Physiological Concentrations Of ATP Research Articles

Novel Small-Molecule ROCK2 Inhibitor GNS-3595 Attenuates Pulmonary Fibrosis in Preclinical Studies.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that leads to respiratory decline caused by scarring and thickening of lung tissues. Multiple pathways contribute to the fibrotic process in this disease, such as inflammation, epithelial-to-mesenchymal transition, and oxidative stress. The Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway is a key regulator of profibrotic signaling, as it affects the organization of actin-myosin and the remodeling of the extracellular matrix. ROCK1/2, a downstream effector of RhoA, is overexpressed in patients with IPF and is a promising target for IPF therapy. However, because of the hypotensive side effects of ROCK1/2 inhibitors, selective ROCK2 compounds are being explored. In this study, we report the discovery of GNS-3595, a potent and selective ROCK2 inhibitor that has ∼80-fold selectivity over ROCK1 at physiological concentrations of ATP. GNS-3595 effectively inhibited ROCK2-mediated phosphorylation of myosin light chain and reduced the expression of fibrosis-related proteins (e.g., collagen, fibronectin, and α-smooth muscle actin) in various invitro cellular models. GNS-3595 also prevented transforming growth factor β-induced fibroblast-to-myofibroblast transition. In addition, in a bleomycin-induced mouse model of pulmonary fibrosis, therapeutic exposure to GNS-3595, suppressed lung fibrosis, stabilized body weight loss, and prevented fibrosis-induced lung weight gain. Transcriptome and protein expression analysis from lung tissues showed that GNS-3595 can revert the fibrosis-related gene expression induced by bleomycin. These results indicate that GNS-3595 is a highly potent, selective, and orally active ROCK2 inhibitor with promising therapeutic efficacy against pulmonary fibrosis.

A Reversible NIR‐II Ratiometric Fluorescent Probe for Real‐Time In Vivo ATP Detection

AbstractATP plays a prominent role in shaping the evolution of immune cell responses to injury, infection, and cancer. However, real‐time monitoring of ATP levels in vivo remains a challenge, due to the lack of reliable tools that work reversibly in deep tissues. Herein, based on the Förster resonance energy transfer (FRET) strategy, a reversible ratiometric second near‐infrared window (NIR‐II) molecular fluorescent probe (CX‐RATP) is designed and synthesized. CX‐RATP exhibits a significant fluorescence ratio change with the existence of 0–10 mM ATP in vitro, which is within the range of physiological and pathological ATP concentrations. Meanwhile, the recognition process can be reversed by apyrase. It can capture the increase and decrease in ATP levels in real time before and after the treatment of acute inflammation. Besides, CX‐RATP is also successfully applied to the hepatocellular carcinoma (HCC) model and achieves in situ imaging of tumor lesions. These results all provide solid proof that CX‐RATP is a powerful tool for in vivo real‐time ATP detection.

Posttranslational modifications of cyclic AMP-dependent protein kinase a affect its binding to the CFTR anion channel.

CFTR is the only ion channel member in the ABC protein superfamily. Mutations of the protein cause malfunction of the pore or retention of the protein in the endoplasmic reticulum leading to the systemic disease cystic fibrosis, the most common lethal genetic disorder in the Caucasian population. CFTR activity relies on an irreversible gating mechanism which involves a cycle of ATP binding and hydrolysis at its cytosolic nucleotide binding domains (NBDs). Upon binding of two molecules of ATP the NBDs form a tight NBD dimer which triggers opening of the channel pore, while ATP hydrolysis closes the channel. ATP-dependent opening of the pore strongly depends on phosphorylation of the channel's regulatory (R) domain by cAMP-dependent protein kinase (PKA). At physiological ATP concentrations the NBDs of the channel are saturated by ligand, hence PKA phosphorylation plays the primary regulatory role. Our group has previously shown (PNAS 117:21740) that direct physical binding of PKA activates CFTR channels independently of, and additively to, phosphorylation. We hypothesized that PKA binding prevents its substrate, the R domain, from returning into an inhibitory position wedged in between the two NBDs, thereby shifting the closed-open equilibrium towards open. In contrast to the protein isolated from bovine heart, heterologously expressed PKA lacks such a binding effect. We therefore studied posttranslational modifications of PKA, and investigated their effects on CFTR activation by comparing the recombinant and tissue-isolated PKA isoforms. Results of the ongoing research will be presented at the meeting.

A Cantú syndrome mutation produces dual effects on KATP channels by disrupting ankyrin B regulation.

ATP-sensitive potassium (KATP) channels composed of Kir6.x and sulfonylurea receptor (SURs) subunits couple cellular metabolism to electrical activity. Cantú syndrome (CS) is a rare disease caused by mutations in the genes encoding Kir6.1 (KCNJ8) and SUR2A (ABCC9) that produce KATP channel hyperactivity due to a reduced channel block by physiological ATP concentrations. We functionally characterized the p.S1054Y SUR2A mutation identified in two CS carriers, who exhibited a mild phenotype although the mutation was predicted as highly pathogenic. We recorded macroscopic and single-channel currents in CHO and HEK-293 cells and measured the membrane expression of the channel subunits by biotinylation assays in HEK-293 cells. The mutation increased basal whole-cell current density and at the single-channel level, it augmented opening frequency, slope conductance, and open probability (Po), and promoted the appearance of multiple conductance levels. p.S1054Y also reduced Kir6.2 and SUR2A expression specifically at the membrane. Overexpression of ankyrin B (AnkB) prevented these gain- and loss-of-function effects, as well as the p.S1054Y-induced reduction of ATP inhibition of currents measured in inside-out macropatches. Yeast two-hybrid assays suggested that SUR2A WT and AnkB interact, while p.S1054Y interaction with AnkB is decreased. The p.E322K Kir6.2 mutation, which prevents AnkB binding to Kir6.2, produced similar biophysical alterations than p.S1054Y. Our results are the first demonstration of a CS mutation whose functional consequences involve the disruption of AnkB effects on KATP channels providing a novel mechanism by which CS mutations can reduce ATP block. Furthermore, they may help explain the mild phenotype associated with this mutation.

Patient mutations in human ATP:cob(I)alamin adenosyltransferase differentially affect its catalytic versus chaperone functions

Human ATP:cob(I)alamin adenosyltransferase (ATR) is a mitochondrial enzyme that catalyzes an adenosyl transfer to cob(I)alamin, synthesizing 5′-deoxyadenosylcobalamin (AdoCbl) or coenzyme B12. ATR is also a chaperone that escorts AdoCbl, transferring it to methylmalonyl-CoA mutase, which is important in propionate metabolism. Mutations in ATR lead to methylmalonic aciduria type B, an inborn error of B12 metabolism. Our previous studies have furnished insights into how ATR protein dynamics influence redox-linked cobalt coordination chemistry, controlling its catalytic versus chaperone functions. In this study, we have characterized three patient mutations at two conserved active site residues in human ATR, R190C/H, and E193K and obtained crystal structures of R190C and E193K variants, which display only subtle structural changes. All three mutations were found to weaken affinities for the cob(II)alamin substrate and the AdoCbl product and increase KM(ATP). 31P NMR studies show that binding of the triphosphate product, formed during the adenosylation reaction, is also weakened. However, although the kcat of this reaction is significantly diminished for the R190C/H mutants, it is comparable with the WT enzyme for the E193K variant, revealing the catalytic importance of Arg-190. Furthermore, although the E193K mutation selectively impairs the chaperone function by promoting product release into solution, its catalytic function might be unaffected at physiological ATP concentrations. In contrast, the R190C/H mutations affect both the catalytic and chaperoning activities of ATR. Because the E193K mutation spares the catalytic activity of ATR, our data suggest that the patients carrying this mutation are more likely to be responsive to cobalamin therapy.

Elucidating specificity of an allosteric inhibitor WNK476 among With-No-Lysine kinase isoforms using molecular dynamic simulations.

Specifically targeting the With-No-Lysine (WNK1) kinase, which is implicated in hypertension, renders a significant challenge in discovering competitive inhibitors due to the highly conserved ATP-binding pocket. However, an allosteric inhibitor may impart high specificity against the WNK kinase isoforms since it targets the less conserved site and can provide greater efficacy even under high physiological ATP concentration. In the current study, we have investigated the structural and energetic basis of the specificity of the allosteric inhibitor WNK476 against WNK kinase isoforms by combining molecular dynamics simulations and free energy calculations using molecular mechanics Poisson-Boltzmann surface area. Our study reveals that the conformational stabilization of αC-helix near the allosteric binding site, including conformational changes in activation and glycine-rich loop regions, favors the specificity of WNK476 toward WNK1. The MM/PBSA calculations suggest that the non-polar contribution from hydrophobic residues and polar solvation energy influences WNK/WNK476 complexation. Despite more favorable electrostatic and van der Waals interactions in WNK2/WNK476, WNK476 is more potent against WNK1 due to the lower contribution of disfavoring components-polar solvation and entropy. Further, we have identified that the hydrophobic residues of DLG, αC-helix, β4 , and β5 regions, and H-bond network near the β4 strand play a critical role in the specificity of WNK476 against WNK1. Finally, our study reveals that residues Leu272 , Val281 , Phe283 , and Leu369 of WNK1 actively contribute to the overall hydrophobic interactions for WNK1/WNK476. Overall, our study might help in the rational design of novel allosteric inhibitors against hypertension.

The Fast and Superprocessive KIF1A Predominately Resides in a Vulnerable One-Head-Bound State during its Chemomechanical Cycle

Kinesin-3 are the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to wild-type KIF1A in BRB80. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and-2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate limiting transition in the KIF1A stepping cycle. The fast speed, superprocessivity and load sensitivity of KIF1A can be explained by a fast rear head detachment rate, a rate-limiting step of tethered head attachment that follows ATP hydrolysis, and a relatively strong microtubule interaction in the post-hydrolysis state.

A kinetic dissection of the fast and superprocessive kinesin-3 KIF1A reveals a predominant one-head-bound state during its chemomechanical cycle

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A's activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.

Negative regulation of RAF kinase activity by ATP is overcome by 14-3-3-induced dimerization.

The RAS-RAF-MEK-ERK signaling axis is frequently activated in human cancers. Physiological concentrations of ATP prevent formation of RAF kinase-domain (RAFKD) dimers that are critical for activity. Here we present a 2.9-Å-resolution crystal structure of human BRAFKD in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that induce an inactive, monomeric conformation of BRAFKD. We also determine how 14-3-3 relieves the negative regulatory effect of ATP through a 2.5-Å-resolution crystal structure of the BRAFKD-14-3-3 complex, in which dimeric 14-3-3 enforces a dimeric BRAFKD assembly to increase BRAF activity. Our data suggest that most oncogenic BRAF mutations alter interactions with ATP and counteract the negative effects of ATP binding by lowering the threshold for RAF dimerization and pathway activation. Our study establishes a framework for rationalizing oncogenic BRAF mutations and provides new avenues for improved RAF-inhibitor discovery.

The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair

DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2–Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2–Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2–Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide–binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2–Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo. The mutant Msh2–Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1–Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1–Pms1-independent and Mlh1–Pms1-dependent MMR reactions in vitro and in activation of the Mlh1–Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2–Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.

2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid (Activator-3) is a potent activator of AMPK

AMPK is considered as a potential high value target for metabolic disorders. Here, we present the molecular modeling, in vitro and in vivo characterization of Activator-3, 2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid, an AMP mimetic and a potent pan-AMPK activator. Activator-3 and AMP likely share common activation mode for AMPK activation. Activator-3 enhanced AMPK phosphorylation by upstream kinase LKB1 and protected AMPK complex against dephosphorylation by PP2C. Molecular modeling analyses followed by in vitro mutant AMPK enzyme assays demonstrate that Activator-3 interacts with R70 and R152 of the CBS1 domain on AMPK γ subunit near AMP binding site. Activator-3 and C2, a recently described AMPK mimetic, bind differently in the γ subunit of AMPK. Activator-3 unlike C2 does not show cooperativity of AMPK activity in the presence of physiological concentration of ATP (2 mM). Activator-3 displays good pharmacokinetic profile in rat blood plasma with minimal brain penetration property. Oral treatment of High Sucrose Diet (HSD) fed diabetic rats with 10 mg/kg dose of Activator-3 once in a day for 30 days significantly enhanced glucose utilization, improved lipid profiles and reduced body weight, demonstrating that Activator-3 is a potent AMPK activator that can alleviate the negative metabolic impact of high sucrose diet in rat model.

The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition.

Cyclic GMP‐AMP synthase (cGAS) is activated by ds‐DNA binding to produce the secondary messenger 2′,3′‐cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds‐DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2′,3′‐cGAMP formation, and interestingly, describe a catalytic mechanism where 2′,3′‐cGAMP may be a minor product of cGAS compared with linear nucleotides.

Novel translocation intermediate allows re-evaluation of roles of ATP, proton motive force and SecG at the late stage of preprotein translocation.

Presecretory proteins such as pOmpA are translocated across the inner membrane of Escherichia coli by Sec translocase powered by ATP and proton motive force (PMF). Translocation activity has been determined by protease protection assaying invitro. We identified a new translocation intermediate at a late stage, which was protected by proteinase K (PK), but became PK sensitive upon urea extraction. At a late stage of pOmpA translocation driven by PMF in the presence of a nonhydrolyzable ATP analogue, the PK-protected materials arose, but were pulled back upon urea extraction, indicating that completion of translocation requires ATP hydrolysis. When inverted membrane vesicles prepared from secG-null strain (ΔSecG IMV) were used in the absence of PMF, the translocation intermediate was accumulated. When the ATP concentration was low in the absence of PMF, the translocation intermediate was also accumulated. Imposition of PMF in the presence of a low ATP concentration caused recovery of pOmpA translocation and resistance to urea extraction for SecG+ IMV, but not for ΔSecG IMV. Thus, analysis of the late translocation intermediate showed that two of three constituents, physiological concentration of ATP, PMF and SecG, are required for the catalytic cycle of preprotein translocation, that is, completion and subsequent initiation of translocation.

Effect of fuel concentration on cargo transport by a team of Kinesin motors

Eukaryotic cells employ specialized proteins called molecular motors for transporting organelles and vesicles from one location to another in a regulated and directed manner. These molecular motors often work collectively in a team while transporting cargos. Molecular motors use cytoplasmic ATP as fuel, which is hydrolyzed to generate mechanical force. While the effect of ATP concentration on cargo transport by single Kinesin motor function is well understood, it is still unexplored, both theoretically and experimentally, how ATP concentration would affect cargo transport by a team of Kinesin motors. For instance, how does fuel concentration affect the travel distances and travel velocities of cargo? How cooperativity of Kinesin motors engaged on a cargo is affected by ATP concentration? To answer these questions, here we develop mechano-chemical models of cargo transport by a team of Kinesin motors. To develop these models we use experimentally-constrained mechano-chemical model of a single Kinesin motor as well as earlier developed mean-field and stochastic models of load sharing for cargo transport. Thus, our new models for cargo transport by a team of Kinesin motors include fuel concentration explicitly, which was not considered in earlier models. We make several interesting predictions which can be tested experimentally. For instance, the travel distances of cargos are very large at limited ATP concentrations in spite of very small travel velocity. Velocities of cargos driven by multiple Kinesin have a Michaelis–Menten dependence on ATP concentration. Similarly, cooperativity among the engaged Kinesin motors on the cargo shows a Michaelis–Menten type dependence, which attains a maximum value near physiological ATP concentrations. Our new results can be potentially useful in controlling artificial nano-molecular shuttles precisely for targeted delivery in various nano-technological applications.

Mechanism of Cooperative Force Generations between Skeletal Myosins

To understand the molecular mechanism of cooperative force generation between skeletal myosin molecules, we measured forces generated by myosin-rod cofilaments, in which approximately 17 myosin molecules interact with single actins at the mixing ratio used in this study. Combined with results from the computational model, three factors are important for synchronization of power strokes between myosin motors. First, strain-dependent kinetics are necessary to couple mechanochemical cycles between myosins. Second, multiple power stroke states further enhance a chance of power stroke synchronization. Finally, the physiological ATP concentration is another important factor to enhance a chance of power stroke synchronization, since the strain-dependent transitions accompanied by the first or second power stroke are the rate limiting steps at higher ATP concentrations. Consequently, our computational model predicts that most of steps were generated by synchronous executions of power strokes between several myosin motors at 1 mM ATP, while they are generated primarily by single myosins. Thus, ensemble average curves of steps obtained from our model were distinctively different between 1 mM and 10 μM ATP. These results were consistent with experimental results, supporting our conclusions.

Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms.

The γ subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different γ isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the γ1, γ2 or γ3 isoform. When assayed at a physiological ATP concentration (5 mM), γ1- and γ2-containing complexes were allosterically activated almost 10-fold by AMP, with EC50 values one to two orders of magnitude lower than the ATP concentration. By contrast, γ3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr(172) phosphorylation, when cells were incubated with mitochondrial inhibitors that increase cellular AMP. With γ1 complexes, activation and Thr(172) phosphorylation induced by the upstream kinase LKB1 [liver kinase B1; but not calmodulin-dependent kinase kinase (CaMKKβ)] in cell-free assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the γ2 and γ3 complexes were small or insignificant. Binding of AMP or ADP protected all three γ subunit complexes against inactivation by Thr(172) dephosphorylation; with γ2 complexes, ADP had similar potency to AMP, but with γ1 and γ3 complexes, ADP was less potent than AMP. Thus, AMPK complexes containing different γ subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles.

Abstract B029: The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment

Abstract Introduction: The phosphoinositide-3-kinase (PI3K) lipid kinases are a family of kinase isoforms that transduce signals in response to various stimuli in different cell types. The PI3K-γ isoform is expressed in immune cells and has limited, if any, expression in epithelial cancer cells. Genetic deletion and kinase-dead knock-in studies highlight a key role for PI3K-γ in the development and function of myeloid-derived cells that constitute a key component of the immune suppressive tumor microenvironment (Joshi Mol Canc Res 2014; Schmid Canc Cell 2011). Targeting PI3K-γ in these tumor-associated myeloid cells could therefore inhibit the immune suppressive tumor microenvironment, enabling the immune system to attack tumor cells more effectively. To date, potent and selective PI3K-γ inhibitors with drug-like properties have not been available to test this hypothesis. We now report the structure, biochemical, cellular, and in vivo properties of a potent and selective, small molecule inhibitor of PI3K-γ, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. Results: Discovery efforts identified a highly selective inhibitor of PI3K-γ, IPI-549, with pharmaceutical properties suitable for further development. Binding studies with IPI-549 revealed a KD value of 0.29 nM for PI3K-γ with >58-fold weaker binding affinity for the other Class I PI3K isoforms. Enzymatic assays utilizing physiological ATP concentrations (3 mM) confirmed the selectivity of IPI-549 for PI3K-γ (>200-fold) over other Class I PI3K isoforms. Cellular assays designed to assess individual Class I PI3K isoform activity demonstrated that IPI-549 is highly potent and specific for PI3K-γ (IC50 of 1.2 nM; >140-fold selectivity). Further selectivity screening revealed that IPI-549 is selective for PI3K-γ over other protein and lipid kinases, receptors, ion channels, and transporters. In vitro functional assays demonstrated that IPI-549 blocked bone marrow derived M2 murine macrophage polarization in response to IL-4 and MCSF1, but did not inhibit ConA-induced T-cell activation. These data indicate the potential for IPI-549 to block immune suppressive macrophage development but not T-cell activity. Pharmacokinetic studies in mice demonstrated IPI-549 to be orally bioavailable with a long plasma half-life enabling selective inhibition of the PI3K-γ isoform relative to the other Class I PI3K isoforms. In an in vivo PI3K-γ-dependent neutrophil migration murine model, IPI-549 blocked neutrophil migration in a dose dependent manner. To evaluate the effect of PI3K-γ inhibition on tumor growth in an immunocompetent animal, IPI-549 was tested in murine syngeneic solid tumor models. Mice treated with IPI-549 demonstrated significant tumor growth inhibition in multiple syngeneic models. Studies to elucidate the mechanism of tumor growth inhibition indicated that IPI-549 affects immune suppressive myeloid cell numbers and/or function, leading to an increase in cytotoxic T-cell activity. Studies in nude or CD8 T-cell depleted mice demonstrated the T-cell dependence of IPI-549 mediated tumor growth inhibition. Finally, in vivo studies with IPI-549 in combination with immune checkpoint inhibitors showed increased tumor growth inhibition compared to either monotherapy. Conclusions: IPI-549 is a potent and selective inhibitor of PI3K-γ with pharmaceutical properties that allow for the selective inhibition of PI3K-γ in vivo. Our findings provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit solid tumor growth in preclinical models. IND-enabling studies with IPI-549 are ongoing to support its initial clinical exploration in the setting of solid tumors. Citation Format: Jeffery Kutok, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Karen McGovern, Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Katherine Walsh, Kerry White, David Winkler, Vito Palombella. The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B029.

Harmonic force spectroscopy measures load-dependent kinetics of individual human β-cardiac myosin molecules.

Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using ‘harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's performance by measuring the force-dependent kinetics of individual human β-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule's ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

Myosin VI deafness mutation prevents the initiation of processive runs on actin

Mutations in the reverse-direction myosin, myosin VI, are associated with deafness in humans and mice. A myosin VI deafness mutation, D179Y, which is in the transducer of the motor, uncoupled the release of the ATP hydrolysis product, inorganic phosphate (Pi), from dependency on actin binding and destroyed the ability of single dimeric molecules to move processively on actin filaments. We observed that processive movement is rescued if ATP is added to the mutant dimer following binding of both heads to actin in the absence of ATP, demonstrating that the mutation selectively destroys the initiation of processive runs at physiological ATP levels. A drug (omecamtiv) that accelerates the actin-activated activity of cardiac myosin was able to rescue processivity of the D179Y mutant dimers at physiological ATP concentrations by slowing the actin-independent release of Pi. Thus, it may be possible to create myosin VI-specific drugs that rescue the function of deafness-causing mutations.