Structure of the MRAS-SHOC2-PP1C phosphatase complex.

RAS-MAPK signalling is fundamental for cell proliferation and is altered in most human cancers1-3. However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF-MEK1-14-3-3 complexes4, the intermediate steps that lead to RAF activation remain unclear. The MRAS-SHOC2-PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF-RAS association3,5,6. MRAS, SHOC2 and PP1C are mutated in rasopathies-developmental syndromes caused by aberrant MAPK pathway activation6-14-and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)-RAS-driven tumours15-18. Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS-SHOC2-PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS-GTP molecules recruit RAF-14-3-3 and SHOC2-PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein-protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS-MAPK signalling, therefore providing the structural basis for therapeutic interventions.

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor's biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

Optimization of novel monobactams with activity against carbapenem-resistant Enterobacteriaceae - Identification of LYS228.

Metallo-ß-lactamases (MBLs), such as New Delhi metallo-ß-lactamase (NDM-1) have spread world-wide and present a serious threat. Expression of MBLs confers resistance in Gram-negative bacteria to all classes of ß-lactam antibiotics, with the exception of monobactams, which are intrinsically stable to MBLs. However, existing first generation monobactam drugs like aztreonam have limited clinical utility against MBL-expressing strains because they are impacted by serine ß-lactamases (SBLs), which are often co-expressed in clinical isolates. Here, we optimized novel monobactams for stability against SBLs, which led to the identification of LYS228 (compound 31). LYS228 is potent in the presence of all classes of ß-lactamases and shows potent activity against carbapenem-resistant isolates of Enterobacteriaceae (CRE).

A NMDA-receptor calcium influx assay sensitive to stimulation by glutamate and glycine/D-serine.

N-methyl-D-aspartate-receptors (NMDARs) are ionotropic glutamate receptors that function in synaptic transmission, plasticity and cognition. Malfunction of NMDARs has been implicated in a variety of nervous system disorders, making them attractive therapeutic targets. Overexpression of functional NMDAR in non-neuronal cells results in cell death by excitotoxicity, hindering the development of cell-based assays for NMDAR drug discovery. Here we report a plate-based, high-throughput approach to study NMDAR function. Our assay enables the functional study of NMDARs with different subunit composition after activation by glycine/D-serine or glutamate and hence presents the first plate-based, high throughput assay that allows for the measurement of NMDAR function in glycine/D-serine and/or glutamate sensitive modes. This allows to investigate the effect of small molecule modulators on the activation of NMDARs at different concentrations or combinations of the co-ligands. The reported assay system faithfully replicates the pharmacology of the receptor in response to known agonists, antagonists, positive and negative allosteric modulators, as well as the receptor's sensitivity to magnesium and zinc. We believe that the ability to study the biology of NMDARs rapidly and in large scale screens will enable the identification of novel therapeutics whose discovery has otherwise been hindered by the limitations of existing cell based approaches.

A screening pattern recognition method finds new and divergent targets for drugs and natural products.

Computational target prediction methods using chemical descriptors have been applied exhaustively in drug discovery to elucidate the mechanisms-of-action (MOAs) of small molecules. To predict truly novel and unexpected small molecule-target interactions, compounds must be compared by means other than their chemical structure alone. Here we investigated predictions made by a method, HTS fingerprints (HTSFPs), that matches patterns of activities in experimental screens. Over 1,400 drugs and 1,300 natural products (NPs) were screened in more than 200 diverse assays, creating encodable activity patterns. The comparison of these activity patterns to an MOA-annotated reference panel led to the prediction of 5,281 and 2,798 previously unknown targets for the NP and drug sets, respectively. Intriguingly, there was limited overlap among the targets predicted; the drugs were more biased toward membrane receptors and the NPs toward soluble enzymes, consistent with the idea that they represent unexplored pharmacologies. Importantly, HTSFPs inferred targets that were beyond the prediction capabilities of standard chemical descriptors, especially for NPs but also for the more explored drug set. Of 65 drug-target predictions that we tested in vitro, 48 (73.8%) were confirmed with AC50 values ranging from 38 nM to 29 µM. Among these interactions was the inhibition of cyclooxygenases 1 and 2 by the HIV protease inhibitor Tipranavir. These newly discovered targets that are phylogenetically and phylochemically distant to the primary target provide an explanation for spontaneous bleeding events observed for patients treated with this drug, a physiological effect that was previously difficult to reconcile with the drug's known MOA.

Ablation of smooth muscle caldesmon affects the relaxation kinetics of arterial muscle.

Smooth muscle caldesmon (h-CaD) is an actin- and myosin-binding protein that reversibly inhibits the actomyosin ATPase activity in vitro. To test the function of h-CaD in vivo, we eliminated its expression in mice. The h-CaD-null animals appeared normal and fertile, although the litter size was smaller. Tissues from the homozygotes lacked h-CaD and exhibited upregulation of the non-muscle isoform, l-CaD, in visceral, but not vascular tonic smooth muscles. While the Ca(2+) sensitivity of force generation of h-CaD-deficient smooth muscle remained largely unchanged, the kinetic behavior during relaxation in arteries was different. Both intact and permeabilized arterial smooth muscle tissues from the knockout animals relaxed more slowly than those of the wild type. Since this difference occurred after myosin dephosphorylation was complete, the kinetic effect most likely resulted from slower detachment of unphosphorylated crossbridges. Detailed analyses revealed that the apparently slower relaxation of h-CaD-null smooth muscle was due to an increase in the amplitude of a slower component of the biphasic tension decay. While the identity of this slower process has not been unequivocally determined, we propose it reflects a thin filament state that elicits fewer re-attached crossbridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.

Direct interaction between caldesmon and cortactin.

Actin polymerization and depolymerization plays a central role in controlling a wide spectrum of cellular processes. There are many actin-binding proteins in eukaryotic cells. Their roles in the remodeling of the actin architecture and whether they work cooperatively await further study. Caldesmon (CaD) is an actin-binding protein present in nearly all mammalian cells. Cortactin is another actin-binding protein found mainly in the cell cortex. There have been no reports suggesting that CaD and cortactin interact with each other or work as partners. Here, we present evidence that CaD binds cortactin directly by overlay, pull-down assays, ELISA, and by column chromatography. The interaction involves the N-terminal region of cortactin and the C-terminal region of CaD, and appears to be enhanced by divalent metal ions. Cortactin competes with both full-length CaD and its C-terminal fragment for actin binding. Binding of cortactin partially alleviates the inhibitory effect of CaD on the actomyosin ATPase activity. Not only can binding be demonstrated in vitro, the two proteins also co-localize in activated cells at the cortex. Whether such interactions bear any functional significance awaits further investigation.

Specific disruption of smooth muscle caldesmon expression in mice.

Caldesmon (CaD) is an actin-binding protein that is capable of inhibiting the actomyosin ATPase activity in vitro. CaD has a single gene that is alternatively spliced to generate the smooth muscle-specific form, h-CaD, and a shorter isoform, l-CaD, that is present only in non-muscle cells. The difference between h- and l-CaD is a highly charged repeating sequence, corresponding to a 35 nm-long single helical region that separates the N-terminal domain from the C-terminal domain of h-CaD. To test whether such an elongated h-CaD is essential for smooth muscles to function properly, we have specifically abrogated its expression in the mouse by targeting h-CaD without affecting the expression of l-CaD. After genotyping, we have obtained homozygous knockout mice that indeed lack h-CaD, but nevertheless express varying amounts of l-CaD in a tissue-dependent fashion. The contractility of smooth muscles isolated from the knockout animals is currently under investigation.

Caldesmon binding to actin is regulated by calmodulin and phosphorylation via different mechanisms.

Smooth muscle caldesmon (CaD) binds F-actin and inhibits actomyosin ATPase activity. The inhibition is reversed by Ca2+/calmodulin (CaM). CaD is also phosphorylated upon stimulation at sites specific for mitogen-activated protein kinases (MAPKs). Because of these properties, CaD is thought to be involved in the regulation of smooth muscle contraction. The molecular mechanism of the reversal of inhibition is not well understood. We have expressed His6-tagged fragments containing the sequence of the C-terminal region of human (from M563 to V793) and chicken (from M563 to P771) CaD as well as a variant of the chicken isoform with a Q766C point mutation. By cleavages with proteases, followed by high-speed cosedimentation with F-actin and mass spectrometry, we found that within the C-terminal region of CaD there are multiple actin contact points forming two clusters. Intramolecular fluorescence resonance energy transfer between probes attached to cysteine residues (the endogenous C595 and the engineered C766) located in these two clusters revealed that the C-terminal region of CaD is elongated, but it becomes more compact when bound to actin. Binding of CaM restores the elongated conformation and facilitates dissociation of the C-terminal CaD fragment from F-actin. When the CaD fragment was phosphorylated with a MAPK, only one of the two actin-binding clusters dissociated from F-actin, whereas the other remained bound. Taken together, these results demonstrate that while both Ca2+/CaM and MAPK phosphorylation govern CaD's function via a conformational change, the regulatory mechanisms are different.