The interaction of a new anti‐tumour drug, KAR‐2 with calmodulin

Abstract

1. KAR-2 (3"-(beta-chloroethyl)-2",4"-dioxo-3,5" -spiro-oxazolidino-4-deacetoxy-vinblastine) is a semisynthetic bis-indol derivative, with high anti-microtubular and anti-tumour activities but with low toxicity. KAR-2, in contrast to other biologically active bis-indols (e.g. vinblastine) did not show anti-calmodulin activity in vitro (enzyme kinetic, fluorescence anisotropy and immunological tests). 2. Direct binding studies (fluorescence resonance energy transfer, circular dichroism) provided evidence for the binding of KAR-2 to calmodulin. The binding affinity of KAR-2 to calmodulin (dissociation constant was about 5 microM) in the presence of Ca2+ was comparable to that of vinblastine. 3. KAR-2 was able to interact with apo-calmodulin as well; in the absence of Ca2+ the binding was of cooperative nature. 4. The effect of drugs on Ca2+ homeostasis in human neutrophil cells was investigated by means of a specific fluorescent probe. Trifluoperazine extensively inhibited the elevation of intracellular Ca2+ level, vinblastine did not appreciably affect it, KAR-2 stimulated the Ca2+ influx and after a transient enhancement the Ca2+ concentration reached a new steady-state level. 5. Comparison of the data obtained with KAR-2 and bis-indols used in chemotherapy suggests that the lack of anti-calmodulin potency resides on the spiro-oxazolidino portion of KAR-2. This character of KAR-2 manifested itself in various systems and might result in its low in vivo toxicity, established in an anti-tumour test.