Molecular Modeling of Single- and Double-Hydrocarbon-Stapled Coiled-Coil Inhibitors against Bcr-Abl: Toward a Treatment Strategy for CML.

The chimeric oncoprotein Bcr-Abl is the causative agent of virtually all chronic myeloid leukemias and a subset of acute lymphoblastic leukemias. As a result of the so-called Philadelphia chromosome translocation t(9;22), Bcr-Abl manifests as a constitutively active tyrosine kinase, which promotes leukemogenesis by activation of cell cycle signaling pathways. Constitutive and oncogenic activation is mediated by an N-terminal coiled-coil oligomerization domain in Bcr (Bcr-CC), presenting a therapeutic target for inhibition of Bcr-Abl activity toward the treatment of Bcr-Abl+ leukemias. Previously, we demonstrated that a rationally designed Bcr-CC mutant, CCmut3, exerts a dominant negative effect upon Bcr-Abl activity by preferential oligomerization with Bcr-CC. Moreover, we have shown that conjugation to a leukemia-specific cell-penetrating peptide (CPP-CCmut3) improves intracellular delivery and activity. However, our full-length CPP-CCmut3 construct (81 aa) is encumbered by an intrinsically high degree of conformational variability and susceptibility to proteolytic degradation relative to traditional small-molecule therapeutics. Here, we iterate a new generation of CCmut3 inhibitors against Bcr-CC-mediated Bcr-Abl assembly designed to address these constraints through incorporation of all-hydrocarbon staples spanning i and i + 7 positions in α-helix 2 (CPP-CCmut3-st). We utilize computational modeling and biomolecular simulation to evaluate single- and double-stapled CCmut3 candidates in silico for dynamics and binding energetics. We further model a truncated system characterized by the deletion of α-helix 1 and the flexible loop linker, which are known to impart high conformational variability. To study the impact of the N-terminal cyclic CPP toward model stability and inhibitor activity, we also model the full-length and truncated systems devoid of the CPP, with a cyclized CPP, and with an open-configuration CPP, for a total of six systems that comprise our library. From this library, we present lead-stapled peptide candidates to be synthesized and evaluated experimentally as our next iteration of inhibitors against Bcr-Abl.

Computational Modeling of Stapled Coiled-Coil Inhibitors Against Bcr-Abl: Toward a Treatment Strategy for CML.

The chimeric oncoprotein Bcr-Abl is the causative agent of virtually all chronic myeloid leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL). As a result of the so-called Philadelphia Chromosome translocation t(9;22), Bcr-Abl manifests as a constitutively active tyrosine kinase which promotes leukemogenesis by activation of cell cycle signaling pathways. Constitutive and oncogenic activation is mediated by an N-terminal coiled-coil oligomerization domain in Bcr (Bcr-CC), presenting a therapeutic target for inhibition of Bcr-Abl activity toward the treatment of Bcr-Abl+ leukemias. Previously, we demonstrated that a rationally designed Bcr-CC mutant, CCmut3, exerts a dominant negative effect upon Bcr-Abl activity by preferential oligomerization with Bcr-CC. Moreover, we have shown conjugation to a leukemia-specific cell-penetrating peptide (CPP-CCmut3) improves intracellular delivery and activity. However, our full-length CPP-CCmut3 construct (81 aa) is encumbered by an intrinsically high degree of conformational variability and susceptibility to proteolytic degradation, relative to traditional small molecule therapeutics. Here, we iterate a new generation of our inhibitor against Bcr-CC mediated Bcr-Abl assembly that is designed to address these constraints through incorporation of all-hydrocarbon staples spanning i, i + 7 positions in helix α2 (CPP-CCmut3-st). We utilize computational modeling and biomolecular simulation to design and characterize single and double staple candidates in silico, evaluating binding energetics and building upon our seminal work modeling single hydrocarbon staples when applied to a truncated Bcr-CC sequence. This strategy enables us to efficiently build, characterize, and screen lead single/double stapled CPP-CCmut3-st candidates for experimental studies and validation in vitro and in vivo. In addition to full-length CPP-CCmut, we model a truncated system characterized by deletion of helix α1 and the flexible-loop linker, which are known to impart high conformational variability. To study the impact of the N-terminal cyclic CPP toward model stability and inhibitor activity, we also model the full-length and truncated systems without CPP, with cyclized CPP, and with linear CPP, for a total of six systems which comprise our library. From this library, we present lead stapled peptide candidates to be synthesized and evaluated experimentally as our next-generation inhibitors against Bcr-Abl.

Reaction mechanism and tautomeric equilibrium of 2-mercaptopyrimidine in the gas phase and in aqueous solution: a combined Monte Carlo and quantum mechanics study.

A combined Monte Carlo and quantum mechanical study was carried out to analyze the tautomeric equilibrium of 2-mercaptopyrimidine in the gas phase and in aqueous solution. Second- and fourth-order Møller-Plesset perturbation theory calculations indicate that in the gas phase thiol (Pym-SH) is more stable than the thione (Pym-NH) by ca. 8 kcal/mol. In aqueous solution, thermodynamic perturbation theory implemented on a Monte Carlo NpT simulation indicates that both the differential enthalpy and Gibbs free energy favor the thione form. The calculated differential enthalpy is DeltaH(SH)(-->)(NH)(solv) = -1.7 kcal/mol and the differential Gibbs free energy is DeltaG(SH)(-->)(NH)(solv) = -1.9 kcal/mol. Analysis is made of the contribution of the solute-solvent hydrogen bonds and it is noted that the SH group in the thiol and NH group in the thione tautomers act exclusively as a hydrogen bond donor in aqueous solution. The proton transfer reaction between the tautomeric forms was also investigated in the gas phase and in aqueous solution. Two distinct mechanisms were considered: a direct intramolecular transfer and a water-assisted mechanism. In the gas phase, the intramolecular transfer leads to a large energy barrier of 34.4 kcal/mol, passing through a three-center transition state. The proton transfer with the assistance of one water molecule decreases the energy barrier to 17.2 kcal/mol. In solution, these calculated activation barriers are, respectively, 32.0 and 14.8 kcal/mol. The solvent effect is found to be sizable but it is considerably more important as a participant in the water-assisted mechanism than the solvent field of the solute-solvent interaction. Finally, the calculated total Gibbs free energy is used to estimate the equilibrium constant.