Identification of key residues involved in mediating the in vivo anti-tumor/anti-endothelial activity of Alphastatin.
BACKGROUND
We have recently shown that Alphastatin, a 24-amino-acid peptide (ADSGEGDFLAEGGGVRGPRVVERH) derived from human fibrinogen has anti-endothelial properties in vitro and in vivo.


OBJECTIVE
The aim of this study was to determine the activity of a terminally modified (stabilized) form of Alphastatin in vitro and in vivo and to identify the key residues required for this activity.


METHODS
The in vitro activity of modified Alphastatin, truncates and mutants was determined by endothelial cell (HuDMEC) tubule formation and migration. Active peptides were then assessed in vivo using syngeneic murine subcutaneous 4T1 mammary carcinomas.


RESULTS
Modified Alphastatin-inhibited HuDMEC migration and tubule formation in response to multiple growth factors and caused a 45% inhibition in tumor growth when administered intravenously at 0.25 mg kg(-1) (three times per week). Intravenous (i.v.) administration proved non-toxic at all doses investigated, whereas oral and intraperitoneal (i.p.) administration demonstrated neither anti-tumor activity nor toxicity. Truncations of Alphastatin revealed an 11-amino-acid peptide (DFLAEGGGVRG), termed AHN419, which inhibited endothelial cell activity in vitro; however, intravenous AHN419 caused a non-significant growth inhibition in vivo. Single amino acid substitutions to alanine along the entire length of Alphastatin indicated that additional residues outside the AHN419 sequence were required for full activity.


CONCLUSIONS
Terminal modification of Alphastatin altered the in vivo efficacy and these studies suggest that a hydrophobic cluster (Phe8, Leu9, Ala10 and Val15) is essential for the biological activity, but additional residues, including Ser3-Gly14, Pro18-Val20 and Arg23 are required for full inhibitory activity of Alphastatin.
Staton
C A
CA
Microcirculation Research Group, University of Sheffield Medical School, Sheffield, UK.
Stribbling
S M
SM
García-Echeverría
C
C
Bury
J P
JP
Tazzyman
S
S
Lewis
C E
CE
Brown
N J
NJ
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D015674:Mammary Neoplasms, Animal / Q000188:drug therapy* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009154:Mutation
D000970:Antineoplastic Agents; C514422:alphastatin; D005340:Fibrinogen