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.

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.

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 up-regulation of the non-muscle isoform, l-CaD, in visceral, but not vascular tonic smooth muscles. While the Ca2+-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 cross-bridges. 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 cross-bridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.

The primary myosin-binding site of caldesmon was thought to be in the N-terminal region of the molecule, but the exact nature of the caldesmon-myosin interaction has not been well characterized. A caldesmon fragment that encompasses residues 1-240 (N240) was found to bind full-length smooth muscle myosin on the basis of co-sedimentation experiments. The interaction between myosin and N240 was not affected by phosphorylation of myosin, but it was weakened by the presence of Ca(2+)/calmodulin. To locate the myosin-binding site, we have designed several synthetic peptides based on the N-terminal caldesmon sequence. We found that a peptide stretch corresponding to the first 27 residues (Met-1 to Tyr-27), but not that of the first 22 residues (Met-1 to Ala-22), exhibited a moderate affinity toward myosin. We also found that a peptide containing the segment from Ile/Leu-25 to Lys-53 bound both myosin and heavy meromyosin more strongly and was capable of displacing caldesmon from myosin. Our results demonstrate that the sequence near the N-terminal extreme of caldesmon harbors a major myosin-binding site of caldesmon, in which both the nonpolar residues and clusters of positively and negatively charged residues confer the specificity and affinity of the caldesmon-myosin interaction.

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.