In various forms, the data in this section have appeared in posters and their abstracts (list in CV), as well as published papers. Therefore, the customary "preliminary data" qualifier may apply. In addition, because some abstracts appeared in copyrighted publications, certain restrictions are applicable, US government exemptions notwithstanding. ©
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Hemiasterlins
Antimitotic Peptides
Anti-Cancer Drug Candidates
("pre-preclinical" as of 1999)
Interactions with tubulin
  Among principal producers of the data here are:
NA Durso, DL Sackett, R Bai, WO Gamble, JH Cardellina, and E Hamel
 
Screening Technology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Frederick Cancer Research & Development Center; Laboratory of Drug Discovery Research and Development; Frederick, Maryland (21702), USA

Hemiasterlins are modified tripeptides isolated from the sponge Siphonochalina sp., among other sources. Our co-workers (principally WO Gamble and JH Cardellina) have noted – by conventional comparative chemical analyses, by prior reports on the same or similar compounds, and by cytotoxicity analyses using a panel of cultured cancer cells – their potential to behave like other cytotoxic compounds having antimitotic mechanisms, likely targeting the microtubule cytoskeleton. We and others have confirmed that it is a potently cytotoxic compound that inhibits mitosis; see, e.g., Gamble, Durso, Fuller, Westergaard, Johnson, Sackett, Hamel, Cardellina, and Boyd; 1999; Bioorganic & Medicinal Chemistry 7 (8), 1611-1615 here, as well as the data below. 
      Hemiasterlin also inhibits the binding of GTP, and at least two other antimitotic agents – dolastatin 10 (a drug already being used in anticancer studies), and vinblastine (long used in anticancer treatments) – to tubulin. It also stabilizes the colchicine-binding activity of tubulin. See Bai, Durso, Sackett, and Hamel; 1999; Biochemistry, 38 (43), 14302-14310 here. 
      These characteristics, coupled with additional preliminary results reported below, indicate that hemiasterlin is likely to interact with tubulin’s vinca/peptide binding region, which is the target for a wide variety of structurally complex natural products. The methods provide evidence regarding whether, how, and at what sites the hemiasterlins bind to purified tubulin and inhibit its assembly in vitro, as well as the mechanism and kinetics by which the compounds poison cells and ultimately cause cell death (apparently programmed; apoptosis, as it turns out). 
      The methods are biochemical, biophysical, and cell biological, and inferences are drawn by comparing the effects of other compounds in parallel assays. A model (below) for the cytotoxic mode of action is essentially that proposed for dolastatin 10 by P. Verdier-Pinard et al. in our lab. 
      The significance of hemiasterlins is that they are structurally the simplest compounds that interact in the vinca region of tubulin, making them amenable to facile chemical modification, which should also prove useful in exploring the essential features of this poorly understood, but important, target for antineoplastic agents.
 

 
Results / Data / Figures
 
click to see figure What are the structures of hemiasterlins, and
How were concentrations of solutions normalized?

 
click to see figure Does hemiasterlin bind to tubulin?
Yes, according to fluorimetric analyses of tryptophanyl moieties, which further indicate conformational changes occurring within a hemiasterlin-tubulin complex.
 
click to see figure Does it bind tightly?
Yes, according to novel comparative Kd analyses exploiting sulfhydryl reactivities of tubulin.
 
Where on tubulin does hemiasterlin bind?
The vinca/peptide site of tubulin, according to...
click to see figurefurther comparative fluorescence analyses;
click to see figurecomparative inhibition studies of drug binding to tubulin;
click to see figureinhibition of GTP exchange on tubulin.
 
What are its effects on tubulin polymer?
click to see figure It inhibits purified tubulin's assembly in vitro (turbidity).
Electron microscopy reveals coils/rings (not shown).
Tubulin oligomers were indicated by...
click to see figureHPLC gel filtration sizing;
click to see figureanalytical ultracentrifugation.
 
click to see figure What do hemiasterlins do to cells, and how quickly?
They are potently cytotoxic, achieving maxima in < 24h.
 
click to see figure How does that cytotoxicity compare to other agents?
High-to-intermediate potency vs tubulin drugs, which are typically very high vs DNA/topoisomerase II drugs. Typical kinetics for drugs (Taxol®/paclitaxel or vinblastine lag).
 
click to see figure Is the cytotoxicity lethal or merely inhibitory?
Preliminary clonogenic assays suggest lethality at 10×IC50s.
 
click to see figure What's the "cause of death" by hemiasterlins?
Chromatin morphologies indicate apoptosis results presumably succeeding mitotic arrest (cf. Taxol®/paclitaxel).
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Model: Why so potent?

Hemiasterlin and dolastatin 10 share several characteristics. In vitro, both induce tubulin oligomers and interact with tubulin's vinca site; in fact, one can inhibit the other's binding to tubulin.  
      As widely understood now for many microtubule depolymerizing agents, stoichiometries on the order of <1:100 suppress dynamics without the mass depolymerization seen at more equi-, or super- molar stoichiometries. 
      In the case of both compounds, the cytotoxic IC50 is ~1000- fold less than the in vitro kd for tubulin binding. An IC50 < kd is not atypical, but the magnitude of the difference in these cases is noteworthy versus some other anti-microtubule agents. This shared characteristic suggests that hemiasterlin's cellular effects may arise from mechanisms quite similar to dolastatin 10's as posited by Verdier-Pinard et al.:

    

By virtue of their small size and hydrophobicity, they enter cells readily; however, efflux is negligible, resulting in cellular accumulation to an extent far in excess of some other tubulin binding agents (e.g., vinblastine). The minimal efflux is effectively explained by the trapping of the drugs in tubulin oligomer complexes induced by the drugs.

  click to see figure
eye
fails

 
  
This explains the potency and apparent irreversibility of hemiasterlin's cellular effects. The sequestration (and inactivation thereby?) of tubulin results in the disruption / loss of the microtubule cytoskeleton composed primarily of tubulin. Cell death (apoptotic) is the ultimate result.
 
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Again, the significance of hemiasterlins is that they are structurally the simplest compounds that interact in the vinca region of tubulin, making them amenable to facile chemical modification, which should also prove useful in exploring the essential features of this poorly understood, but important, target for antineoplastic agents.



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