Design, synthesis and biological evaluation of novel 9-methyl-9H-purine and thieno[3, 2-d]pyrimidine derivatives as potent mTOR inhibitors.

The mammalian target of rapamycin (mTOR) has been proved to be an effective target for cancer therapy. Two kinds of mTOR inhibitors, the rapalogs and mTOR kinase inhibitors (TORKi), have been developed and clinically validated in several types of malignancies. Compared with rapalogs, TORKi can exert better antitumor activity by inhibiting both mTORC1 and mTORC2, but the clinical development of current TORKi candidates has been relative slow, more TORKi with novel scaffold need to be developed to expand the current pipelines. In this study, a series of 9-methyl-9H-purine and thieno[3, 2-d]pyrimidine derivatives were designed, synthesized and biological evaluation. Most of these compounds exhibited good mTOR kinase inhibitory activity and selectivity over PI3Kα. Subsequent antiproliferative assay allowed us to identify the lead compound 15i, which display nanomolar to low micromolar IC50s against six human cancer cell lines. 15i could induce cell cycle arrest of MCF-7, PC-3 and A549 cells at the G0/G1 phase and suppress the migration and invasion of these cancer cells by suppressing the phosphorylation of AKT and P70S6 kinase. It could also regulate autophagy-related proteins to induce autophagy. Therefore, 15i would be a starting point for the development of new TORKi as anticancer drug.

Phase Behavior of Ion-Containing Polymers in Polar Solvents: Predictions from a Liquid-State Theory with Local Short-Range Interactions.

The thermodynamic phase behavior of charged polymers is a crucial property underlying their role in biology and various industrial applications. A complete understanding of the phase behaviors of such polymer solutions remains challenging due to the multi-component nature of the system and the delicate interplay among various factors, including the translational entropy of each component, excluded volume interactions, chain connectivity, electrostatic interactions, and other specific interactions. In this work, the phase behavior of partially charged ion-containing polymers in polar solvents is studied by further developing a liquid-state (LS) theory with local shortrange interactions. This work is based on the LS theory developed for fully-charged polyelectrolyte solutions. Specific interactions between charged groups of the polymer and counterions, between neutral segments of the polymer, and between charged segments of the polymer are incorporated into the LS theory by an extra Helmholtz free energy from the perturbed-chain statistical associating fluid theory (PC-SAFT). The influence of the sequence structure of the partially charged polymer is modeled by the number of connections between bonded segments. The effects of chain length, charge fraction, counterion valency, and specific short-range interactions are explored. A computational App for salt-free polymer solutions is developed and presented, which allows easy computation of the binodal curve and critical point by specifying values for the relevant model parameters.

Computational Design of Extraordinarily Stable Peptide Structures through Side-Chain-Locked Knots.

Extraordinarily stable protein and peptide structures are critically demanded in many applications. Typical approaches to enhance protein and peptide stability are strengthening certain interactions. Here, we develop a very different approach: stabilizing peptide structures through side-chain-locked knots. More specifically, a peptide core consists of a knot, which is prevented from unknotting and unfolding by large side chains of amino acids at knot boundaries. These side chains impose free energy barriers for unknotting. The free energy barriers are quantified using all-atom and coarse-grained simulations. The barriers become infinitely high for large side chains and tight knot cores, resulting in stable peptide structures, which never unfold unless one chemical bond is broken. The extraordinary stability is essentially kinetic stability. Our new approach lifts the thermodynamic restriction in designing peptide structures, provides extra freedom in selecting sequence and structural motifs that are thermodynamically unstable, and should expand the functionality of peptides. This work also provides a bottom-up understanding of how knotting enhances protein stability.

Quantifying the effects of slit confinement on polymer knots using the tube model.

Knots can spontaneously form in DNA, proteins, and other polymers and affect their properties. These knots often experience spatial confinement in biological systems and experiments. While confinement dramatically affects the knot behavior, the physical mechanisms underlying the confinement effects are not fully understood. In this work, we provide a simple physical picture of the polymer knots in slit confinement using the tube model. In the tube model, the polymer segments in the knot core are assumed to be confined in a virtual tube due to the topological restriction. We first perform Monte Carlo simulation of a flexible knotted chain confined in a slit. We find that with the decrease of the slit height from H=+∞ (the 3D case) to H=2a (the 2D case), the most probable knot size L_{knot}^{*} dramatically shrinks from (L_{knot}^{*})_{3D}≈140a to (L_{knot}^{*})_{2D}≈26a, where a is the monomer diameter of the flexible chain. Then we quantitatively explain the confinement-induced knot shrinking and knot deformation using the tube model. Our results for H=2a can be applied to a polymer knot on a surface, which resembles DNA knots measured by atomic force microscopy under the conditions that DNA molecules are weakly absorbed on the surface and reach equilibrium 2D conformations. This work demonstrates the effectiveness of the tube model in understanding polymer knots.

Systematic Analysis of Tyrosine Kinase Inhibitor Response to RET Gatekeeper Mutations in Thyroid Cancer.

The proto-oncogene protein RET is a receptor tyrosine kinase that plays an important role in the development and progress of various human cancers. Currently, targeting RET with small-molecule tyrosine kinase inhibitors (TKIs) has been established as promising therapeutic strategy for thyroid carcinoma (TC). However, two gatekeeper mutations V804M and V804L in RET kinase domain have been frequently observed to cause drug resistance during the targeted therapy, largely limiting the application of reversible TKIs in TC. Here, we described an integrative protocol that combined literature curation, computational analysis, and in vitro kinase assay to systematically investigate the response profile of 9 approved RET TKIs to the two clinical RET gatekeeper mutations. It was revealed that the two mutations exhibit a similar energetic behavior to influence TKI binding, which can moderately decrease competitive inhibitor affinity and modestly increase substrate ATP affinity simultaneously. However, the binding potency of few second-generation kinase inhibitors such as Ponatinib and Alectinib can be improved to overcome the increased ATP affinity, thus restoring their inhibitory activity against the kinase mutants. Subsequently, the established protocol was employed to investigate the response profile of 4 commercially available RET TKIs that are under preclinical or clinical development. Three out of the four TKIs were found to become resistant upon the mutations due to steric hindrance effect introduced by the mutated residues, while the remaining one was moderately sensitized by the mutations since the mutated residues can form additional hydrophobic and van der Waals interactions with the inhibitor.