Publications

Reversible covalent inhibitors have drawn increasing attention in drug design, as they are likely more potent than noncovalent inhibitors and less toxic than covalent inhibitors. Despite those advantages, the computational prediction of reversible covalent binding presents a formidable challenge because the binding process consists of multiple steps and quantum mechanics (QM) level calculation is needed to estimate the covalent binding free energy. It has been shown that the dissociation rates and the equilibrium dissociation constants vary significantly even with similar warheads, due to noncovalent interactions. We have previously used a simplistic two-state model for predicting the relative binding selectivity of reversible covalent inhibitors ( J. Am. Chem. Soc. 2017, 139 , 17945 ). Here we go beyond binding selectivity and demonstrate that it is possible to use free energy perturbation (FEP) molecular dynamics (MD) to calculate the overall reversible covalent binding using a specially designed thermodynamic cycle. We show that FEP can predict the varying binding free energies of the analogs sharing a common warhead. More importantly, our results revealed that the chemical modification away from warhead alters the binding affinity at both noncovalent and covalent binding states, and the computational prediction can be improved by considering the binding free energy of both states. Furthermore, we explored the possibility of using a more rapid computational method, site-identification by ligand competitive saturation (SILCS), to rank the same set of reversible covalent inhibitors. We found that the fragment docking to a set of precomputed fragment maps produces a reasonable ranking. In conclusion, two independent approaches provided consistent results that the covalent binding state is suitable for the initial ranking of the reversible covalent drug candidates. For lead-optimization, the FEP approach designed here can provide more rigorous and detailed information regarding how much the covalent and noncovalent binding states are contributing to the overall binding affinity, thus offering a new avenue for fine-tuning the noncovalent interactions for optimizing reversible covalent drugs.

Relapse remains the main cause of MLL-rearranged (MLL-r) acute lymphoblastic leukemia (ALL) treatment failure resulting from persistence of drug-resistant clones after conventional chemotherapy treatment or targeted therapy. Thus, defining mechanisms underlying MLL-r ALL maintenance is critical for developing effective therapy. PRMT1, which deposits an asymmetric dimethylarginine mark on histone/non-histone proteins, is reportedly overexpressed in various cancers. Here, we demonstrate elevated PRMT1 levels in MLL-r ALL cells and show that inhibition of PRMT1 significantly suppresses leukemic cell growth and survival. Mechanistically, we reveal that PRMT1 methylates Fms-like receptor tyrosine kinase 3 (FLT3) at arginine (R) residues 972 and 973 (R972/973), and its oncogenic function in MLL-r ALL cells is FLT3 methylation dependent. Both biochemistry and computational analysis demonstrate that R972/973 methylation could facilitate recruitment of adaptor proteins to FLT3 in a phospho-tyrosine (Y) residue 969 (Y969) dependent or independent manner. Cells expressing R972/973 methylation-deficient FLT3 exhibited more robust apoptosis and growth inhibition than did Y969 phosphorylation-deficient FLT3-transduced cells. We also show that the capacity of the type I PRMT inhibitor MS023 to inhibit leukemia cell viability parallels baseline FLT3 R972/973 methylation levels. Finally, combining FLT3 tyrosine kinase inhibitor PKC412 with MS023 treatment enhanced elimination of MLL-r ALL cells relative to PKC412 treatment alone in patient-derived mouse xenografts. These results indicate that abolishing FLT3 arginine methylation through PRMT1 inhibition represents a promising strategy to target MLL-r ALL cells.

Resveratrol, a natural compound found in red wine and various vegetables, has drawn increasing interest due to its reported benefit in cardiovascular protection, neurodegenerative disorders, and cancer therapy. The mechanism by which resveratrol exerts such pleiotropic effects remains unclear. It remains as one of the most discussed polyphenol compounds in the debating "French Paradox". In this study, using molecular dynamics simulations of dipalmitoyl phosphatidylcholine (DPPC) bilayer with resveratrol, we generated a free energy map of resveratrol's location and orientation of inside the lipid bilayer. We found that resveratrol increases the surface area per lipid and decreases membrane thickness, which is the opposite effect of the well-studied cholesterol on liquid phase DPPC. Most importantly, based on the simulation observation that resveratrol has a high probability of forming hydrogen bonds with sn-1 and sn-2 ester groups, we discovered a new mechanism using experimental approach, in which resveratrol protects both sn-1 and sn-2 ester bonds of DPPC and distearoyl phosphatidylcholine (DSPC) from phospholipase A1 (PLA1) and phospholipase A2 (PLA2) cleavage. Our study elucidates the new molecular mechanism of potential health benefits of resveratrol and possibly other similar polyphenols and provides a new paradigm for drug design based on resveratrol and its analogs.

The connexins are members of a family of integral membrane proteins that form gap junction channels between apposed cells and/or hemichannels across the plasma membranes. The importance of the arginine at position 76 (Arg(76)) in the structure and/or function of connexin 46 (Cx46) is highlighted by its conservation across the entire connexin family and the occurrence of pathogenic mutations at this (or the corresponding homologous) residue in a number of human diseases. Two mutations at Arg(76) in Cx46 are associated with cataracts in humans, highlighting the importance of this residue. We examined the expression levels and macroscopic and single-channel properties of human Cx46 and compared them with those for two pathogenic mutants, namely R76H and R76G. To gain further insight into the role of charge at this position, we generated two additional nonnaturally occurring mutants, R76K (charge conserving) and R76E (charge inverting). We found that, when expressed exogenously in Neuro2a cells, all four mutants formed membrane hemichannels, inducing membrane permeability at levels comparable to those recorded in cells expressing the wild-type Cx46. In contrast, the number of gap-junction plaques and the magnitude of junctional coupling were reduced by all four mutations. To gain further insight into the role of Arg(76) in the function of Cx46, we performed homology modeling of Cx46 and in silico mutagenesis of Arg(76) to Gly, His, or Glu. Our studies suggest that the loss of interprotomeric interactions has a significant effect on the extracellular domain conformation and dynamics, thus affecting the hemichannel docking required for formation of cell-cell channels.

BACKGROUND: The nuclear factor erythroid 2-related factor 2 (Nrf2) is a potential molecular target for cancer chemoprevention. Si-Wu-Tang (SWT), a popular traditional Chinese medicine for women's health, was reported with a novel activity of cancer prevention. PURPOSE: The present study was aimed to identify the bioactive constituents in SWT responsible for the Nrf2 activating and cancer preventive activity and explore the pharmacological mechanisms. METHODS: Nine compounds detectable from various batches of SWT were ranked using in silico molecular docking based on their ability to interfere the forming of Nrf2-Keap1 complex. The predicted Nrf2 activating effect was validated using the antioxidant response element (ARE) luciferase reporter assay and quantitative RT-PCR analysis for select Nrf2 regulated genes Hmox1, Nqo1 and Slc7a11. The antimutagenic activity of the compounds were determined by the Ames test. The chemopreventive activity of these compounds were assessed on EGF-induced neoplastic transformation of JB6 P+ cells, an established non-cancerous murine epidermal model for studying tumor promotion and identifying cancer preventive agents. These compounds were further characterized using luciferase reporter assay on EGF-induced activation of AP-1, a known transcription factor mediating carcinogenesis. RESULTS: Three of the nine compounds predicted as Nrf2 activators by molecular docking, gallic acid (GA), Z-liguistilide (LIG), and senkyunolide A (SA), were confirmed with highest potency of increasing the Nrf2/ARE promoter activity and upregulating the expression of Hmox1, Nqo1 and Slc7a11. In addition, GA, LIG and SA exhibited an antimutagenic activity against the direct mutagen 2-nitrofluorene while no mutagenic effects were observed at the same time in Ames test. At nontoxic concentrations, GA, LIG, and SA inhibited EGF-induced neoplastic transformation of JB6 P+ cells. Combined treatment of GA, LIG and SA, in the same ratio as detected in SWT, showed enhanced effect against JB6 transformation compared with that of the single compound alone. GA, LIG and SA, alone or in combination, suppressed EGF-induced activation of AP-1. CONCLUSION: We identified three bioactive constituents in SWT responsible for the Nrf2 activating and cancer preventive activity. This study provides evidence supporting novel molecular basis of SWT in cancer prevention.

Piezo proteins are transmembrane ion channels which transduce many forms of mechanical stimuli into electrochemical signals. Their pore, formed by the assembly of three identical subunits, opens by an unknown mechanism. Here, to probe this mechanism, we investigate the interaction of Piezo1 with the small molecule agonist Yoda1. By engineering chimeras between mouse Piezo1 and its Yoda1-insensitive paralog Piezo2, we first identify a minimal protein region required for Yoda1 sensitivity. We next study the effect of Yoda1 on heterotrimeric Piezo1 channels harboring wild type subunits and Yoda1-insensitive mutant subunits. Using calcium imaging and patch-clamp electrophysiology, we show that hybrid channels harboring as few as one Yoda1-sensitive subunit exhibit Yoda1 sensitivity undistinguishable from homotrimeric wild type channels. Our results show that the Piezo1 pore remains fully open if only one subunit remains activated. This study sheds light on the gating and pharmacological mechanisms of a member of the Piezo channel family.

Type 1 Serine/Threonine Kinase Receptors (STKR1) transduce a wide spectrum of biological signals mediated by TGF-beta superfamily members. The STKR1 activity is tightly controlled by their regulatory glycine-serine rich (GS) domain adjacent to the kinase domain. Despite decades of studies, it remains unknown how physiological or pathological GS domain modifications are coupled to STKR1 kinase activity. Here, by performing molecular dynamics simulations and free energy calculation of Activin-Like Kinase 2 (ALK2), we found that GS domain phosphorylation, FKBP12 dissociation, and disease mutations all destabilize a D354-R375 salt-bridge, which normally acts as an electrostatic lock to prevent coordination of adenosine triphosphate (ATP) to the catalytic site. We developed a WAFEX-guided principal analysis and unraveled how phosphorylation destabilizes this highly conserved salt-bridge in temporal and physical space. Using current-flow betweenness scores, we identified an allosteric network of residue-residue contacts between the GS domain and the catalytic site that controls the formation and disruption of this salt bridge. Importantly, our novel network analysis approach revealed how certain disease-causing mutations bypass FKBP12-mediated kinase inhibition to produce leaky signaling. We further provide experimental evidence that this salt-bridge lock exists in other STKR1s, and acts as a general safety mechanism in STKR1 to prevent pathological leaky signaling. In summary, our study provides a compelling and unifying allosteric activation mechanism in STKR1 kinases that reconciles a large number of experimental studies and sheds light on a novel therapeutic avenue to target disease-related STKR1 mutants.

Reversible covalent inhibitors have many clinical advantages over noncovalent or irreversible covalent drugs. However, apart from selecting a warhead, substantial efforts in design and synthesis are needed to optimize noncovalent interactions to improve target-selective binding. Computational prediction of binding affinity for reversible covalent inhibitors presents a unique challenge since the binding process consists of multiple steps, which are not necessarily independent of each other. In this study, we lay out the relation between relative binding free energy and the overall reversible covalent binding affinity using a two-state binding model. To prove the concept, we employed free energy perturbation (FEP) coupled with lambda-exchange molecular dynamics method to calculate the binding free energy of a series of alpha-ketoamide analogues relative to a common warhead scaffold, in both noncovalent and covalent binding states, and for two highly homologous proteases, calpain-1 and calpain-2. We conclude that covalent binding state alone, in general, can be used to predict reversible covalent binding selectivity. However, exceptions may exist. Therefore, we also discuss the conditions under which the noncovalent binding step is no longer negligible and propose to combine the relative FEP calculations with a single QM/MM calculation of warhead to predict the binding affinity and binding kinetics. Our FEP calculations also revealed that covalent and noncovalent binding states of an inhibitor do not necessarily exhibit the same selectivity. Thus, investigating both binding states, as well as the kinetics will provide extremely useful information for optimizing reversible covalent inhibitors.

Aberrant opening of nonjunctional connexin hemichannels at the plasma membrane is associated with many diseases, including ischemia and muscular dystrophy. Proper control of hemichannel opening is essential to maintain cell viability and is achieved by physiological levels of extracellular Ca(2+), which drastically reduce hemichannel activity. Here we examined the role of conserved charged residues that form electrostatic networks near the extracellular entrance of the connexin pore, a region thought to be involved in gating rearrangements of hemichannels. Molecular dynamics simulations indicate discrete sites for Ca(2+) interaction and consequent disruption of salt bridges in the open hemichannels. Experimentally, we found that disruption of these salt bridges by mutations facilitates hemichannel closing. Two negatively charged residues in these networks are putative Ca(2+) binding sites, forming a Ca(2+)-gating ring near the extracellular entrance of the pore. Accessibility studies showed that this Ca(2+)-bound gating ring does not prevent access of ions or small molecules to positions deeper into the pore, indicating that the physical gate is below the Ca(2+)-gating ring. We conclude that intra- and intersubunit electrostatic networks at the extracellular entrance of the hemichannel pore play critical roles in hemichannel gating reactions and are tightly controlled by extracellular Ca(2+) Our findings provide a general mechanism for Ca(2+) gating among different connexin hemichannel isoforms.

A signal property of connexin channels is the ability to mediate selective diffusive movement of molecules through plasma membrane(s), but the energetics and determinants of molecular movement through these channels have yet to be understood. Different connexin channels have distinct molecular selectivities that cannot be explained simply on the basis of size or charge of the permeants. To gain insight into the forces and interactions that underlie selective molecular permeation, we investigated the energetics of two uncharged derivatized sugars, one permeable and one impermeable, through a validated connexin26 (Cx26) channel structural model, using molecular dynamics and associated analytic tools. The system is a Cx26 channel equilibrated in explicit membrane/solvent, shown by Brownian dynamics to reproduce key conductance characteristics of the native channel. The results are consistent with the known difference in permeability to each molecule. The energetic barriers extend through most of the pore length, rather than being highly localized as in ion-specific channels. There is little evidence for binding within the pore. Force decomposition reveals how, for each tested molecule, interactions with water and the Cx26 protein vary over the length of the pore and reveals a significant contribution from hydrogen bonding and interaction with K(+). The flexibility of the pore width varies along its length, and the tested molecules have differential effects on pore width as they pass through. Potential sites of interaction within the pore are defined for each molecule. The results suggest that for the tested molecules, differences in hydrogen bonding and entropic factors arising from permeant flexibility substantially contribute to the energetics of permeation. This work highlights factors involved in selective molecular permeation that differ from those that define selectivity among atomic ions.