Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Background

Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results

As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion

Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.     

Synthesis and pharmacology of 1-alkyl-3-(1-naphthoyl)indoles: steric and electronic effects of 4- and 8-halogenated naphthoyl substituents

To develop SAR at both the cannabinoid CB(1) and CB(2) receptors for 3-(1-naphthoyl)indoles bearing moderately electron withdrawing substituents at C-4 of the naphthoyl moiety, 1-propyl and 1-pentyl-3-(4-fluoro, chloro, bromo and iodo-1-naphthoyl) derivatives were prepared. To study the steric and electronic effects of substituents at the 8-position of the naphthoyl group, the 3-(4-chloro, bromo and iodo-1-naphthoyl)indoles were also synthesized. The affinities of both groups of compounds for the CB(1) and CB(2) receptors were determined and several of them were evaluated in vivo in the mouse. The effects of these substituents on receptor affinities and in vivo activity are discussed and structure-activity relationships are presented. Although many of these compounds are selective for the CB(2) receptor, only three JWH-423, 1-propyl-3-(4-iodo-1-naphthoyl)indole, JWH-422, 2-methyl-1-propyl-3-(4-iodo-1-naphthoyl)indole, the 2-methyl analog of JWH-423 and JWH-417, 1-pentyl-3-(8-iodo-1-naphthoyl)indole, possess the desirable combination of low CB(1) affinity and good CB(2) affinity.     

STAT-3 activation is necessary for ischemic preconditioning in hypertrophied myocardium

The JAK-STAT pathway is activated in the early and late phases of ischemic preconditioning (IPC) in normal myocardium. The role of this pathway and the efficacy of IPC in hypertrophied hearts remain largely unknown. We hypothesized that phosphorylated STAT-3 (pSTAT-3) is necessary for effective IPC in pressure-overload hypertrophy. Male Sprague-Dawley rats 8 wk after thoracic aortic constriction (TAC) or sham operation underwent echocardiography and Langendorff perfusion. Randomized hearts were subjected to 30 min of global ischemia and 120 min of reperfusion with or without IPC in the presence or absence of the JAK-2 inhibitor AG-490 (AG). Functional recovery and STAT activation were assessed. TAC rats had a 31% increase in left ventricular mass (1,347 +/- 58 vs. 1,028 +/- 43 mg, TAC vs. sham, P < 0.001), increased anterior and posterior wall thickness but no difference in ejection fraction compared with sham-operated rats. In TAC, IPC improved end-reperfusion maximum first derivative of developed pressure (+dP/dt(max); 4,648 +/- 309 vs. 2,737 +/- 343 mmHg/s, IPC vs. non-IPC, P < 0.05) and minimum -dP/dt (-dP/dt(min); -2,239 +/- 205 vs. -1,215 +/- 149 mmHg/s, IPC vs. non-IPC, P < 0.05). IPC increased nuclear pSTAT-1 and pSTAT-3 in sham-operated rats but only pSTAT-3 in TAC. AG in TAC significantly attenuated +dP/dt(max) (4,648 +/- 309 vs. 3,241 +/- 420 mmHg/s, IPC vs. IPC + AG, P < 0.05) and -dP/dt(min) (-2,239 +/- 205 vs. -1,323 +/- 85 mmHg/s, IPC vs. IPC + AG, P < 0.05) and decreased only nuclear pSTAT-3. In myocardial hypertrophy, JAK-STAT signaling is important in IPC and exhibits a pattern of STAT activation distinct from nonhypertrophied myocardium. Limiting STAT-3 activation attenuates the efficacy of IPC in hypertrophy.     

Synthesis and pharmacology of 11-nor-1-methoxy-9-hydroxyhexahydrocannabinols and 11-nor-1-deoxy-9-hydroxyhexahydrocannabinols: new selective ligands for the cannabinoid CB2 receptor

Fourteen novel CB2 receptor selective cannabinoids were synthesized via initial Lewis acid catalyzed rearrangement of resorcinol precursors to obtain the cannabinoid moiety. These are the 1-methoxy-9-hydroxyhexahydrocannabinols and the 1-deoxy-9-hydroxyhexahydrocannabinols, with 1',1'-dimethylalkyl side chains of four to seven carbon atoms at C-3 of the cannabinoid nucleus. The cannabinols synthesized and described in this paper all exhibit greater affinity for the CB2 receptor than for the CB1 receptor. Exceptionally high CB2 affinity was observed for 1-deoxy-9beta-hydroxy-dimethylhexylhexahydrocannabinol (JWH-361, 9, n = 3) K(i) = 2.7 nM and 1-deoxy-9beta-hydroxydimethylpentylhexahydrocannabinol (JWH-300, 9, n = 2) K(i) = 5.3 nM. In general, the stereochemistry of the 9-hydroxy group is important and the beta-orientation enhances both CB2 receptor affinity and selectivity.     

1-Bromo-3-(1',1'-dimethylalkyl)-1-deoxy-Δ(8)-tetrahydrocannabinols: New selective ligands for the cannabinoid CB(2) receptor

Δ(8)-Tetrahydrocannabinol (26), 3-(1',1'-dimethylbutyl)- (12), 3-(1',1'-dimethylpentyl)- (13), 3-(1',1'-dimethylhexyl)- (14) and 3-(1',1'-dimethylheptyl)-Δ(8)-tetrahydrocannabinol (15) have been converted into the corresponding 1-bromo-1-deoxy-Δ(8)-tetrahydrocannabinols (25, 8-11). This was accomplished using a protocol developed in our laboratory in which the trifluoromethanesulfonate of a phenol undergoes palladium mediated coupling with pinacolborane. Reaction of this dioxaborolane with aqueous-methanolic copper(II) bromide provides the aryl bromide. The affinities of these bromo cannabinoids for the cannabinoid CB(1) and CB(2) receptors were determined. All of these compounds showed selectivity for the CB(2) receptor and one of them, 1-bromo-1-deoxy-3-(1',1'-dimethylhexyl)-Δ(8)-tetrahydrocannabinol (10), exhibits 52-fold selectivity for this receptor with good (28nM) affinity.     

The small regulatory RNA molecule MicA is involved in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium biofilm formation

Background  

LuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules.     

Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

Background  

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters.     

A nucleic acid sequence-based amplification assay for real-time detection of norovirus genogroup II

AIMS: To use molecular beacon based nucleic acid sequence-based amplification (NASBA) to develop a rapid, sensitive, specific detection method for norovirus (NV) genogroupII (GII). METHODS AND RESULTS: A method to detect NV GII from environmental samples using real-time NASBA was developed. This method was routinely sensitive to 100 copies of target RNA and intermittent amplification occurred with as few as 10 copies. Quantitative estimates of viral load were possible over at least four orders of magnitude. CONCLUSIONS: The NASBA method described here is a reliable and sensitive assay for the detection of NV. This method has the potential to be linked to a handheld NASBA device that would make this real-time assay a portable and inexpensive alternative to bench-top, lab-based assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of the real-time NASBA assay described here has resulted in a simple, rapid (<1 h), convenient testing format for NV. To our knowledge, this is the first example of a molecular beacon based NASBA assay for NV.     

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1–5) have high affinity (K_i=<20 nM) for the CB₂ receptor. Four of them (2, n=1–4) also have little affinity for the CB₁ receptor (K_i=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K_i=3.4±1.0 nM) and little affinity for the CB₁ receptor (K_i=677±132 nM).

Scheme 3. (a) (C₆H₅)₃PCH₃⁺ Br⁻, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, −78 to 25°C then H₂O₂/NaOH.     

Quassinoids from the twigs and thorns of Castela polyandra

The structures of six new C20 quassinoids and one new C19 quassinoid, all isolated from the twigs and thorns of Castela polyandra, were established by a combination of spectroscopic and single-crystal X-ray analysis. Five known quassinoids and one known sterol were also identified.