for small-molecule activation of the cardiac sarcoplasmic reticulum Ca2+-ATPase, a pharmacologically important pump that transports Ca2+ at the expense of adenosine triphosphate (ATP) hydrolysis. We first tested whether the dissociation of sarcoplasmic reticulum Ca2+-ATPase from its regulatory protein phospholamban is required for small-molecule activation. We found that CDN1163, a validated sarcoplasmic reticulum Ca2+-ATPase activator, does not have significant effects on the stability of the sarcoplasmic reticulum Ca2+-ATPase-phospholamban complex. Time-correlated single photon-counting imaging experiments using the nonhydrolyzable ATP analog ß,γ-Methyleneadenosine 5'-triphosphate (AMP-PCP) showed ATP is an allosteric modulator of sarcoplasmic reticulum Ca2+-ATPase, increasing the fraction of catalytically competent structures at physiologically relevant Ca2+ concentrations. Unlike ATP, CDN1163 alone has no significant effects on the Ca2+-dependent shifts in the structural populations of sarcoplasmic reticulum Ca2+-ATPase, and it does not increase the pump's affinity for Ca2+ ions. However, we found that CDN1163 enhances the ATP-mediated modulatory effects to increase the population of catalytically competent sarcoplasmic reticulum Ca2+-ATPase structures. Importantly, this structural shift occurs within the physiological window of Ca2+ concentrations at which sarcoplasmic reticulum Ca2+-ATPase operates. We demonstrated that ATP is both a substrate and modulator of sarcoplasmic reticulum Ca2+-ATPase and showed that CDN1163 and ATP act synergistically to populate sarcoplasmic reticulum Ca2+-ATPase structures that are primed for phosphorylation. This study provides novel insights into the structural mechanisms for sarcoplasmic reticulum Ca2+-ATPase activation by its substrate and a synthetic allosteric modulator.

Primitive Phospholamban- and Sarcolipin-like Peptides Inhibit the Sarcoplasmic Reticulum Calcium Pump SERCA.

Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.

The sarco-plasmic reticulum calcium pump (SERCA) plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.

An in silico pipeline for the discovery of multitarget ligands: A case study for epi-polypharmacology based on DNMT1/HDAC2 inhibition.

The search for novel therapeutic compounds remains an overwhelming task owing to the time-consuming and expensive nature of the drug development process and low success rates. Traditional methodologies that rely on the one drug-one target paradigm have proven insufficient for the treatment of multifactorial diseases, leading to a shift to multitarget approaches. In this emerging paradigm, molecules with off-target and promiscuous interactions may result in preferred therapies. In this study, we developed a general pipeline combining machine learning algorithms and a deep generator network to train a dual inhibitor classifier capable of identifying putative pharmacophoric traits. As a case study, we focused on dual inhibitors targeting DNA methyltransferase 1 (DNMT) and histone deacetylase 2 (HDAC2), two enzymes that play a central role in epigenetic regulation. We used this approach to identify dual inhibitors from a novel large natural product database in the public domain. We used docking and atomistic simulations as complementary approaches to establish the ligand-interaction profiles between the best hits and DNMT1/HDAC2. By using the combined ligand- and structure-based approaches, we discovered two promising novel scaffolds that can be used to simultaneously target both DNMT1 and HDAC2. We conclude that the flexibility and adaptability of the proposed pipeline has predictive capabilities of similar or derivative methods and is readily applicable to the discovery of small molecules targeting many other therapeutically relevant proteins.

Linking Biochemical and Structural States of SERCA: Achievements, Challenges, and New Opportunities.

Sarcoendoplasmic reticulum calcium ATPase (SERCA), a member of the P-type ATPase family of ion and lipid pumps, is responsible for the active transport of Ca2+ from the cytoplasm into the sarcoplasmic reticulum lumen of muscle cells, into the endoplasmic reticulum (ER) of non-muscle cells. X-ray crystallography has proven to be an invaluable tool in understanding the structural changes of SERCA, and more than 70 SERCA crystal structures representing major biochemical states (defined by bound ligand) have been deposited in the Protein Data Bank. Consequently, SERCA is one of the best characterized components of the calcium transport machinery in the cell. Emerging approaches in the field, including spectroscopy and molecular simulation, now help integrate and interpret this rich structural information to understand the conformational transitions of SERCA that occur during activation, inhibition, and regulation. In this review, we provide an overview of the crystal structures of SERCA, focusing on identifying metrics that facilitate structure-based categorization of major steps along the catalytic cycle. We examine the integration of crystallographic data with different biophysical approaches and computational methods to link biochemical and structural states of SERCA that are populated in the cell. Finally, we discuss the challenges and new opportunities in the field, including structural elucidation of functionally important and novel regulatory complexes of SERCA, understanding the structural basis of functional divergence among homologous SERCA regulators, and bridging the gap between basic and translational research directed toward therapeutic modulation of SERCA.

Structural Basis for the Limited Response to Oxidative and Thiol-Conjugating Agents by Triosephosphate Isomerase From the Photosynthetic Bacteria Synechocystis.

In plants, the ancestral cyanobacterial triosephosphate isomerase (TPI) was replaced by a duplicated version of the cytosolic TPI. This isoform acquired a transit peptide for chloroplast localization and functions in the Calvin-Benson cycle. To gain insight into the reasons for this gene replacement in plants, we characterized the TPI from the photosynthetic bacteria Synechocystis (SyTPI). SyTPI presents typical TPI enzyme kinetics profiles and assembles as a homodimer composed of two subunits that arrange in a (ß-α)8 fold. We found that oxidizing agents diamide (DA) and H2O2, as well as thiol-conjugating agents such as oxidized glutathione (GSSG) and methyl methanethiosulfonate (MMTS), do not inhibit the catalytic activity of SyTPI at concentrations required to inactivate plastidic and cytosolic TPIs from the plant model Arabidopsis thaliana (AtpdTPI and AtcTPI, respectively). The crystal structure of SyTPI revealed that each monomer contains three cysteines, C47, C127, and C176; however only the thiol group of C176 is solvent exposed. While AtcTPI and AtpdTPI are redox-regulated by chemical modifications of their accessible and reactive cysteines, we found that C176 of SyTPI is not sensitive to redox modification in vitro. Our data let us postulate that SyTPI was replaced by a eukaryotic TPI, because the latter contains redox-sensitive cysteines that may be subject to post-translational modifications required for modulating TPI's enzymatic activity.

The Ca2+-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum.

Ca2+ transport across the sarco/endoplasmic reticulum (SR) plays an essential role in intracellular Ca2+ homeostasis, signalling, cell differentiation and muscle contractility. During SR Ca2+ uptake and release, proton fluxes are required to balance the charge deficit generated by the exchange of Ca2+ and other ions across the SR. During Ca2+ uptake by the SR Ca2+-ATPase (SERCA), two protons are countertransported from the SR lumen to the cytosol, thus partially compensating for the charge moved by Ca2+ transport. Studies have shown that protons are also transported from the cytosol to the lumen during Ca2+ release, but a transporter that facilitates proton transport into the SR lumen has not been described. In this article we propose that SERCA forms pores that facilitate bidirectional proton transport across the SR. We describe the location and structure of water-filled pores in SERCA that form cytosolic and luminal pathways for protons to cross the SR membrane. Based on this structural information, we suggest mechanistic models for proton translocation to the cytosol during active Ca2+ transport, and into the SR lumen during SERCA inhibition by endogenous regulatory proteins. Finally, we discuss the physiological consequences of SERCA-mediated bidirectional proton transport across the SR membrane of muscle and non-muscle cells.

Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation.

We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa's BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel ß-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

High-resolution structural characterization of Noxa, an intrinsically disordered protein, by microsecond molecular dynamics simulations.

High-resolution characterization of the structure and dynamics of intrinsically disordered proteins (IDPs) remains a challenging task. Consequently, a detailed understanding of the structural and functional features of IDPs remains limited, as very few full-length disordered proteins have been structurally characterized. We have performed microsecond-long molecular dynamics (MD) simulations of Noxa, the smallest member of the large Bcl-2 family of apoptosis regulating proteins, to characterize in atomic-level detail the structural features of a disordered protein. A 2.5 µs MD simulation starting from an unfolded state of the protein revealed the formation of a central antiparallel ß-sheet structure flanked by two disordered segments at the N- and C-terminal ends. This topology is in reasonable agreement with protein disorder predictions and available experimental data. We show that this fold plays an essential role in the intracellular function and regulation of Noxa. We demonstrate that unbiased MD simulations in combination with a modern force field reveal structural and functional features of disordered proteins at atomic-level resolution.

Aromatic residues link binding and function of intrinsically disordered proteins.

We have searched for intermolecular aromatic pairs in 77 protein-protein complexes of intrinsically disordered proteins (IDPs) to understand the role of π-π interactions in protein-protein interactions involving IDPs. We found that 40% of the complexes possess at least one intermolecular pair of aromatic residues. Analysis of composition, characteristics, location and the contribution to the free energy of binding showed that π-π interactions substantially contribute to binding by working as anchor residues, conformational locks, and ready-made recognition motifs required for specific binding. By using available experimental data we show that π-π interactions play a variety of roles that link binding of IDPs and their function in the cell. The results presented in this study pave the way to understand in atomic detail the inner workings of IDPs interaction networks.

Dynamic optimization of signal transduction via intrinsic disorder.

It is widely accepted that the inherent flexibility of intrinsically disordered proteins (IDPs) correlates with essential functions in the cell such as signaling. However, the mechanisms by which disorder dynamically facilitates and optimizes signal transduction remain unclear. In this study, we have used a computational protocol to evaluate the interplay between the intrinsic disorder of p27(kip1) and the collective motions of its binding partners, cyclin dependent kinase 2 (CDK2) and cyclin A (CA). We found that the synergy between intrinsic disorder of p27(kip1) and the essential collective motions of the CDK2-CA complex introduces a set of sequential steps to dynamically optimize signal transduction. Our observations indicate that optimized p27(kip1)-mediated signaling originates from a combination of adaptive folding, and the cooperativity between its residual disorder and the functional collective motions of the CDK2-CA complex.

Atomic-level characterization of the activation mechanism of SERCA by calcium.

We have performed molecular dynamics (MD) simulations to elucidate, in atomic detail, the mechanism by which the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) is activated by Ca(2+). Crystal structures suggest that activation of SERCA occurs when the cytoplasmic head-piece, in an open (E1) conformation stabilized by Ca(2+), undergoes a large-scale open-to-closed (E1 to E2) transition that is induced by ATP binding. However, spectroscopic measurements in solution suggest that these structural states (E1 and E2) are not tightly coupled to biochemical states (defined by bound ligands); the closed E2 state predominates even in the absence of ATP, in both the presence and absence of Ca(2+). How is this loose coupling consistent with the high efficiency of energy transduction in the Ca(2+)-ATPase? To provide insight into this question, we performed long (500 ns) all-atom MD simulations starting from the open crystal structure, including a lipid bilayer and water. In both the presence and absence of Ca(2+), we observed a large-scale open-to-closed conformational transition within 400 ns, supporting the weak coupling between structural and biochemical states. However, upon closer inspection, it is clear that Ca(2+) is necessary and sufficient for SERCA to reach the precise geometrical arrangement necessary for activation of ATP hydrolysis. Contrary to suggestions from crystal structures, but in agreement with solution spectroscopy, the presence of ATP is not required for this activating transition. Principal component analysis showed that Ca(2+) reshapes the free energy landscape of SERCA to create a path between the open conformation and the activated closed conformation. Thus the malleability of the free energy landscape is essential for SERCA efficiency, ensuring that ATP hydrolysis is tightly coupled to Ca(2+) transport. These results demonstrate the importance of real-time dynamics in the formation of catalytically competent conformations of SERCA, with broad implications for understanding enzymatic catalysis in atomic detail.

Tyr74 is essential for the formation, stability and function of Plasmodium falciparum triosephosphate isomerase dimer.

Plasmodium falciparum triosephosphate isomerase (PfTIM) is known to be functional only as a homodimer. Although many studies have shown that the interface Cys13 plays a major role in the stability of the dimer, a few reports have demonstrated that structurally conserved Tyr74 may be essential for the stability of PfTIM dimer. To understand the role of Tyr74, we have performed molecular dynamics (MD) simulations of monomeric and dimeric PfTIM mutated to glycine and cysteine at position 74. Simulations of the monomer revealed that mutant Tyr74Gly does not produce changes in folding and stability of the monomer. Interestingly, comparison of the flexibility of Tyr74 in the monomer and dimer revealed that this residue possesses an intrinsic restricted mobility, indicating that Tyr74 is an anchor residue required for homodimerization. Tyr74 also appears to play an important role in binding by facilitating the disorder-to-order transitions of loops 1 and 3, which allows Cys13 to form favorable interactions with loop 3 and Lys12 to be locked in a favorable position for catalysis. High-temperature MD simulations of the wild-type and Tyr74Gly PfTIM dimers showed that the aromatic moiety of Tyr74 is necessary to preserve the geometry and native contacts between loops 1 and 3 at the interface of the dimer. Disulfide cross-linking between mutant Tyr74Cys and Cys13 further revealed that Tyr74 stabilizes the geometry of loop 1 (which contains the catalytic residue Lys12) and the interactions between loops 1 and 3 via aromatic-aromatic interactions with residues Phe69, Tyr101, and Phe102. Principal component analysis showed that Tyr74 is also necessary to preserve the collective motions in the dimer that contribute to the catalytic efficiency of PfTIM dimer. We conclude that Tyr74 not only plays a role in the stability of the dimer, but also participates in the dimerization process and collective motions via coupled disorder-to-order transitions of intrinsically disordered regions, necessary for efficiency in the catalytic function of PfTIM.

Leucine-rich hydrophobic clusters promote folding of the N-terminus of the intrinsically disordered transactivation domain of p53.

Molecular dynamics simulations have been performed on the intrinsically disordered 39-residue N-terminal transactivation domain of p53 (p53(1-39)). Simulations not only revealed that p53(1-39) is natively compact, but also possesses a folded structure. Furthermore, leucine-rich hydrophobic clusters were found to play a crucial role in the formation and stabilization of the folded structure of p53(1-39). Collapsing in the sub-microsecond timescale might allow for rapid conformational turnovers of p53(1-39), necessary for its efficient transactivation activity and modulation. Fast collapsing might be the result of unique conformational landscapes, featuring several energy minima separated by small energy barriers. It is suggested that IDPs with highly specialized functions in the cell, such as transactivation, possibly display more ordered patterns than their less specialized counterparts.

Aromatic-aromatic interactions in the formation of the MDM2-p53 complex.

Although the molecular interaction of MDM2 with the transactivation domain of p53 has been thoroughly studied, there is very limited information regarding the steps involved in the recognition mechanism between these proteins. On this basis, we performed four high-temperature molecular dynamics simulations in explicit solvent to gain insight into the interactions involved in the fist contact toward the formation of the complex. We found that the presence of specific intermolecular aromatic pairs at the interface of the complex, around the native-like state of MDM2, is consistent among independent molecular dynamics runs. This observation suggests that aromatic-aromatic interactions are closely related to the first contact between MDM2 and p53. Thus, we propose that aromatic-aromatic interactions are an important, and probably essential, requirement for the formation and stabilization of the MDM2-p53 complex.

Transient stability of the helical pattern of region F19-L22 of the N-terminal domain of p53: a molecular dynamics simulation study.

Two molecular dynamics simulations of the region E17-N29 of p53 (p53(17-29)) at different temperatures were performed for a total time of 0.2 micros, to study the conformational landscape of this region. Previous studies have suggested that this region displays different structural motifs, such as helix of a double beta-turn, and that its secondary structure might be transiently stable. Interestingly, in this study it was found that the region F19-L25, and particularly its fragment F19-L22, display a stable, transient helical pattern at sub-microsecond periods. The region F19-L22, which contains one of the most important residues needed for the interaction of p53 with MDM2, seems to be formed and stabilized by the existence of one hydrophobic and one aromatic cluster. The main function of these clusters is to help their surrounding area to desolvate, to allow the hydrogen bond network, therefore favoring the formation of a stable helix. This preliminary study would be useful for a better understanding of the structure and function of the N-terminal domain of p53 and its implications for the control of different types of cancer.

Targeting MDM2 by the small molecule RITA: towards the development of new multi-target drugs against cancer.

BACKGROUND: The use of low-molecular-weight, non-peptidic molecules that disrupt the interaction between the p53 tumor suppressor and its negative regulator MDM2 has provided a promising alternative for the treatment of different types of cancer. Among these compounds, RITA (reactivation of p53 and induction of tumor cell apoptosis) has been shown to be effective in the selective induction of apoptosis, and this effect is due to its binding to the p53 tumor suppressor. Since biological systems are highly dynamic and MDM2 may bind to different regions of p53, new alternatives should be explored. On this basis, the computational "blind docking" approach was employed in this study to see whether RITA would bind to MDM2. RESULTS: It was observed that RITA binds to the MDM2 p53 transactivation domain-binding cleft. Thus, RITA can be used as a lead compound for designing improved "multi-target" drugs. This novel strategy could provide enormous benefits to enable effective anti-cancer strategies. CONCLUSION: This study has demonstrated that a single molecule can target at least two different proteins related to the same disease.