Characterization of compound 584, an Abl kinase inhibitor with lasting effects.
BACKGROUND
Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584).


METHODS
To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor.


RESULTS
cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib.


CONCLUSIONS
The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.
Puttini
Miriam
M
School of Pharmaceutical Sciences, University of Geneva, Quai Ernest-Ansermet 30, 1211 Genève 4, Switzerland.
Redaelli
Sara
S
Moretti
Loris
L
Brussolo
Stefania
S
Gunby
Rosalind H
RH
Mologni
Luca
L
Marchesi
Edoardo
E
Cleris
Loredana
L
Donella-Deana
Arianna
A
Drueckes
Peter
P
Sala
Elisa
E
Lucchini
Vittorio
V
Kubbutat
Michael
M
Formelli
Franca
F
Zambon
Alfonso
A
Scapozza
Leonardo
L
Gambacorti-Passerini
Carlo
C
D000813:Anilides / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001549:Benzamides / Q000737:chemistry; D045744:Cell Line, Tumor; D002626:Chemistry, Pharmaceutical / Q000379:methods; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D000068877:Imatinib Mesylate; D007938:Leukemia / Q000188:drug therapy*; D051379:Mice; D009368:Neoplasm Transplantation; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D016315:Proto-Oncogene Proteins c-abl / Q000037:antagonists & inhibitors*; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology*
C531555:3-chloro-4-(4-methylpiperazin-1-ylmethyl)-N-(4-methyl-3-(4-pyridin-3-ylpyrimidin-2-ylamino)phenyl)benzamide; D000813:Anilides; D000970:Antineoplastic Agents; D001549:Benzamides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D016315:Proto-Oncogene Proteins c-abl