Activating point mutations in the MET kinase domain represent a unique molecular subset of lung cancer and other malignancies targetable with MET inhibitors.

Activating point mutations in the MET tyrosine kinase domain (TKD) are oncogenic in a subset of papillary renal cell carcinomas (PRCC). Here, using comprehensive genomic profiling among >600,000 patients, we identify activating MET TKD point mutations as putative oncogenic driver across diverse cancers, with a frequency of ~0.5%. The most common mutations in the MET TKD defined as oncogenic or likely oncogenic according to OncoKB resulted in amino acid substitutions at positions H1094, L1195, F1200, D1228, Y1230, M1250, and others. Preclinical modeling of these alterations confirmed their oncogenic potential, and also demonstrated differential patterns of sensitivity to type I and type II MET inhibitors. Two patients with metastatic lung adenocarcinoma harboring MET TKD mutations (H1094Y, F1200I) and no other known oncogenic drivers achieved confirmed partial responses to a type I MET inhibitor. Activating MET TKD mutations occur in multiple malignancies and may confer clinical sensitivity to currently available MET inhibitors.

PI(4,5)P2 and Cholesterol: Synthesis, Regulation, and Functions.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is the most abundant membrane phosphoinositide and cholesterol is an essential component of the plasma membrane (PM). Both lipids play key roles in a variety of cellular functions including as signaling molecules and major regulators of protein function. This chapter provides an overview of these two important lipids. Starting from a brief description of their structure, synthesis, and regulation, the chapter continues to describe the primary functions and signaling processes in which PI(4,5)P2 and cholesterol are involved. While PI(4,5)P2 and cholesterol can act independently, they often act in concert or affect each other's impact. The chapters in this volume on "Cholesterol and PI(4,5)P2 in Vital Biological Functions: From Coexistence to Crosstalk" focus on the emerging relationship between cholesterol and PI(4,5)P2 in a variety of biological systems and processes. In this chapter, the next section provides examples from the ion channel field demonstrating that PI(4,5)P2 and cholesterol can act via common mechanisms. The chapter ends with a discussion of future directions.

Protein Binding Pocket Optimization for Virtual High-Throughput Screening (vHTS) Drug Discovery.

The virtual high-throughput screening (vHTS) approach has been widely used for large database screening to identify potential lead compounds for drug discovery. Due to its high computational demands, docking that allows receptor flexibility has been a challenging problem for virtual screening. Therefore, the selection of protein target conformations is crucial to produce useful vHTS results. Since only a single protein structure is used to screen large databases in most vHTS studies, the main challenge is to reduce false negative rates in selecting compounds for in vitro tests. False negatives are most likely to occur when using apo structures or homology models of protein targets due to the small volume of the binding pocket formed by incorrect side-chain conformations. Even holo protein structures can exhibit high false negative rates due to ligand-induced fit effects, since the shape of the binding pocket highly depends on its bound ligand. To reduce false negative rates and improve success rates for vHTS in drug discovery, we have developed a new Monte Carlo-based approach that optimizes the binding pocket of protein targets. This newly developed Monte Carlo pocket optimization (MCPO) approach was assessed on several datasets showing promising results. The binding pocket optimization approach could be a useful tool for vHTS-based drug discovery, especially in cases when only apo structures or homology models are available.

The small molecule GAT1508 activates brain-specific GIRK1/2 channel heteromers and facilitates conditioned fear extinction in rodents.

G-protein-gated inwardly-rectifying K+ (GIRK) channels are targets of Gi/o-protein-signaling systems that inhibit cell excitability. GIRK channels exist as homotetramers (GIRK2 and GIRK4) or heterotetramers with nonfunctional homomeric subunits (GIRK1 and GIRK3). Although they have been implicated in multiple conditions, the lack of selective GIRK drugs that discriminate among the different GIRK channel subtypes has hampered investigations into their precise physiological relevance and therapeutic potential. Here, we report on a highly-specific, potent, and efficacious activator of brain GIRK1/2 channels. Using a chemical screen and electrophysiological assays, we found that this activator, the bromothiophene-substituted small molecule GAT1508, is specific for brain-expressed GIRK1/2 channels rather than for cardiac GIRK1/4 channels. Computational models predicted a GAT1508-binding site validated by experimental mutagenesis experiments, providing insights into how urea-based compounds engage distant GIRK1 residues required for channel activation. Furthermore, we provide computational and experimental evidence that GAT1508 is an allosteric modulator of channel-phosphatidylinositol 4,5-bisphosphate interactions. Through brain-slice electrophysiology, we show that subthreshold GAT1508 concentrations directly stimulate GIRK currents in the basolateral amygdala (BLA) and potentiate baclofen-induced currents. Of note, GAT1508 effectively extinguished conditioned fear in rodents and lacked cardiac and behavioral side effects, suggesting its potential for use in pharmacotherapy for post-traumatic stress disorder. In summary, our findings indicate that the small molecule GAT1508 has high specificity for brain GIRK1/2 channel subunits, directly or allosterically activates GIRK1/2 channels in the BLA, and facilitates fear extinction in a rodent model.

On the mechanism of GIRK2 channel gating by phosphatidylinositol bisphosphate, sodium, and the Gßγ dimer.

G protein-gated inwardly rectifying K+ (GIRK) channels belong to the inward-rectifier K+ (Kir) family, are abundantly expressed in the heart and the brain, and require that phosphatidylinositol bisphosphate is present so that intracellular channel-gating regulators such as Gßγ and Na+ ions can maintain the channel-open state. However, despite high-resolution structures (GIRK2) and a large number of functional studies, we do not have a coherent picture of how Gßγ and Na+ ions control gating of GIRK2 channels. Here, we utilized computational modeling and all-atom microsecond-scale molecular dynamics simulations to determine which gates are controlled by Na+ and Gßγ and how each regulator uses the channel domain movements to control gate transitions. We found that Na+ ions control the cytosolic gate of the channel through an anti-clockwise rotation, whereas Gßγ stabilizes the transmembrane gate in the open state through a rocking movement of the cytosolic domain. Both effects alter the way in which the channel interacts with phosphatidylinositol bisphosphate and thereby stabilizes the open states of the respective gates. These studies of GIRK channel dynamics present for the first time a comprehensive structural model that is consistent with the great body of literature on GIRK channel function.

A V-to-F substitution in SK2 channels causes Ca2+ hypersensitivity and improves locomotion in a C. elegans ALS model.

Small-conductance Ca2+-activated K+ (SK) channels mediate medium afterhyperpolarization in the neurons and play a key role in the regulation of neuronal excitability. SK channels are potential drug targets for ataxia and Amyotrophic Lateral Sclerosis (ALS). SK channels are activated exclusively by the Ca2+-bound calmodulin. Previously, we identified an intrinsically disordered fragment that is essential for the mechanical coupling between Ca2+/calmodulin binding and channel opening. Here, we report that substitution of a valine to phenylalanine (V407F) in the intrinsically disordered fragment caused a ~6 fold increase in the Ca2+ sensitivity of SK2-a channels. This substitution resulted in a novel interaction between the ectopic phenylalanine and M411, which stabilized PIP2-interacting residue K405, and subsequently enhanced Ca2+ sensitivity. Also, equivalent valine to phenylalanine substitutions in SK1 or SK3 channels conferred Ca2+ hypersensitivity. An equivalent phenylalanine substitution in the Caenorhabditis elegans (C. elegans) SK2 ortholog kcnl-2 partially rescued locomotion defects in an existing C. elegans ALS model, in which human SOD1G85R is expressed at high levels in neurons, confirming that this phenylalanine substitution impacts channel function in vivo. This work for the first time provides a critical reagent for future studies: an SK channel that is hypersensitive to Ca2+ with increased activity in vivo.

Molecular determinants of Kv7.1/KCNE1 channel inhibition by amitriptyline.

Amitriptyline (AMIT) is a compound widely prescribed for psychiatric and non-psychiatric conditions including depression, migraine, chronic pain, and anorexia. However, AMIT has been associated with risks of cardiac arrhythmia and sudden death since it can induce prolongation of the QT interval on the surface electrocardiogram and torsade de pointes ventricular arrhythmia. These complications have been attributed to the inhibition of the rapid delayed rectifier potassium current (IKr). The slow delayed rectifier potassium current (IKs) is the main repolarizing cardiac current when IKr is compromised and it has an important role in cardiac repolarization at fast heart rates induced by an elevated sympathetic tone. Therefore, we sought to characterize the effects of AMIT on Kv7.1/KCNE1 and homomeric Kv7.1 channels expressed in HEK-293H cells. Homomeric Kv7.1 and Kv7.1/KCNE1 channels were inhibited by AMIT in a concentration-dependent manner with IC50 values of 8.8 ±â€¯2.1 µM and 2.5 ±â€¯0.8 µM, respectively. This effect was voltage-independent for both homomeric Kv7.1 and Kv7.1/KCNE1 channels. Moreover, mutation of residues located on the P-loop and S6 domain along with molecular docking, suggest that T312, I337 and F340 are the most important molecular determinants for AMIT-Kv7.1 channel interaction. Our experimental findings and modeling suggest that AMIT preferentially blocks the open state of Kv7.1/KCNE1 channels by interacting with specific residues that were previously reported to be important for binding of other compounds, such as chromanol 293B and the benzodiazepine L7.

Synthetic Aß peptides acquire prion-like properties in the brain.

In transmission studies with Alzheimer's disease (AD) animal models, the formation of Aß plaques is proposed to be initiated by seeding the inoculated amyloid ß (Aß) peptides in the brain. Like the misfolded scrapie prion protein (PrPSc) in prion diseases, Aß in AD shows a certain degree of resistance to protease digestion while the biochemical basis for protease resistance of Aß remains poorly understood. Using in vitro assays, histoblotting, and electron microscopy, we characterize the biochemical and morphological features of synthetic Aß peptides and Aß isolated from AD brain tissues. Consistent with previous observations, monomeric and oligomeric Aß species extracted from AD brains are insoluble in detergent buffers and resistant to digestions with proteinase K (PK). Histoblotting of AD brain tissue sections exhibits an increased Aß immunoreactivity after digestion with PK. In contrast, synthetic Aß40 and Aß42 are soluble in detergent buffers and fully digested by PK. Electron microscopy of Aß40 and Aß42 synthetic peptides shows that both species of Aß form mature fibrils. Those generated from Aß40 are longer but less numerous than those made of Aß42. When spiked into human brain homogenates, both Aß40 and Aß42 acquire insolubility in detergent and resistance to PK. Our study favors the hypothesis that the human brain may contain cofactor(s) that confers the synthetic Aß peptides PrPSc-like physicochemical properties.

Comparative Study of Prions in Iatrogenic and Sporadic Creutzfeldt-Jakob Disease.

Differentiating iatrogenic Creutzfeldt-Jakob disease (iCJD) from sporadic CJD (sCJD) would be useful for the identification and prevention of human-to-human prion transmission. Currently, the diagnosis of iCJD depends on identification of a recognized source of contamination to which patients have been exposed, in addition to fulfilling basic requirements for the establishment of diagnosis of CJD. Attempts to identify differences in clinical manifestations, neuropathological changes and pathological prion protein (PrPSc) between iCJD and sCJD have been unsuccessful. In the present study, using a variety of more sophisticated methods including sucrose step gradient sedimentation, conformational stability immunoassay, protein misfolding cyclic amplification (PMCA), fragment-mapping, and transmission study, we show no significant differences in gel profiles, oligomeric state, conformational stability and infectivity of PrPSc between iCJD and sCJD. However, using PMCA, we find that convertibility and amplification efficiency of PrPSc is greater in iCJD than in sCJD in a polymorphism-dependent manner. Moreover, two protease-resistant PrP C-terminal fragments (termed PrP-CTF12/13) were detected in all 9 cases of sCJD but not in 6 of 8 cases of iCJD tested in this study. The use of fragment mapping- and PMCA-based assays thus provides a means to distinguish most cases of iCJD from sCJD.