Mary A. Napier

Neocarzinostatin: Spectral characterization and separation of a non-protein chromophore

Abstract Chromatographically purified neocarzinostatin exhibits absorption, fluorescence, magnetic circular dichroic and circular dichroic spectral characteristics above and below 300 nm atypical for a protein with its reported aminoacid composition, indicating the presence of a non-protein chromophore. The drug complex, stable at acidic pH, can be dissociated by treatment with reducing or denaturing agents at neutral or basic pH. Chromatography of the dissociated complex, or more conveniently, methanol extraction of the lyophilized drug, separates a protein with an amino-acid composition identical to neocarzinostatin and a highly fluorescent chromophore free of amino-acids.

Kistrin, a polypeptide platelet GPIIb/IIIa receptor antagonist, enhances and sustains coronary arterial thrombolysis with recombinant tissue-type plasminogen activator in a canine preparation.

BackgroundKistrin is a 68-amino acid polypeptide from the venom of the Malayan pit viper Agkistrodon rhodostoma, which inhibits the platelet GPIIb/IIIa receptor. Its effect on thrombolysis, reocclusion, and bleeding associated with administration of recombinant tissue-type plasminogen activator (rt-PA) was studied in a canine model of coronary artery thrombosis. Methods and ResultsCoronary patency was monitored for 2 hours by ulltrasonic flow probe and repeated coronary angiography. The rt-PA was given as 0.45-mg/kg bolus injections at 15-minute intervals until recanalization or to a maximum of four boluses. Four groups of four or five dogs were studied: a control group that received intravenous heparin (4,000-unit bolus and 1,000 units each hour) and three groups that received heparin and 0.48, 0.24, or 0.12 mg/kg kistrin, administered as a 10% bolus injection and an infusion during a 60-minute period. In the control group, reflow occurred in four of five dogs within 37±47 minutes but was followed by cyclic reflow and reocclusion. Kistrin at a dose of 0.48 and 0.24 mg/kg reduced the time to reflow to 6±5 and 10±3 minutes, respectively, and abolished reocclusion. With 0.12 mg/kg kistrin, reflow occurred in all four animals, within 27±23 minutes, and reocclusion occurred in two animals. Kistrin induced a dose-related prolongation of the template bleeding time: with 0.48 mg/kg kistrin, the bleeding time was prolonged from 3.8±1.3 minutes before infusion to 29±2 minutes during infusion, but it was shortened to 8.3 ±2.6 minutes at 90 minutes after the end of infusion. Kistrin also caused a dose-related inhibition of platelet aggregation with ADP and collagen: with 0.48 mg/kg kistrin, platelet aggregation was abolished during the infusion but had partially recovered toward the end of the observation period. Pathological examination of recanalized coronary arterial segments of dogs given 0.48 or 0.24 mg/kg kistrin revealed widely patent arteries with some platelets layered on the damaged intimal surface. ConclusionsKistrin increases the rate and extent of thrombolysis with a reduced dose of rt-PA, and it prevents reocclusion. At an effective dose, it is associated with a transient prolongation of the bleeding time and inhibition of platelet aggregation. Kistrin may offer promise as adjunctive treatment to thrombolytic agents in patients with acute myocardial infarction. (Circulation 1991;83:1038–1047)

Neocarzinostatin chromophore: Presence of a highly strained ether ring and its reaction with mercaptan and sodium borohydride

Spectroscopic evidence suggests the presence of a highly strained ether ring (Fig. 1) (possibly an epoxide) in the C12-subunit of the previously determined partial structure 2a (Fig. 2) of the major neocarzinostatin chromophore (NCS-Chrom A) which completes assignment of all the oxygens in the molecule. The main product from mercaptan treatment suggests opening of the ether ring involving the addition of one molecule of mercaptan as well as reduction of the C12-substructure, whereas a parallel two-step reduction occurs on NaBH4 treatment. Both reactions occur with rearrangement of the C12-substructure and the implication for the mechanism of action of NCS-Chrom A in DNA strand scission activity is discussed. The evidence suggests a downward revision of the molecular formula for NCS-Chrom A as well as minor components B and C by two protons.

Neocarzinostatin: Chemical characterization and partial structure of the non-protein chromophore

Abstract The molecular formula C35H35NO12 (mol.wt. 661) is proposed for the biologically active chromophoric component of neocarzinostatin. The partial structure 2 is proposed based on 1 H NMR and mass spectral data and consists, in part, of a 2,6-dideoxy-2-methylamino-galactose moiety and a naphthoic acid derivative. Special treatments required to obtain spectral data of the labile chromophore are described.

Neocarzinostatin chromophore: Presence of a cyclic carbonate subunit and its modification in the structure of other biologically active forms

Abstract On the basis of spectroscopic evidence, opening of a five-membered cyclic carbonate ring (1,3-dioxolan-2-one) in the C15-subunit of the previously determined partial structure 1 (Fig. 1) of the major neocarzinostatin chromophore (NCS-Chrom A ), is proposed to account for its base-catalyzed methanolysis to NCS-Chrom C . NCS-Chrom B , apparently an authentic natural product present as a minor component in all preparations of NCS studied, was found to be formally equivalent to the hydrolysis/decarboxylation product of the cyclic carbonate functionality in NCS-Chrom A . The mercaptan-dependent DNA strand-scission activity, equivalent for NCS-Chrom A , B and C , is independent of the integrity of the cyclic carbonate ring system and implicates a secondary site in the C15-substructure for mercaptan activation.

Effects of G4120, a Arg-Gly-Asp containing synthetic platelet glycoprotein IIB/IIIA receptor antagonist, on arterial and venous thrombolysis with recombinant tissue-type plasminogen activator in dogs

Abstract The effects of G4120, L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-α-aspartyl-cyclic(l->5)-sulfide, 5-oxide, a novel synthetic cyclic RGD-containing pentapeptide, on thrombolysis with rt-PA were investigated in a combined arterial and venous thrombosis model in the dog. In a blinded study, design dogs were randomly assigned to 0.5 mg/kg rt-PA + 0.1 mg/kg G4120 (group I, n = 5), 0.5 mg/kg rt-PA + placebo (II, n = 5), 0.25mg/kg rt-PA + 0.1 mg/kg G4120 (III, n = 5) or 0.25 mg/kg rt-PA + placebo (IV, n = 5). Cyclic reflow and reocclusion occurred in 8 out of 10 animals given rt-PA alone versus 2 out of 10 dogs given rt-PA + G4120 (p

DNA as a Target for a Protein Antibiotic: Molecular Basis of Action

The antitumor antibiotic neocarzinostatin (NSC), isolated from the culture filtrates of Streptomyces carzinostaticus variant F-41 (Ishida et al. 1965), is an acidic single-chain polypeptide with a molecular weight of 10,700 (Meienhofer et al. 1972a). The protein has been purified to homogeneity and its amino acid sequence (Fig. 1) (Meienhofer et al. 1972a, b; Maeda et al. 1974; Samy et al. 1977) and physical properties (Maeda et al. 1973; Samy and Meienhofer 1974) have been determined. There are high degrees of homology of some regions of NCS and the protein antibiotics actinoxanthin (Khokhlov et al. 1969, 1976) and macromomycin (Sawyer et al. 1979). NCS exists in a tight, proteolysis-resistant conformation with an antiparallel β-pleated sheet structure (Samy et al. 1974). It possesses two reduction-resistant disulfide bridges and lacks methionine and histidine. The positions of the disulfides have not yet been unambiguously assigned. NCS contains two tryptophan residues in positions 46 (buried) and 79 and one buried tyrosine residue at position 32. Oxidation of tryptophan 79 does not result in loss of biological activity (Samy et al. 1974). Similarly, acylation of the amino groups (alanine 1 and lysine 20) does not affect the activity of NCS (Maeda 1974; Samy 1977). On the other hand, modification of the carboxyl groups results in loss of activity (Samy 1977). Further, spontaneous deamidation of asparagine 83 at a weakly acidic pH generates “preneocarzinostatin” which lacks biological activity (Maeda and Kuromizu 1977). The chemically deamidated compound is thought to be the same as the material isolated from culture filtrates that antagonizes NCS activity (Kikuchi et al. 1974).