14 research outputs found
Small molecule inhibitors of PSD95-nNOS protein-protein interactions as novel analgesics.
Aberrant increases in NMDA receptor (NMDAR) signaling contributes to central nervous system sensitization and chronic pain by activating neuronal nitric oxide synthase (nNOS) and generating nitric oxide (NO). Because the scaffolding protein postsynaptic density 95kDA (PSD95) tethers nNOS to NMDARs, the PSD95-nNOS complex represents a therapeutic target. Small molecule inhibitors IC87201 (EC5O: 23.94 µM) and ZL006 (EC50: 12.88 µM) directly inhibited binding of purified PSD95 and nNOS proteins in AlphaScreen without altering binding of PSD95 to ErbB4. Bot
Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s)
ACKNOWLEDGMENTS The work was supported by National Institutes of Health grants DA027113 and EY024717 to G.A.T. and DA09158 to A.M. A portion of this work was submitted in 2011 by A. Kulkarni in partial fulfillment of M.S. degree requirements from Northeastern University, Boston, MA.Peer reviewedPublisher PD
Stability analysis of the GAL regulatory network in Saccharomyces cerevisiae and Kluyveromyces lactis
<p>Abstract</p> <p>Background</p> <p>In the yeast <it>Saccharomyces cerevisiae</it>, interactions between galactose, Gal3p, Gal80p, and Gal4p determine the transcriptional status of the genes required for the galactose utilization. Increase in the cellular galactose concentration causes the galactose molecules to bind onto Gal3p which, via Gal80p, activates Gal4p, which induces the GAL3 and GAL80 gene transcription. Recently, a linear time-invariant multi-input multi-output (MIMO) model of this GAL regulatory network has been proposed; the inputs being galactose and Gal4p, and the outputs being the active Gal4p and galactose utilization. Unfortunately, this model assumes the cell culture to be homogeneous, although it is not so in practice. We overcome this drawback by including more biochemical reactions, and derive a quadratic ordinary differential equation (ODE) based model.</p> <p>Results</p> <p>We show that the model, referred to above, does not exhibit bistability. We establish sufficiency conditions for the domain of attraction of an equilibrium point of our ODE model for the special case of full-state feedback controller. We observe that the GAL regulatory system of <it>Kluyveromyces lactis </it>exhibits an aberration of monotone nonlinearity and apply the Rantzer multipliers to establish a class of stabilizing controllers for this system.</p> <p>Conclusion</p> <p>Feedback in a GAL regulatory system can be used to enhance the cellular memory. We show that the system can be modeled as a quadratic nonlinear system for which the effect of feedback on the domain of attraction of the equilibrium point can be characterized using <it>linear matrix inequality </it>(LMI) conditions that are easily implementable in software. The benefit of this result is that a mathematically sound approach to the synthesis of full-state and partial-state feedback controllers to regulate the cellular memory is now possible, irrespective of the number of state-variables or parameters of interest.</p
Allosteric Cannabinoid Receptor 1 (CB1) Ligands Reduce Ocular Pain and Inflammation
Cannabinoid receptor 1 (CB1) activation has been reported to reduce transient receptor potential cation channel subfamily V member 1 (TRPV1)-induced inflammatory responses and is anti-nociceptive and anti-inflammatory in corneal injury. We examined whether allosteric ligands, can modulate CB1 signaling to reduce pain and inflammation in corneal hyperalgesia. Corneal hyperalgesia was generated by chemical cauterization of cornea in wildtype and CB2 knockout (CB2−/−) mice. The novel racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229 were examined alone or in combination with the orthosteric CB1 agonist Δ8-tetrahydrocannabinol (Δ8-THC). Pain responses were assessed following capsaicin (1 µM) stimulation of injured corneas at 6 h post-cauterization. Corneal neutrophil infiltration was also analyzed. GAT228, but not GAT229 or GAT211, reduced pain scores in response to capsaicin stimulation. Combination treatments of 0.5% GAT229 or 1% GAT211 with subthreshold Δ8-THC (0.4%) significantly reduced pain scores following capsaicin stimulation. The anti-nociceptive effects of both GAT229 and GAT228 were blocked with CB1 antagonist AM251, but remained unaffected in CB2−/− mice. Two percent GAT228, or the combination of 0.2% Δ8-THC with 0.5% GAT229 also significantly reduced corneal inflammation. CB1 allosteric ligands could offer a novel approach for treating corneal pain and inflammation
Enantiomer-specific positive allosteric modulation of CB1 signaling in autaptic hippocampal neurons
Research was supported by NIH grants DA-009789 (RR), DA-026449 (RR), DA-003672 (RR), CIHR Proof of Principal Phase 1 grant 288645 (RR), EY24625 (AS), EY24717 (GT & AS), DA011322 (KM) and DA021696 (KM).Peer reviewedPostprin
Mapping Cannabinoid 1 Receptor Allosteric Site(s): Critical Molecular Determinant and Signaling Profile of GAT100, a Novel, Potent, and Irreversibly Binding Probe
One of the most abundant
G-protein coupled receptors (GPCRs) in
brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic
target for treating diverse psychobehavioral and somatic disorders.
Adverse on-target effects associated with small-molecule CB1R orthosteric
agonists and inverse agonists/antagonists have plagued their translational
potential. Allosteric CB1R modulators offer a potentially safer modality
through which CB1R signaling may be directed for therapeutic benefit.
Rational design of candidate, druglike CB1R allosteric modulators
requires greater understanding of the architecture of the CB1R allosteric
endodomain(s) and the capacity of CB1R allosteric ligands to tune
the receptor’s information output. We have recently reported
the synthesis of a focused library of rationally designed, covalent
analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative
allosteric modulators (NAMs). Among the novel, pharmacologically active
CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead
by virtue of its exceptional potency in the [<sup>35</sup>S]GTPγS
and β-arrestin signaling assays and its ability to label CB1R
as a covalent allosteric probe with significantly reduced inverse
agonism in the [<sup>35</sup>S]GTPγS assay as compared to Org27569.
We report here a comprehensive functional profiling of GAT100 across
an array of important downstream cell-signaling pathways and analysis
of its potential orthosteric probe-dependence and signaling bias.
The results demonstrate that GAT100 is a NAM of the orthosteric CB1R
agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and
anandamide for β-arrestin1 recruitment, PLCβ3 and ERK1/2
phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A
cells overexpressing CB1R and in Neuro2a and ST<i>Hdh</i><sup>Q7/Q7</sup> cells endogenously expressing CB1R. Distinctively,
GAT100 was a more potent and efficacious CB1R NAM than Org27569 and
PSNCBAM-1 in all signaling assays and did not exhibit the inverse
agonism associated with Org27569 and PSNCBAM-1. Computational docking
studies implicate C7.38(382) as a key feature of GAT100 ligand-binding
motif. These data help inform the engineering of newer-generation,
druggable CB1R allosteric modulators and demonstrate the utility of
GAT100 as a covalent probe for mapping structure–function correlates
characteristic of the druggable CB1R allosteric space