Targeting tumors using peptides

To penetrate solid tumors, low molecular weight (Mw < 10 KDa) compounds have an edge over antibodies: their higher penetration because of their small size. Because of the dense stroma and high interstitial fluid pressure of solid tumors, the penetration of higher Mw compounds is unfavored and being small thus becomes an advantage. This review covers a wide range of peptidic ligands—linear, cyclic, macrocyclic and cyclotidic peptides—to target tumors: We describe the main tools to identify peptides experimentally, such as phage display, and the possible chemical modifications to enhance the properties of the identified peptides. We also review in silico identification of peptides and the most salient non-peptidic ligands in clinical stages. We later focus the attention on the current validated ligands available to target different tumor compartments: blood vessels, extracelullar matrix, and tumor associated macrophages. The clinical advances and failures of these ligands and their therapeutic conjugates will be discussed. We aim to present the reader with the state-of-the-art in targeting tumors, by using low Mw molecules, and the tools to identify new ligands.Fil: Scodeller, Pablo David. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Tartu; EstoniaFil: Asciutto, Eliana Karina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentin

Physical Interactions Driving the Activation/Inhibition of Calcium/Calmodulin Dependent Protein Kinase II

CaMKII is a protein kinase whose function is regulated by the binding of the Calcium/Calmodulin complex (Ca2+ /CaM). It is a major player in the Long Term Potentiation process where it acts as a molecular switch, oscillating between inhibited and active conformations. The mechanism for the switching is thought to be initiated by Ca2+/CaM binding, which allows the trans-phosphorylation of a subunit of CaMKII by a neighboring kinase, leading to the active state of the system. A combination of all-atom and coarse-grained MD simulations with free energy calculations, led us to reveal an interplay of electrostatic forces exerted by Ca2+/CaM on CaMKII, which initiate the activation process. The highly electrically charged Ca2+/CaM neutralizes basic regions in the linker domain of CaMKII, facilitating its opening and consequent activation. The emerging picture of CaMKII's behavior highlights the preponderance of electrostatic interactions, which are modulated by the presence of Ca2+/CaM and the phosphorylation of key sites.Fil: Asciutto, Eliana Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias Físicas. - Universidad Nacional de San Martín. Instituto de Ciencias Físicas; ArgentinaFil: Pantano, Sergio. Universidad Nacional de San Martín; Argentina. Instituto Pasteur de Montevideo; UruguayFil: General, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias Físicas. - Universidad Nacional de San Martín. Instituto de Ciencias Físicas; Argentin

Sodium versus potassium effects on the glutamic acid side-chains interaction on a heptapeptide

Equilibrium peptide conformations in solution, especially in the presence of salts, has been of interest for several decades. The fundamental interactions that determine the dominant peptide conformations in solution have been experimentally and computationally probed; however, a uni¯ed understanding has not yet emerged. In a previous study, we performed metadynamics simulations on the heptapeptide AEAAAEA in Sodium Chloride (NaCl) and Potassium Chloride (KCl) solutions at concentrations ranging from 0.5–2.0 M. Using a three-dimensional collective variable coordinate system, we computed the free energy landscapes in each saline environment as well as in pure water. We found that the presence of Naþ and Kþ ions induces some changes in the stability of the conformers that de¯ne the state space, but does not alter the overall energetics between conformers and does not favor helical conformations. We investigate here, how the presence of salts (NaCl and KCl) a®ects the glutamic–glutamic interaction and its consequences on the stability of each equilibrium conformation. We perform this study through ¯xed backbone simulations for the most populated conformations identi¯ed in our previous work: the -helix, 310-helix, -helix, the extended polyproline II (PPII) and 2.51-helix conformations. It was found that for each conformation, there exists stable substates determined by the glutamic acid side-chains distance and orientation, and that Naþ and Kþ cations (de) stabilize preferentially each conformation. It was also found that intramolecular single water mediated hydrogen bonds play a crucial role in the observed (de) stabilization of each equilibrium conformation.Fil: Asciutto, Eliana Karina. Duquesne University. Department of Chemistry and Biochemistry; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gaborek, Timothy. Duquesne University. Department of Chemistry and Biochemistry; Estados UnidosFil: Madura, Jeffry D.. Duquesne University. Department of Chemistry and Biochemistry; Estados Unido

Pl1 peptide engages acidic surfaces on tumor-associated fibronectin and tenascin isoforms to trigger cellular uptake

Tumor extracellular matrix (ECM) is a high-capacity target for the precision delivery of affinity ligand-guided drugs and imaging agents. Recently, we developed a PL1 peptide (sequence: PPRRGLIKLKTS) for systemic targeting of malignant ECM. Here, we map the dynamics of PL1 binding to its receptors Fibronectin Extra Domain B (FN-EDB) and Tenascin C C-isoform (TNC-C) by computational modeling and cell-free binding studies on mutated receptor proteins, and study cellular binding and internalization of PL1 nanoparticles in cultured cells. Molecular dynamics simulation and docking analysis suggested that the engagement of PL1 peptide with both receptors is primarily driven by electrostatic interactions. Substituting acidic amino acid residues with neutral amino acids at predicted PL1 binding sites in FN-EDB (D52N-D49N-D12N) and TNC-C (D39N-D45N) resulted in the loss of binding of PL1 nanoparticles. Remarkably, PL1-functionalized nanoparticles (NPs) were not only deposited on the target ECM but bound the cells and initiated a robust cellular uptake via a pathway resembling macropinocytosis. Our studies establish the mode of engagement of the PL1 peptide with its receptors and suggest applications for intracellular delivery of nanoscale payloads. The outcomes of this work can be used for the development of PL1-derived peptides with improved stability, affinity, and specificity for precision targeting of the tumor ECM and malignant cells.Fil: Lingasamy, Prakash. University of Tartu; EstoniaFil: Põnograjeva, Kristina. University of Tartu; EstoniaFil: Kopanchuk, Sergei. University of Tartu; EstoniaFil: Tobi, Allan. University of Tartu; EstoniaFil: Rinken, Ago. University of Tartu; EstoniaFil: General, Ignacio. University of California; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Asciutto, Eliana Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias Físicas. - Universidad Nacional de San Martín. Instituto de Ciencias Físicas; ArgentinaFil: Teesalu, Tambet. University of Tartu; Estoni

Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450cam (CYP101A1)

Cytochrome P450cam (CYP101A1) catalyzes the stereospecific 5-exo hydroxylation of d-camphor by molecular oxygen. Previously, residual dipolar couplings measured for backbone amide 1H–15N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamic simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling molecular dynamic of the residual dipolar coupling-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed.Fil: Asciutto, Eliana Karina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pochapsky, Thomas C.. Universidad Brandeis; Estados Unido

Hydroxylation Regiochemistry Is Robust to Active Site Mutations in Cytochrome P450cam(CYP101A1)

Cytochrome P450cam(CYP101A1) catalyzes the hydroxylation of d-camphor by molecular oxygen. The enzyme-catalyzed hydroxylation exhibits a high degree of regioselectivity and stereoselectivity, with a single major product, d-5-exo-hydroxycamphor, suggesting that the substrate is oriented to facilitate this specificity. In previous work, we used an elastic network model and perturbation response scanning to show that normal deformation modes of the enzyme structure are highly responsive not only to the presence of a substrate but also to the substrate orientation. This work examines the effects of mutations near the active site on substrate localization and orientation. The investigated mutations were designed to promote a change in substrate orientation and/or location that might give rise to different hydroxylation products, while maintaining the same carbon and oxygen atom balances as in the wild type (WT) enzyme. Computational experiments and parallel in vitro site-directed mutations of CYP101A1 were used to examine reaction products and enzyme activity. 1H-15N TROSY-HSQC correlation maps were used to compare the computational results with detectable perturbations in the enzyme structure and dynamics. We found that all of the mutant enzymes retained the same regio- and stereospecificity of hydroxylation as the WT enzyme, with varying degrees of efficiency, which suggests that large portions of the enzyme have been subjected to evolutionary pressure to arrive at the appropriate sequence-structure combination for efficient 5-exo hydroxylation of camphor.Fil: Alvarez, Guadalupe. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Ciencias Fisicas. - Universidad Nacional de San Martin. Instituto de Ciencias Fisicas.; ArgentinaFil: Le, Thu. Brandeis University; Estados UnidosFil: Wong, Nathan. Brandeis University; Estados UnidosFil: Echave, Julian. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Ciencias Fisicas. - Universidad Nacional de San Martin. Instituto de Ciencias Fisicas.; ArgentinaFil: Pochapsky, Thomas C.. Brandeis University; Estados UnidosFil: Asciutto, Eliana Karina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Ciencias Fisicas. - Universidad Nacional de San Martin. Instituto de Ciencias Fisicas.; Argentin

Phage-Display-Derived Peptide Binds to Human CD206 and Modeling Reveals a New Binding Site on the Receptor

We recently identified a tumor-homing peptide (mUNO, sequence: "CSPGAK") that specifically interacts with mouse CD206 to target CD206/MRC1-expressing tumor-associated macrophages in mice. Here, we report studies on the binding of mUNO to human recombinant CD206 (hCD206) and on modeling the mUNO/hCD206 interaction by computational analysis. Fluorescence anisotropy analysis demonstrated that fluorophore-labeled mUNO interacts with hCD206. Microsecond time-scale molecular dynamics simulations and docking predictions showed that mUNO binds to a newly identified epitope between C-type lectin domains 1 and 2. The physical mechanisms that contribute to the docking interactions of mUNO include electrostatic interactions, aromatic interactions, and hydrogen bonds. We also demonstrate the selectivity of FAM-mUNO for CD206+-cultured human macrophages. The peptide mUNO appears to be the first ligand capable of interacting with this epitope of hCD206, for which no ligands have been reported. Our study has implications for targeting human M2-like tumor-associated macrophages, a subpopulation of immune cells with a major protumoral role.Fil: Asciutto, Eliana Karina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kopanchuk, Sergei. University of Tartu; EstoniaFil: Lepland, Anni. University of Tartu; EstoniaFil: Simón-Gracia, Lorena. University of Tartu; EstoniaFil: Aleman, Carlos. Universidad Politécnica de Catalunya; EspañaFil: Teesalu, Tambet. University of Tartu; Estonia. Sanford-burnham Medical Research Institute; Estados Unidos. University of California; Estados UnidosFil: Scodeller, Pablo David. University of Tartu; Estonia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin