Search

Development of Grb2–SH2 domain antagonists is considered to be an effective and non-cytotoxic strategy to develop new antiproliferative agents because of their potential to shut down the Ras signaling pathway. We developed a concise route for the efficient synthesis of G1TE analogs on solid phase. Using this route, a series of cyclic peptides that do not rely on phosphotyrosine or its mimics were designed and synthesized based upon the phage library-derived cyclopeptide, G1TE. Considering that Gly7 plays prominent roles for G1TE binding to the Grb2–SH2 domain, we introduced different amino acids in the 7th position. The d -Ala7-containing peptide 3 demonstrates improved binding affinity by adopting favorable conformation for protein binding. This can be rationalized by molecular modeling. The optimization at the Leu2 position was also studied, and the resulting cyclopeptides exhibited remarkably improved binding affinity. Based upon these global modifications, a highly potent peptide ligand 9 was discovered with a Kd = 17 nM, evaluated by Biacore binding assay. This new analog is one of the most potent non-phosphorus-containing Grb2–SH2 antagonists reported to date. This potent peptidomimetic provides a new template for the development of non-pTyr containing Grb2–SH2 domain antagonists and acts as a chemotherapeutic lead for the treatment of erbB2-related cancer.

The growth factor receptor-bound protein 2 (Grb2) is an SH2 domain-containing docking module that participates in the signaling of numerous oncogenic growth factor receptor protein-tyrosine kinases (PTKs). Presented herein is a 5-methylindolyl-containing macrocyclic tetrapeptide mimetic (5) that binds to Grb2 SH2 domain protein with K(d)=75 pM. This represents the highest affinity yet reported for a synthetic inhibitor against any SH2 domain. In whole cell assays this novel analogue is able to effectively block the association of Grb2 to cognate cytoplasmic erbB-2 at IC(50)10nM without prodrug derivatization or the addition of carrier peptide motifs. Anti-mitogenic effects against erbB-2-dependent breast cancers are achieved at non-cytotoxic concentrations (IC(50)=0.6 microM). Macrocycle 5 may be representative of a new class of therapeutically relevant Grb2 SH2 domain-directed agents.

The crucial functions of HIV-1 nucleocapsid-p7 protein (NC-p7) at different stages of HIV replication are dependent on its nucleic acid binding properties. In this study, a search has been made to identify antagonists of the interaction between NC-p7 and d(TG)(4). A chemical library of approximately 2000 small molecules (the NCI Diversity Set) was screened, of the 26 active inhibitors that were identified, five contained a xanthenyl ring structure. Further analysis of 63 structurally related compounds led to the identification of 2,3,4,5-tetrachloro-6-(4('),5('),6(')-trihydroxy-3(')-oxo-3H-xanthen-9(')-yl)benzoic acid, which binds to NC-p7 stoichiometrically. This compound exerted a significant anti-HIV activity in vitro with an IC(50) of 16.6+/-4.3 microM (means+/-SD). Synthetic variants lacking the two hydroxyls at positions 4(') and 5(') in the xanthenyl ring system failed to bind NC-p7 and showed significantly less protection against HIV infection. Molecular modeling predicts that these hydroxyl groups would bind to the amide nitrogen of Gly(35) with other contacts at the carbonyl oxygens of Gly(40) and Lys(33).

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of Escherichia coli by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of E. coli , was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD+ and NADP+, but NADPH served to accelerate the rate of inhibition.

Summary The stimulation of the energy dependent transhydrogenase (reduction of NADP by NADH) driven by oxidative energy was linear with oligomycin in the ammonia-EDTA depleted (AE) submitochondrial particle but sigmoidal in the more severely depleted ammonia-Sephadex-urea (ASU) and silicotungstate (STA) particles. The optimal activities were quite high even in the more depleted particles. The mitochondrial energy transfer factor C (Factor C) stimulated this activity of the AE particle alone and in combination with oligomycin. Oligomycin also induced substantial activity in the ASU particle in NAD reduction by succinate driven by ascorbatetetramethyl-p-phenylenediamine oxidation in the presence of antimycin. The results show that these particles are not significantly depleted in components involved in the production of the non-phosphorylated high energy intermediate from respiration.

The aerobic and anaerobic energy linked transhydrogenase reactions have been demonstrated in membrane preparations from E. , coli . The aerobic transhydrogenase activity could be enhanced by an energy transfer factor prepared from rat-liver mitochondria. These reactions were sensitive to 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole, an uncoupler of oxidative phosphorylation.

The ATP-Pi exchange activity of highly purified preparations of ‘ATP synthetase’ was inhibited by F1-antiserum, Pullman inhibitor, azide and also by avidin (See You and Hatefi, 1973) . The inhibition produced by the first three was relieved in the presence of ADP, and the avidin sensitivity was lost on pretreatment on the avidin with biotin. It is concluded that the ATP-Pi exchange resulted from the combined action of Factor A and a contaminating avidin-sensitive enzyme. The ADP necessary for the exchange reaction catalyzed by the latter was generated by the ATPase activity of Factor A.