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Supplementary Fig. S1 from The Aurora kinase inhibitor SNS-314 shows broad therapeutic potential with chemotherapeutics and synergy with microtubule-targeted agents in a colon carcinoma model

Aurora kinases play key roles in regulating centrosome maturation, mitotic spindle formation, and cytokinesis during cell division, and are considered promising drug targets due to their frequent overexpression in a variety of human cancers. SNS-314 is a selective and potent pan Aurora inhibitor currently in a dose escalation phase 1 clinical trial for the treatment of patients with advanced solid tumors. Here, we report the antiproliferative effects of SNS-314 in combination with common chemotherapeutics in cell culture and xenograft models. The HCT116 colorectal carcinoma cell line, with intact or depleted p53 protein levels, was treated with SNS-314 and a cytotoxic chemotherapeutic from a panel comprised of gemcitabine, 5-fluorouracil (5-FU), carboplatin, daunomycin, SN-38 (the active metabolite of irinotecan), docetaxel, and vincristine. Combinations were administered under either concurrent or sequential schedules. SNS-314 has predominantly additive effects when administered concurrently with commonly used anticancer agents. Sequential administration of SNS-314 with chemotherapeutic compounds showed additive antiproliferative effects with carboplatin, gemcitabine, 5-FU, daunomycin, and SN-38, and synergy was observed in combination with gemcitabine, docetaxel, or vincristine. The most profound antiproliferative effects were observed with sequential administration of SNS-314 followed by docetaxel or vincristine. In vivo, SNS-314 potentiated the antitumor activity of docetaxel in xenografts. Both the in vitro synergies observed between SNS-314 and agents that target the mitotic spindle and the potentiation seen with docetaxel in vivo are consistent with a mechanism of action in which Aurora inhibition bypasses the mitotic spindle assembly checkpoint and prevents cytokinesis, augmenting subsequent spindle toxin–mediated mitotic catastrophe and cell death. [Mol Cancer Ther 2009;8(4):930–9]

Aurora kinases play key roles in regulating centrosome maturation, mitotic spindle formation, and cytokinesis during cell division, and are considered promising drug targets due to their frequent overexpression in a variety of human cancers. SNS-314 is a selective and potent pan Aurora inhibitor currently in a dose escalation phase 1 clinical trial for the treatment of patients with advanced solid tumors. Here, we report the antiproliferative effects of SNS-314 in combination with common chemotherapeutics in cell culture and xenograft models. The HCT116 colorectal carcinoma cell line, with intact or depleted p53 protein levels, was treated with SNS-314 and a cytotoxic chemotherapeutic from a panel comprised of gemcitabine, 5-fluorouracil (5-FU), carboplatin, daunomycin, SN-38 (the active metabolite of irinotecan), docetaxel, and vincristine. Combinations were administered under either concurrent or sequential schedules. SNS-314 has predominantly additive effects when administered concurrently with commonly used anticancer agents. Sequential administration of SNS-314 with chemotherapeutic compounds showed additive antiproliferative effects with carboplatin, gemcitabine, 5-FU, daunomycin, and SN-38, and synergy was observed in combination with gemcitabine, docetaxel, or vincristine. The most profound antiproliferative effects were observed with sequential administration of SNS-314 followed by docetaxel or vincristine. In vivo, SNS-314 potentiated the antitumor activity of docetaxel in xenografts. Both the in vitro synergies observed between SNS-314 and agents that target the mitotic spindle and the potentiation seen with docetaxel in vivo are consistent with a mechanism of action in which Aurora inhibition bypasses the mitotic spindle assembly checkpoint and prevents cytokinesis, augmenting subsequent spindle toxin–mediated mitotic catastrophe and cell death. [Mol Cancer Ther 2009;8(4):930–9]

Lysine 2,3-aminomutase from Clostridium SB4 has been studied by electron paramagnetic resonance (EPR) spectroscopy at 77 K. Although the reaction catalyzed by this enzyme is similar to rearrangements catalyzed by enzymes requiring adenosylcobalamin, lysine 2,3-aminomutase does not utilize this cofactor. The enzyme instead contains iron-sulfur clusters, cobalt, and pyridoxal phosphate and is activated by S-adenosylmethionine. Subsequent to a reductive incubation procedure that is required to activate the enzyme, EPR studies reveal the appearance of an organic radical signal (g = 2.001) upon addition of both L-lysine and S-adenosylmethionine. The radical signal is complex, having multiple hyperfine transitions. The total radical concentration is proportional to enzyme activity and decreases in parallel with the approach to chemical equilibrium between alpha-lysine and beta-lysine. The signal changes over the time course of the reaction in a way that suggests the presence of more than one radical species, with different relative proportions of species in the steady state and equilibrium state. Isotopic substitution experiments show that unpaired spin density resides on the molecular framework of lysine and that solvent-exchangeable protons do not participate in strong hyperfine coupling to the radical. The results indicate that lysine radicals participate in the rearrangement mechanism.

Radicals have been detected in the steady states of most of the adenosylcobalamin-dependent reactions. Although in most cases the structures of these radicals have not been fully established, the thiyl-protein radical in B 12 -dependent ribonucleotide reductase has been characterized. Radical intermediates in monooxygenase reactions—such as those of cytochrome P450s and methane monooxygenase—have been postulated. However, the assignment of radical mechanisms is controversial in these cases because of the indirect nature of the evidence for the participation of organic radicals. The most direct evidence for a radical mechanism is the observation of transient radicals by electron paramagnetic resonance spectroscopy (EPR). In these cases, the question of kinetic competence can be addressed experimentally by rapid-mix freeze-quench EPR spectroscopy. This technique has allowed the kinetic competence of the thiyl-protein radical in the B l2 -dependent ribonucleotide reductase to be proven through studies in the past. The method described in the chapter should be generally applicable to the evaluation of kinetic competence for any enzymatic radical that can be labeled with deuterium or carbon-13 to perturb the EPR spectrum. Increasing numbers of such radicals are being observed in the rapidly expanding world of enzymatic radicals.

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-lysine and L-beta-lysine. The enzyme contains iron-sulfur clusters and is activated by pyridoxal 5'-phosphate and S-adenosylmethionine, all of which participate in catalysis. Current spectroscopic evidence implicates two substrate-based organic radicals as intermediates in the mechanism. One of these species, the radical N3-(5'-phosphopyridoxylidene)-beta-lysin-2-yl (3), appears in the steady state of the reaction of lysine and has been definitively characterized by EPR and ESEEM spectroscopy. The 2-deuterio form of this radical, 3-2-d, which is generated in the reaction of L-[2-2H]lysine, can be distinguished by line shape analysis from 3. The rate at which the signal for 3-2-d is transformed into that for 3 has been measured by rapid mix-freeze quench kinetic analysis. The rate constant for this process is 24 +/- 8 s-1 at 21 degrees C. This is the rate constant for the turnover of radical 3 and is indistinguishable from the turnover number of lysine 2,3-aminomutase. Therefore, radical 3 is kinetically competent as an intermediate in the reaction of lysine 2,3-aminomutase.

The role of pyridoxal 5'-phosphate (PLP) in the radical-mediated amino group migration catalyzed by lysine 2,3-aminomutase from Clostridia SB4 has been investigated by electron spin echo envelope modulation (ESEEM) spectroscopy. This pulsed electron paramagnetic resonance (EPR) method was used to estimate the distance between the unpaired electron in the alpha-radical of beta-lysine, a steady-state intermediate in the reaction, and deuterium at the C4' position of the cofactor, PLP. [4'-2H]PLP was synthesized and exchanged into the enzyme. The steady-state radical was generated in the labeled samples and in samples with unlabeled PLP by addition of L-lysine.H2SO4 to activated enzyme. ESEEM spectra of the samples prepared with [4'-2H]PLP exhibited distinctive low-frequency modulations that were not present in spectra of matched samples with unlabeled PLP. Fourier transformation of the modulations yielded a prominent doublet signal centered about the Larmor frequency of deuterium. The magnitude of the doublet splitting of the 2H ESEEM signal exhibited angle selection across the CW EPR powder pattern. The observed angle selection, as well as simulation of the time domain spectra, indicated that the doublet splitting was due to the combined effects of the 2H hyperfine and nuclear quadrupole interactions. The influences of the quadrupole interaction and of isotropic and dipolar hyperfine interactions were explored by simulations of the ESEEM spectra. The analysis indicates a distance of < 3.5 A between the 2H at C4' of PLP and the radical center at C alpha lysine. The data are most compatible with an aldimine linkage between PLP and the beta-nitrogen of beta-lysine.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron paramagnetic resonance (EPR) spectroscopy has been used to characterize an organic radical that appears in the steady state of the reaction catalyzed by lysine 2,3-aminomutase from Clostridium SB4. Results of a previous electron paramagnetic resonance (EPR) study [Ballinger, M. D., Reed, G. H., & Frey, P. A. (1992) Biochemistry 31, 949-953] demonstrated the presence of EPR signals from an organic radical in reaction mixtures of the enzyme. The materialization of these signals depended upon the presence of the enzyme, all of its cofactors, and the substrate, lysine. Changes in the EPR spectrum in response to deuteration in the substrate implicated the carbon skeleton of lysine as host for the radical center. This radical has been further characterized by EPR measurements on samples with isotopically substituted forms of lysine and by analysis of the hyperfine splittings in resolution-enhanced spectra by computer simulations. Changes in the hyperfine splitting patterns in EPR spectra from samples with [2-2H]lysine and [2-13C]-lysine show that the paramagnetic species is a pi-radical with the unpaired spin localized primarily in a p orbital on C2 of beta-lysine. In the EPR spectrum of this radical, the alpha-proton, the beta-nitrogen, and the beta-proton are responsible for the hyperfine structure. Analysis of spectra for reactions initiated with L-lysine, [3,3,4,4,5,5,6,6-2H8]lysine, [2-2H]lysine, perdeuteriolysine, [alpha-15N]lysine, and [alpha-15N,2-2H]lysine permit a self-consistent assignment of hyperfine splittings.(ABSTRACT TRUNCATED AT 250 WORDS)