Targeting the XIAP/caspase-7 complex selectively kills caspase-3-deficient malignancies.

Caspase-3 downregulation (CASP3/DR) in tumors frequently confers resistance to cancer therapy and is significantly correlated with a poor prognosis in cancer patients. Because CASP3/DR cancer cells rely heavily on the activity of caspase-7 (CASP7) to initiate apoptosis, inhibition of activated CASP7 (p19/p12-CASP7) by X-linked inhibitor of apoptosis protein (XIAP) is a potential mechanism by which apoptosis is prevented in those cancer cells. Here, we identify the pocket surrounding the Cys246 residue of p19/p12-CASP7 as a target for the development of a protein-protein interaction (PPI) inhibitor of the XIAP:p19/p12-CASP7 complex. Interrupting this PPI directly triggered CASP7-dependent apoptotic signaling that bypassed the activation of the apical caspases and selectively killed CASP3/DR malignancies in vitro and in vivo without adverse side effects in nontumor cells. Importantly, CASP3/DR combined with p19/p12-CASP7 accumulation correlated with the aggressive evolution of clinical malignancies and a poor prognosis in cancer patients. Moreover, targeting of this PPI effectively killed cancer cells with multidrug resistance due to microRNA let-7a-1-mediated CASP3/DR and resensitized cancer cells to chemotherapy-induced apoptosis. These findings not only provide an opportunity to treat CASP3/DR malignancies by targeting the XIAP:p19/p12-CASP7 complex, but also elucidate the molecular mechanism underlying CASP3/DR in cancers.

Fluorescent substrate analog for monitoring chain elongation by undecaprenyl pyrophosphate synthase in real time.

Farnesyl pyrophosphate (FPP) is a common substrate for a variety of prenyltransferases for synthesizing isoprenoid compounds. In this study, (2E,6E)-8-O-(N-methyl-2-aminobenzoyl)-3,7-dimethyl-2,6-octandien-1-pyrophosphate (MANT-O-GPP), a fluorescent analog of FPP, was synthesized and demonstrated as a satisfactory substrate for Escherichia coli undecaprenyl pyrophosphate synthase (UPPS) with a K(m) of 1.5 µM and a k(cat) of 1.2s(-1) based on [(14)C]IPP consumption. Interesting, we found that its emission fluorescence intensity at 420 nm increased remarkably during chain elongation, thereby useful for real-time monitoring kinetics of UPPS to yield a K(m) of 1.1 µM and a k(cat) of 1.0 s(-1), consistent with those measured using radiolabeled substrate. Using this assay, the IC(50) of a known UPPS inhibitor farnesyl thiopyrophosphate (FsPP) was confirmed. Our studies provide a convenient and environmentally friendly alternative for kinetics and inhibition studies on UPPS drug target.

Mechanism of cis-prenyltransferase reaction probed by substrate analogues.

Undecaprenyl pyrophosphate synthase (UPPS) is a cis-type prenyltransferases which catalyzes condensation reactions of farnesyl diphosphate (FPP) with eight isopentenyl pyrophosphate (IPP) units to generate C(55) product. In this study, we used two analogues of FPP, 2-fluoro-FPP and [1,1-(2)H(2)]FPP, to probe the reaction mechanism of Escherichia coli UPPS. The reaction rate of 2-fluoro-FPP with IPP under single-turnover condition is similar to that of FPP, consistent with the mechanism without forming a farnesyl carbocation intermediate. Moreover, the deuterium secondary KIE of 0.985±0.022 measured for UPPS reaction using [1,1-(2)H(2)]FPP supports the associative transition state. Unlike the sequential mechanism used by trans-prenyltransferases, our data demonstrate E. coli UPPS utilizes the concerted mechanism.

Intracellular beta-tubulin/chaperonin containing TCP1-beta complex serves as a novel chemotherapeutic target against drug-resistant tumors.

In the present study, treatment of HEK-293 cells with the synthetic small molecule N-iodoacetyl-tryptophan (I-Trp) at submicromolar concentrations efficiently induced cell apoptosis as judged from the accumulation of sub-G(0) cells and intracellular DNA fragmentation. Activation of all intracellular caspases, except caspase-1, was detected in I-Trp-treated cells. Proteomic analysis revealed that beta-tubulin acted as a specific intracellular target of I-Trp. Protein fingerprinting analysis indicated that the Cys(354) residue in the peptide fragment TAVCDIPPR of beta-tubulin, which is located at the binding interface with chaperonin containing TCP1-beta (CCT-beta), was alkylated by I-Trp. Moreover, site-directed mutagenesis of Cys(354) (Cys-Ala) abolished the incorporation of I-Trp into beta-tubulin, suggesting Cys(354) is indeed the targeting site of I-Trp. Immunoprecipitation showed that the beta-tubulin/CCT-beta complex was constitutively formed but disrupted after treatment with I-Trp. Overexpression of the truncated beta-tubulin (T351-S364) or treatment with I-Trp or the synthetic peptide Myr-TAVCDIPPRG caused more severe cell apoptosis in multidrug-resistant MES-SA/Dx5 cancer cells due to higher levels of CCT-beta relative to wild-type MES-SA cancer cells. Silencing the expression of CCT-beta rendered MES-SA/Dx5 cells less sensitive to I-Trp-induced apoptotic cell death. These findings suggest that the beta-tubulin/CCT-beta complex may serve as an effective chemotherapeutic target for treating clinical tubulin-binding agent-resistant or CCT-beta-overexpressing tumors.