Targeting inhibitor of apoptosis proteins (IAPs) for cancer therapy

Since cell death by apoptosis plays a key role in the regulation of tissue homeostasis, dysregulation of the cell's intrinsic death program may foster tumor formation and progression. "Inhibitor of apoptosis proteins" (IAPs) block apoptosis at the core of the apoptotic machinery by inhibiting effector caspases. Aberrant expression and/or function of IAPs are found in many human cancers and have been implied in resistance to current treatment approaches. Recent insights into the role of IAPs have provided the basis for various exciting discoveries that aim at modulating expression or function of IAPs. Thus, targeting IAPs, e.g. by antisense approaches or small molecule inhibitors, presents a promising novel approach for future drug development and may proof to be a successful strategy to overcome apoptosis resistance of human cancers.     

Targeting inhibitor of apoptosis proteins (IAPs) for diagnosis and treatment of human diseases

Since cell death by apoptosis plays a key role in the regulation of tissue homeostasis, any defect in this intrinsic death program may result in tumor formation. "Inhibitor of apoptosis proteins" (IAPs) block apoptosis at the core of the apoptotic machinery by inhibiting effector caspases. Aberrant expression and/or function of IAPs have been implied to be involved in the pathogenesis and progression of various human diseases including cancer, autoimmune disorders or neurodegeneration. Recent insights into the regulation of IAPs have provided the basis for various exciting discoveries aimed at modulating expression or dysfunction of IAPs. Thus, targeting IAPs, e.g. by antisense approaches, RNA interference or small molecules, may proof to be a novel strategy for the diagnosis and treatment of human diseases.     

凋亡抑制蛋白(IAP):抗癌新靶点

凋亡抑制蛋白 (IAP)能够抑制细胞内凋亡作用 ,是与肿瘤发生、发展密切相关的关键分子。近年来 ,以IAP为靶点研制有基因特异性的拮抗剂 ,寻找高效低毒的基因治疗策略倍受关注。IAP家族中survivin及livin特异表达于肿瘤组织的特性以及IAP抑制剂Smac的发现 ,为肿瘤治疗提供了新的发展方向。     

Structure-based design, synthesis, evaluation, and crystallographic studies of conformationally constrained Smac mimetics as inhibitors of the X-linked inhibitor of apoptosis protein (XIAP)

Small molecules designed to mimic the binding of Smac protein to X-linked inhibitor of apoptosis protein (XIAP) are being pursued as a promising new class of anticancer drugs. Herein, we report the design, synthesis, and comprehensive structure-activity relationship studies of a series of conformationally constrained bicyclic Smac mimetics. Our studies led to the discovery of a number of highly potent and cell-permeable Smac mimetics and yielded important new insights into their structure-activity relationship for their binding to XIAP and for their activity in inhibition of cancer cell growth. Determination of the crystal structure of one potent Smac mimetic, compound 21, in complex with XIAP BIR3 provides the structural basis for its high-affinity binding to XIAP and for the design of highly potent Smac mimetics.     

Irreversible binding of an anticancer compound (BI-94) to plasma proteins

Abstract

1.?We investigated the mechanisms responsible for the in vivo instability of a benzofurazan compound BI-94 (NSC228148) with potent anti-cancer activity.

2.?BI-94 was stable in MeOH, water, and in various buffers at pHs 2.5–5, regardless of the buffer composition. In contrast, BI-94 was unstable in NaOH and at pHs 7–9, regardless of the buffer composition. BI-94 disappeared immediately after spiking into mice, rat, monkey, and human plasma. BI-94 stability in plasma can be only partially restored by acidifying it, which indicated other mechanisms in addition to pH for BI-94 instability in plasma.

3.?BI-94 formed adducts with the trapping agents, glutathione (GSH) and N-acetylcysteine (NAC), in vivo and in vitro via nucleophilic aromatic substitution reaction. The kinetics of adduct formation showed that neutral or physiological pHs enhanced and accelerated GSH and NAC adduct formation with BI-94, whereas acidic pHs prevented it. Therefore, physiological pHs not only altered BI-94 chemical stability but also enhanced adduct formation with endogenous nucleophiles. In addition, adduct formation with human serum albumin-peptide 3 (HSA-T3) at the Cys34 position was demonstrated.

4.?In conclusion, BI-94 was unstable at physiological conditions due to chemical instability and irreversible binding to plasma proteins.     

A potent and orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in clinical development for cancer treatment

We report the discovery and characterization of SM-406 (compound 2), a potent and orally bioavailable Smac mimetic and an antagonist of the inhibitor of apoptosis proteins (IAPs). This compound binds to XIAP, cIAP1, and cIAP2 proteins with K(i) of 66.4, 1.9, and 5.1 nM, respectively. Compound 2 effectively antagonizes XIAP BIR3 protein in a cell-free functional assay, induces rapid degradation of cellular cIAP1 protein, and inhibits cancer cell growth in various human cancer cell lines. It has good oral bioavailability in mice, rats, non-human primates, and dogs, is highly effective in induction of apoptosis in xenograft tumors, and is capable of complete inhibition of tumor growth. Compound 2 is currently in phase I clinical trials for the treatment of human cancer.     

Theoretical studies of the binding of trifluoperazine derivatives to site (82-93) of calmodulin: effect of lengthenings of the methylene linker chain on the binding affinity

A theoretical study was performed of the comparative binding affinities to fragment (82-93) of calmodulin (CaM) of trifluoperazine (TFP) and three derivatives, in which the methylene chain linking the phenothiazine ring and the piperazinium group was lengthened by addition of one to three methylenes. The backbone of the oligopeptide was held in the alpha-helical conformation. The computations were performed with the SIBFA procedures (sum of interactions between fragments computed ab initio), which use empirical formulas based on ab initio self-consistent field computations. The interaction energy is the sum of the intermolecular phenothiazine derivative-oligopeptide interaction energy and of the separate intramolecular energy variations of the ligand, on the one hand, and of the oligopeptide, on the other hand, upon relaxing the conformations of side chains Glu 84, Glu 87, Phe 89 and Phe 92 due to complex formation. All three derivatives were found to display a higher binding affinity than did TFP itself, an optimal affinity being found for a four- and a five-methylene linker chain. In as much as fragment (82-93) of CaM is a plausible candidate receptor site for phenothiazines, these results imply that two such compounds should be endowed with a significantly greater anti-CaM activity than TFP itself.     

Depsipeptide (FK228) as a novel histone deacetylase inhibitor: mechanism of action and anticancer activity

Depsipeptide (FK228), a new histone deacetylase inhibitor, has been recently introduced into clinical trials. This agent shows interesting metabolic properties, novel mechanism of action, and is undergoing phase I-II clinical studies in hematopoietic malignancies and solid tumors. Mechanism of action, pharmacokinetics and anticancer activity of depsipeptide is the subject of this review.     

Targeting IAP (inhibitor of apoptosis) proteins for therapeutic intervention in tumors

Apoptosis, or programmed cell death, is a cell suicide process with a major role in development and homeostasis in vertebrates and invertebrates. Dysregulation of apoptosis leading to early cell death or the absence of normal cell death contributes to a number of disease conditions including neurodegenerative diseases and cancer. Inhibition of apoptosis enhances the survival of cancer cells and facilitates their escape from immune surveillance and cytotoxic therapies. Inhibitor of apoptosis (IAP) proteins, a family of anti-apoptotic regulators that block cell death in response to diverse stimuli through interactions with inducers and effectors of apoptosis are among the principal molecules contributing to this phenomenon. IAP proteins are expressed in the majority of human malignancies at elevated levels and play an active role in promoting tumor maintenance through the inhibition of cellular death and participation in signaling pathways associated with malignancies. Herein, the role of IAP proteins in cancer and strategies toward targeting IAP proteins for therapeutic intervention will be discussed.     

Synergistic effects of chemotherapeutic drugs in lymphoma cells are associated with down-regulation of inhibitor of apoptosis proteins (IAPs), prostate-apoptosis-response-gene 4 (Par-4), death-associated protein (Daxx) and with enforced caspase activation

Cytotoxic drugs mediate apoptotic tumor cell death by influencing key regulator proteins of programmed cell death. In clinical practice cytotoxic drug combinations are desired to potentiate tumor cell kill and to minimize side effects. Nevertheless, the molecular mechanisms underlying synergistic and antagonistic effects on tumor cells are still poorly understood. In order to elucidate these molecular mechanisms we established models of synergistic and antagonistic drug combinations within the same lymphoma cell lines. By combination index method we demonstrated that bendamustine in combination with either doxorubicin or mitoxantrone caused antagonistic effects on disruption of mitochondrial membrane potential as well as on the rate of apoptosis. In contrast the combination of bendamustine with cladribine acted synergistically on these parameters. By using the IC(50) (dosages causing 50% rate of apoptosis) the synergistic effect of the combination of bendamustine and cladribine was associated with an enhanced mitochondrial release of cytochrome c and Smac/DIABLO, by down-regulation of x-linked inhibitor of apoptosis (XIAP), cIAP1, Par-4 and Daxx as well as by a significantly increased activation of caspases-3, -6, -7, -8 and -9. At the same rate of apoptosis (IC(50)), the antagonistic combinations did not increase the release of cytochrome c or Smac/DIABLO, nor down-regulate the expression of XIAP, cIAP1, Par-4 and Daxx, nor increase the activation of caspases. The role of down-regulation of IAPs and of enforced caspase activation for synergism in this model was supported by the observation, that broad spectrum inhibition of caspases re-established expression of XIAP. Our study is the first to outline the molecular alterations caused by synergistic and antagonistic drug combinations within the same lymphoma cell model. The above described mechanisms were already assessable at a point where the effects of synergistic or antagonistic combinations could not yet be discriminated quantitatively by the level of apoptosis rate of the lymphoma cells.     

Physiologically based pharmacokinetic and pharmacodynamic modeling of an antagonist (SM‐406/AT‐406) of multiple inhibitor of apoptosis proteins (IAPs) in a mouse xenograft model of human breast cancer

The inhibitors of apoptosis proteins (IAPs) are a class of key apoptosis regulators overexpressed or dysregulated in cancer. SM‐406/AT‐406 is a potent and selective small molecule mimetic of Smac that antagonizes the inhibitor of apoptosis proteins (IAPs). A physiologically based pharmacokinetic and pharmacodynamic (PBPK‐PD) model was developed to predict the tissue concentration–time profiles of SM‐406, the related onco‐protein levels in tumor, and the tumor growth inhibition in a mouse model bearing human breast cancer xenograft. In the whole body physiologically based pharmacokinetic (PBPK) model for pharmacokinetics characterization, a well stirred (perfusion rate‐limited) model was used to describe SM‐406 pharmacokinetics in the lung, heart, kidney, intestine, liver and spleen, and a diffusion rate‐limited (permeability limited) model was used for tumor. Pharmacodynamic (PD) models were developed to correlate the SM‐406 concentration in tumor to the cIAP1 degradation, pro‐caspase 8 decrease, CL‐PARP accumulation and tumor growth inhibition. The PBPK‐PD model well described the experimental pharmacokinetic data, the pharmacodynamic biomarker responses and tumor growth. This model may be helpful to predict tumor and plasma SM‐406 concentrations in the clinic. Copyright © 2013 John Wiley & Sons, Ltd.     

On the binding of diisopropyl-phosphoro-fluoridate (DFP) to plasma proteins

Archives of Toxicology - Sephadex® gel filtration has been employed for the quantitative determination of the binding of3H-labeIled diisopropyl-phosphoro-fluoridate (DFP) in guinea-pig plasma.     

Covalent binding of benzo[a]pyrene to rat liver cytosolic proteins and its effect on the binding to microsomal proteins

Benzo[a]pyrene will bind covalently to rat liver cytosolic proteins when incubated with microsomes and NADPH. The binding is most extensive when microsomes from 3-methylcholanthrene-treated rather than phenobarbital-treated or control rats are used. The binding to cytosolic proteins increases when incubations are performed with increasing concentrations of cytosol. At the same time the covalent binding of benzo[a]pyrene to microsomal proteins decreases. Two cytosolic polypeptides are the main targets for benzo[a]pyrene. These have the same mobility in polyacrylamide gels as the subunits of purified glutathione S-transferase B. These subunits also react covalently with benzo[a]pyrene when the transferase is incubated with microsomes and NADPH.     

凋亡抑制蛋白家族与肿瘤治疗的研究进展

1前言　　目前认为肿瘤治疗失败的主要原因是癌细胞对化疗、放疗及细胞毒T细胞和自然杀伤细胞介导的免疫疗法不敏感,事实上,这些看似不同的治疗方法是通过相同的生物学的机制即细胞凋亡的方式来消除肿瘤的.     

Dicationic bis(9-methylphenazine-1-carboxamides): relationships between biological activity and linker chain structure for a series of potent topoisomerase targeted anticancer drugs

Bis(9-methylphenazine-1-carboxamides) joined by a variety of dicationic (CH(2))(n)()NR(CH(2))(m)NR(CH(2))(n) linkers of varying length (carboxamide N-N distances from 11.0 to 18.4 A) and rigidity were prepared by reaction of 9-methylphenazine-1-carboxylic acid imidazolide with the appropriate polyamines. The compounds were evaluated for growth inhibitory properties in P388 leukemia, Lewis lung carcinoma, and wild-type (JL(C)) and mutant (JL(A) and JL(D)) forms of human Jurkat leukemia with low levels of topoisomerase II (topo II). The compounds all had IC(50) ratios of <1 in the resistant Jurkat lines, consistent with topo II inhibition not being the primary mechanism of action. Analogues joined by an (CH(2))(2)NR(CH(2))(2)NR(CH(2))(2) linker were extremely potent cytotoxins, with selectivity toward the human cell lines, but absolute potencies declined sharply from R = H through R = Me to R = Pr and Bu. In contrast, (CH(2))(2)NR(CH(2))(3)NR(CH(2))(2) compounds showed reverse effects, with the R = Me analogue being more potent than the R = H one as well as being the most potent in the series [IC(50) in JL(C) cells 0.08 nM; superior to that for the clinical bis(naphthalimide) LU 79553]. Overall, the IC(50)s of analogues with linker chains (CH(2))(n)NH(CH(2))(m)NH(CH(2))(n) were inversely proportional to linker length. Constraining the rigidity of the linker chain by incorporating a piperazine ring did not decrease potency significantly. A representative compound bound tightly to DNA with high selectivity for GC sites, compatible with recent work suggesting that compounds of this type place their side chains in the major groove, making specific contacts with guanine bases. Representative compounds were susceptible to transport mediated resistance, being much less effective in cells that overexpressed P-glycoprotein. Overall the results suggest these compounds have a similar mode of action, mediated primarily by poisoning of topo I (possibly with some involvement of topo II). The bis(9-methylphenazine-1-carboxamides) show very high in vitro growth inhibitory potencies compared to their monomeric analogues. Two compounds showed in vivo activity in murine colon 38 syngeneic and HT29 human colon tumor xenograft models using intraperitoneal dosing.     

The histone deacetylase inhibitor butyroyloxymethyl diethylphosphate (AN-7) protects normal cells against toxicity of anticancer agents while augmenting their anticancer activity

The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) has been shown to synergize doxorubicin (Dox) anticancer activity while attenuating its cardiotoxicity. In this study we further explored the selectivity of AN-7’s action in several cancer and normal cells treated with anticancer agents. The cells studied were murine mammary 4T1, human breast T47D and glioblastoma U251 cancer cell lines, neonatal rat cardiomyocytes, cardiofibroblasts and astrocytes, and immortalized cardiomyocyte H9C2 cells. Cell death, ROS production and changes in protein expression were measured and in vivo effects were evaluated in Balb-c mice. AN-7 synergized Dox and anti-HER2 cytotoxicity against mammary carcinoma cells with combination indices of 0.74 and 0.79, respectively, while it protected cardiomyocytes against their toxicity. Additionally AN-7 protected astrocytes from Dox-cytoxicity. Cell-type specific changes in the expression of proteins controlling survival, angiogenesis and inflammation by AN-7 or AN-7+Dox were observed. In mice, the protective effect of AN-7 against Dox cardiotoxicity was associated with a reduction in inflammatory factors. In summary, AN-7 augmented the anticancer activity of Dox and anti-HER2 and attenuated their toxicity against normal cells. AN-7 modulation of c-Myc, thrombospondin-1, lo-FGF-2 and other proteins were cell type specific. The effects of AN-7, Dox and their combination were preserved in vivo indicating the potential benefit of combining AN-7 and Dox for clinical use.     

Potent bivalent thrombin inhibitors: replacement of the scissile peptide bond at P(1)-P(1)' with arginyl ketomethylene isosteres.

We have designed highly potent synthetic bivalent thrombin inhibitors, which consist of an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. The bivalent inhibitors bind to the active site and the fibrinogen recognition exosite simultaneously. As a result, the inhibitors showed much higher affinity for thrombin than the individual blocking segments. Various arginyl ketomethylene isosteres ArgPsi[CO-CH(2)-X]P(1)' were incorporated into the bivalent inhibitors as P(1)-P(1)' segment to eliminate the scissile bond. The P(1)' residue is a natural or unnatural amino acid; specifically, the incorporation of mercaptoacetic acid exhibited superiority in synthesis and affinity for thrombin. Inhibitor 16, (D-cyclohexylalanine)-Pro-ArgPsi[CO-CH(2)-S]Gly-(Gly)(4)-Asp-Tyr-G lu- Pro-Ile-Pro-Glu-Glu-Tyr-cyclohexylalanine-(D-Glu)-OH, showed the lowest K(i) value of 3.5 +/- 0.5 x 10(-13) M, which is comparable to that (K(i) = 2.3 x 10(-13) M) of recombinant hirudin. Consequently we successfully reduced the size of the inhibitor from approximately 7 kDa of recombinant hirudin to approximately 2 kDa without losing the affinity.     

Folate-targeted dinitrophenyl hapten immunotherapy: effect of linker chemistry on antitumor activity and allergic potential

Targeting of malignancies with folate-linked therapeutics has proven to be a promising endeavor due to the preferential expression of folate receptors (FR) on human tumors. We have shown that folic acid (pteroyl-glutamate) can be used to deliver an antigenic hapten, fluorescein, to the surface of tumor cells to promote their opsonization within a fluorescein-immunized host. Here, we investigate structure-activity relationships among members of another class of folate-hapten conjugates ( EC57, EC63, EC0293, and EC0294), namely, those containing the dinitrophenyl (DNP) group as the antigenic hapten. We report that despite exhibiting similar affinities for the FR, the antitumor activity and allergic potential of these DNP conjugates varied depending on their linker chemistries and abilities to bind anti-DNP IgG/IgE antibodies. Unlike EC57 and EC63, both EC0293 and EC0294 (i) share the identical DNP bridging chemistry to that found in keyhole limpet hemocyanin (KLH)-DNP (i.e., the immunogen), (ii) efficiently recognize DNP-specific IgG, and (iii) mediate more pronounced antitumor responses. However, EC0293 and EC0294 were also found to recognize DNP-specific IgE, and they displayed a greater risk of allergy when evaluated in a passive cutaneous anaphylaxis assay. Nonetheless, upon co-stimulation with the appropriate cytokines (IL-2/IFN-alpha), the folate-targeted "haptenization" process allowed for tumor rejection and protective antitumor immunity without causing any visible allergy in immunized mice. Our data further support the concept that folate-hapten-targeted immunotherapy may offer an effective therapeutic option for treatment of FR-positive cancers, but such treatment should proceed with caution given the risk of a potential allergic reaction.     

The effect of sodium salicylate on the binding of L-tryptophan to serum proteins

In human serum, l -tryptophan is the only amino-acid bound to protein. Salicylate causes a release of tryptophan from its binding sites on human serum proteins and bovine albumin. Some implications of this finding are discussed.