A Role for Both Conformational Selection and Induced Fit in Ligand Binding by the LAO Protein

Molecular recognition is determined by the structure and dynamics of both a protein and its ligand, but it is difficult to directly assess the role of each of these players. In this study, we use Markov State Models (MSMs) built from atomistic simulations to elucidate the mechanism by which the Lysine-, Arginine-, Ornithine-binding (LAO) protein binds to its ligand. We show that our model can predict the bound state, binding free energy, and association rate with reasonable accuracy and then use the model to dissect the binding mechanism. In the past, this binding event has often been assumed to occur via an induced fit mechanism because the protein's binding site is completely closed in the bound state, making it impossible for the ligand to enter the binding site after the protein has adopted the closed conformation. More complex mechanisms have also been hypothesized, but these have remained controversial. Here, we are able to directly observe roles for both the conformational selection and induced fit mechanisms in LAO binding. First, the LAO protein tends to form a partially closed encounter complex via conformational selection (that is, the apo protein can sample this state), though the induced fit mechanism can also play a role here. Then, interactions with the ligand can induce a transition to the bound state. Based on these results, we propose that MSMs built from atomistic simulations may be a powerful way of dissecting ligand-binding mechanisms and may eventually facilitate a deeper understanding of allostery as well as the prediction of new protein-ligand interactions, an important step in drug discovery

Candimine from Hippeastrum escoipense (Amaryllidaceae): Anti-Trypanosoma cruzi activity and synergistic effect with benznidazole

Background: Chagas disease (CD), caused by Trypanosoma cruzi, represents a healththreat to around 20 million people worldwide. Side effects of benznidazole (Bzn)cause 15-20% of patients to discontinue their treatment. Evidence has increased infavour of the use of drug combinations to improve the efficacy and tolerance of thetreatment. Natural products are well known to provide structures that could serve asnew drugs or scaffolds for CD treatment. Spp of the Amaryllidoideae sub family ofAmaryllidaceae family are known by its bioactives alkaloids, which have a reportedantiparasitic activity.Purpose: To evaluate the anti-T. cruzi activity of the isolated alkaloid candimine(Cnd) from Hippeastrum escoipense Slanis & Huaylla; and to assess the combinationeffect between Cnd and Bzn against different life stages of T. cruzi parasites.Methods: The chemical profile of H. escoipense alkaloids extract (AE-H. escoipense),including quantitation of Cnd was performed through GC/MS and UPLC-MS/MStechniques. Subsequently, Cnd was isolated using Shephadex LH20. Then, the AE-H.escoipense and Cnd were tested against T. cruzi, (epimastigotes, trypomastigotes, andamastigotes) by in vitro proliferation and viability assays. The cytotoxicity wasevaluated against Vero and HepG2 mammalian cells. The ultrastructural analysis wasperform by Transmission Electron Microcopy (TEM) and mitochondrial activity wascarried out by MTT assay. Drug combination assay between Cnd and Bzn wasevaluated using the Chou-Talalay method.4Results: The AE-H. escoipense and Cnd showed high and specific anti-T. cruziactivity, comparable to Bzn. Cnd induces ultrastructural changes in T. cruzi, such asvacuolization, membrane blebs and increases the mitochondrial activity. Regarding,the interaction between Cnd and Bzn generates synergism in the combinations of0.25×IC50 in epimastigotes, 2×IC50 in trypomastigotes+amastigotes, and 0.25, 2, and4×IC50 in amastigotes.Conclusion: The synergism between Cnd and Bzn indicates that the combination atthe concentration of 4×IC50 could be useful as an effective new therapy against CD inthe chronic stage. Thus, Cnd isolated from the leaves of the H. escoipense emerges aspotential candidate for the development of a new drug for the treatment of CD.Fil: Ortiz, Javier Esteban. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan; ArgentinaFil: Piñeiro Gomez, Mauricio Daniel. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan; ArgentinaFil: Martines Peinado, Nieves. Universidad de Barcelona; EspañaFil: Barrera, Patricia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Sosa, Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Bastida, Jaume. Universidad de Barcelona. Facultad de Farmacia; EspañaFil: Alonso Padilla, Julio. Universidad de Barcelona; EspañaFil: Feresin, Gabriela Egly. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan; Argentin

Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family. Demethylation of Alu elements occurs in aging and cancer processes and has been associated with gene reactivation and genomic instability. By targeting the unmethylated SmaI site within the Alu sequence as a surrogate marker, we have quantified and identified unmethylated Alu elements on the genomic scale. Normal colon epithelial cells contain in average 25 486 ± 10 157 unmethylated Alu's per haploid genome, while in tumor cells this figure is 41 995 ± 17 187 (P = 0.004). There is an inverse relationship in Alu families with respect to their age and methylation status: the youngest elements exhibit the highest prevalence of the SmaI site (AluY: 42%; AluS: 18%, AluJ: 5%) but the lower rates of unmethylation (AluY: 1.65%; AluS: 3.1%, AluJ: 12%). Data are consistent with a stronger silencing pressure on the youngest repetitive elements, which are closer to genes. Further insights into the functional implications of atypical unmethylation states in Alu elements will surely contribute to decipher genomic organization and gene regulation in complex organisms

Tumor cell dormancy

Metastasis is the primary cause of death in cancer patients and current treatments fail to provide durable responses. Efforts to treat metastatic disease are hindered by the fact that metastatic cells often remain dormant for prolonged intervals of years, or even decades. Tumor dormancy reflects the capability of disseminated tumor cells (DTCs), or micrometastases, to evade treatment and remain at low numbers after primary tumor resection. Unfortunately, dormant cells will eventually produce overt metastasis. Innovations are needed to understand metastatic dormancy and improve cancer detection and treatment. Currently, few models exist that faithfully recapitulate metastatic dormancy and metastasis to clinically relevant tissues, such as the bone. Herein, we discuss recent advances describing genetic cell-autonomous and systemic or local changes in the microenvironment that have been shown to endow DTCs with properties to survive and eventually colonize distant organs

Bisindolylmaleimides New Ligands of CaM Protein

In the present study, we reported the interactions at the molecular level of a series of compounds called Bisindolylmaleimide, as potential inhibitors of the calmodulin protein. Bisindolylmaleimide compounds are drug prototypes derived from Staurosporine, an alkaloid with activity for cancer treatment. Bisindolylmaleimide compounds II, IV, VII, X, and XI, are proposed and reported as possible inhibitors of calmodulin protein for the first time. For the above, a biotechnological device was used (fluorescent biosensor hCaM M124C-mBBr) to directly determine binding parameters experimentally (Kd and stoichiometry) of these compounds, and molecular modeling tools (Docking, Molecular Dynamics, and Chemoinformatic Analysis) to carry out the theoretical studies and complement the experimental data. The results indicate that this compound binds to calmodulin with a Kd between 193–248 nM, an order of magnitude lower than most classic inhibitors. On the other hand, the theoretical studies support the experimental results, obtaining an acceptable correlation between the ΔGExperimental and ΔGTheoretical (r2 = 0.703) and providing us with complementary molecular details of the interaction between the calmodulin protein and the Bisindolylmaleimide series. Chemoinformatic analyzes bring certainty to Bisindolylmaleimide compounds to address clinical steps in drug development. Thus, these results make these compounds attractive to be considered as possible prototypes of new calmodulin protein inhibitors

Redesign of LAOBP to bind novel L amino acid ligands

Computational protein design is still a challenge for advancing structure-function relationships. While recent advances in this field are promising, more information for genuine predictions is needed. Here, we discuss different approaches applied to install novel glutamine (Gln) binding into the Lysine/Arginine/Ornithine binding protein (LAOBP) from Salmonella typhimurium. We studied the ligand binding behavior of two mutants: a binding pocket grafting design based on a structural superposition of LAOBP to the Gln binding protein QBP from Escherichia coli and a design based on statistical coupled positions. The latter showed the ability to bind Gln even though the protein was not very stable. Comparison of both approaches highlighted a nonconservative shared point mutation between LAOBP_graft and LAOBP_sca. This context dependent L117K mutation in LAOBP turned out to be sufficient for introducing Gln binding, as confirmed by different experimental techniques. Moreover, the crystal structure of LAOBP_L117K in complex with its ligand is reported

Application of a Fluorescent Biosensor in Determining the Binding of 5-HT to Calmodulin

Here, we show the utility of the fluorescent biosensor hCaM-M124C-mBBr in detecting and determining the affinity of serotonin (5-HT). We obtained a Kd of 5-HT (0.71 μm) for the first time, the same order of magnitude as most anti-CaM drugs. This data can contribute to understanding the direct and indirect modulation of CaM on its binding proteins when the 5-HT concentration varies in different tissues or explain some of the side effects of anti-CaM drugs. On the other hand, molecular modeling tools help the rational design of biosensors and adequately complement the experimental results. For example, the docking study indicates that 5-HT binds at the same site as chlorpromazine (site 1) with a theoretical Ki of 2.84 μM; while the molecular dynamics simulations indicate a stability of the CaM–5-HT complex with a theoretical ΔG of −4.85 kcal mol−1, where the enthalpy contribution is greater. Thus, the combination of biotechnology and bioinformatics helps in the design and construction of more robust biosensors