Anti-Cancer Drug Candidates
("pre-preclinical" as of 1999)
Interactions with tubulin
Among principal producers of the data here
NA Durso, DL Sackett, R Bai, WO Gamble, JH Cardellina, and E
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
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
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),
These characteristics, coupled with additional
preliminary results reported below, indicate that hemiasterlin is likely to
interact with tubulins 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
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
What are the structures of
How were concentrations of solutions normalized?
Does hemiasterlin bind to
Yes, according to fluorimetric analyses of tryptophanyl moieties, which further
indicate conformational changes occurring within a hemiasterlin-tubulin
Does it bind
Yes, according to novel comparative Kd analyses
exploiting sulfhydryl reactivities of tubulin.
on tubulin does hemiasterlin bind?
|The vinca/peptide site
of tubulin, according to...
studies of drug binding to tubulin;
||inhibition of GTP exchange
are its effects on tubulin polymer?
| It inhibits
purified tubulin's assembly in vitro (turbidity).
Electron microscopy reveals coils/rings (not shown).
Tubulin oligomers were indicated by...
||HPLC gel filtration
What do hemiasterlins do to
cells, and how quickly?
They are potently cytotoxic, achieving maxima in < 24h.
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).
Is the cytotoxicity lethal
or merely inhibitory?
Preliminary clonogenic assays suggest lethality at 10×IC50s.
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?
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
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.
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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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