Potent non-benzoquinone ansamycin heat shock protein 90 inhibitors from genetic engineering of Streptomyces hygroscopicus

Article abstract of DOI:10.1021/jm900012a

Inhibition of the protein chaperone Hsp90 is a promising new approach to cancer therapy. We describe the preparation of potent non-benzoquinone ansamycins. One of these analogues, generated by feeding 3-amino-5-chlorobenzoic acid to a genetically engineer

Full text of DOI:10.1021/jm900012a

1518
J. Med. Chem. 2009, 52, 1518–1521
The biosynthesis of geldanamycin is initiated by the starter
unit 3-amino-5-hydroxybenzoic acid (AHBA). Subsequent
extension of this starter unit by the polyketide synthase (PKS)
and cyclization produces a macrolactam, which is then subjected
to post-PKS carbamoylation, 4,5-dehydrogenation, and several
oxidation steps to produce the final product.13-15 The non-
benzoquinone ansamycins reblastatin and autolytimycin have
been reported, presumably resulting from natural mutations or
deletions in post-PKS modification genes.16-18 Genetic engi-
neering previously resulted in non-benzoquinone geldanamycin
analogues such as KOS-1559 and autolytimycin (KOS-1806)
through oxidase knockout or PKS modification,13,14 but this
approach necessarily produces a very limited number of new
compounds, most of which have poor in vitro cytostatic activity.
During this previous work, an AHBA-deficient mutant of the
geldanamycin producing strain was generated by the Kosan
research group to positively identify the geldanamycin biosyn-
thetic gene cluster and the genes involved in its accessory
pathways.14 We surmised that a broader range of diversity could
be generated by feeding analogues of the AHBA starter unit to
such an AHBA-deficient mutant strain. A panel of ∼30
substituted 3-aminobenzoic acids and related heterocycles was
prepared (Supporting Information Figure 1) and fed to cultures
of the AHBA-deficient mutant strain K554-161. Ten of the
AHBA analogues were incorporated into novel non-benzo-
quinone ansamycins as identified by LC/MS/MS analyses of
the culture broths (Supporting Information Table 1). Generally,
AHBA analogues having small hydrophobic substituents were
accepted by the geldanamycin PKS to produce non-benzo-
quinone ansamycins, while feeding of AHBA analogues with
more polar substituents or heterocycles did not lead to detectable
ansamycin products. In many cases, more than one product was
obtained because of partial post-PKS processing.
Potent Non-Benzoquinone Ansamycin Heat
Shock Protein 90 Inhibitors from Genetic
Engineering of Streptomyces hygroscopicus
Hugo G. Menzella,*,† Thomas-Toan Tran, John R. Carney,
Janice Lau-Wee, Jorge Galazzo, Christopher D. Reeves,
Christopher Carreras, Sophie Mukadam, Sara Eng,
Ziyang Zhong, Pieter B. M. W. M. Timmermans,
Sumati Murli, and Gary W. Ashley
Kosan Biosciences Incorporated, 3832 Bay Center Place,
Hayward, California 94545
ReceiVed January 7, 2009
Abstract: Inhibition of the protein chaperone Hsp90 is a promising
new approach to cancer therapy. We describe the preparation of potent
non-benzoquinone ansamycins. One of these analogues, generated by
feeding 3-amino-5-chlorobenzoic acid to a genetically engineered strain
of Streptomyces hygroscopicus, shows high accumulation and long
residence time in tumor tissue, is well-tolerated upon intravenous dosing,
and is highly efficacious in the COLO205 mouse tumor xenograft
model.
Heat Shock Protein 90 (Hsp90a) is a protein chaperone that
acts to stabilize and/or activate a number of proteins required
for cellular signaling pathways, including several known
determinants of carcinogenesis.1-3 Hsp90 is effectively inhibited
by benzoquinone ansamycins such as geldanamycin, herbimycin,
macbecin, and many of their derivatives, which bind to the ATP
binding site in the N-terminal domain.4,5 Recent results from
clinical trials using the geldanamycin derivatives tanespimycin
and alvespimycin, also known as 17-AAG (17-allylamino-17-
demethoxygeldanamycin) and 17-DMAG (17-(2-dimethylami-
no)ethylamino-17-demethoxygeldanamycin), respectively, sug-
gest that inhibition of Hsp90 could be an important therapeutic
mechanism for cancer treatment.6 The antiproliferative activity
of several benzoquinone Hsp90 inhibitors against cancer cells
has been shown in various models to be dependent on reductive
activation to the hydroquinone by the enzyme NAD(P)H/
quinone oxidoreductase 1 (NQO1).7-10 The activity of this
enzyme in cancer patients is variable. The frequency of the most
common mutant allele P187S differs in different ethnic groups,
but the number of homozygous individuals can be as high as
25% in some populations, resulting in a lack of NQO1 activity
in these patients.11,12
Several detected ansamycin products were fermented on a
larger scale to provide purified compounds for full structural
characterization and further study (Table 1). These compounds
were completely characterized by detailed NMR analysis
(Supporting Information compound data). Examination of
binding to the purified N-terminal domain of human Hsp90R
using isothermal titration calorimetry or a scintillation proximity
assay19,20 demonstrated that the non-benzoquinone ansamycins
are tight binders to the ATP binding site, having affinities
between 10 and 103 times greater than those of the quinone
form of tanespimycin (Table 1).
Cell growth inhibition assays demonstrated activities similar
to those of tanespimycin and alvespimycin for three of the five
isolated molecules (1, 3, and 5) against a panel of cancer cell
lines (Table 1). There is no obvious correlation between the
binding affinity to the purified N-terminal domain of human
Hsp90R and cytostatic activity, in keeping with earlier
observations,21-23 suggesting that binding to the purified
N-terminal domain is necessary but not sufficient for activity.
Molecules that retain the exquisite specificity of tanespimycin
and alvespimycin for the ATP binding pocket of Hsp90 while
obviating the need for activation by NQO1 could be very
attractive candidates for drug development. Here we report
potent, efficacious nonbenzoquinone ansamycin Hsp90 inhibitors
that retain robust activity against NQO1 deficient tumor cell
lines, produced by genetic engineering of Streptomyces hygro-
scopicus NRRL3602.
As previously reported, cell growth inhibitory activity of the
quinone-containing Hsp90 inhibitors tanespimycin and alvespi-
mycin against NQO1-deficient cell lines is relatively poor.8
However, the non-benzoquinone analogues described above
maintain potency against the NQO1-deficient cell lines MDA-
468 and NCI-H596 (Table 1). Further, addition of the NQO1
inhibitor dicumarol to the NQO1 positive cell lines SKBr3 and
MCF7 had no effect on the cytostatic potency of 1, despite
significantly reducing the potency of tanespimycin and alvespi-
* To whom correspondence should be addressed. Phone: (54) (341)
4350661, ext 117. Fax: (54) (341) 4390465. E-mail: menzella@ibr.gov.ar.
†
Current address: IBR, CONICET, Facultad de Ciencias Bioqu´ımicas
y Farmace´uticas, Universidad Nacional de Rosario, Suipacha 531,
(S2002LRK) Rosario, Argentina.
a Abbreviations: Hsp90, heat shock protein 90; NQO1, NAD(P)H/quinone
oxidoreductase 1; AHBA, 3-amino-5-hydroxybenzoic acid; PKS, polyketide
synthase; 17-AAG, 17-allylamino-17-demethoxygeldanamycin; 17-DMAG,
17-(2-dimethylamino)ethylamino-17-demethoxygeldanamycin.
10.1021/jm900012a CCC: $40.75
2009 American Chemical Society
Published on Web 02/20/2009

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1519
Table 1. Products isolated from fermentations of K554-161 strain fed with either 3-amino-5-chlorobenzoic acid or 3-aminobenzoic acida
Kd (nM)
IC50 (nM)
MCF-7
SPA
ITC
SKBr3
SKOV-3
HCT-116
MDA-468 (NQ01-)
NCI-H596 (NQ01-)
tanespimycin
alvespimycin
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
760
500
50
160
87
44
58
55
394
96
835
83
240
122
141
350
320
840
158
202
57
58
71
57
330
139
610
87
1500
1600
89
443
190
420
160
1600
1100
190
710
390
n.d.
n.d.
5.4
0.8
2.5
120
422
240
1050
179
n.d
n.d.
n.d.
n.d.
1200
325
a Hsp90 binding was determined by isothermal titration calorimetry (ITC) or scintillation proximity assay (SPA) using purified recombinant human
Hsp90R N terminal domain, and growth inhibition effects on indicated cancer cell lines were determined after 72 h exposure to the drugs using the CellTiter-
Glo cell viability assay from Promega Co. Values shown are means of three independent determinations. The standard deviations were in all the cases less
than 10% of the corresponding means. n.d., not determined.
Figure 1. In vivo efficacy of 1. COLO205 tumor bearing mice were dosed iv with vehicle or 1 or ip, with tanespimycin as indicated. (a) Tumors
were collected 4 or 24 h after a single dose of tanespimycin or 1 to determine drug levels in the tumors by LC/MS analysis. Tumor samples were
analyzed for levels of Hsp70 (b) or ErbB2 (c) by ELISA assay 4 or 24 h after a single dose of 90 mg/kg tanespimycin or 30 mg/kg 1. (d) The
impact on tumor growth of iv dosing COLO205 tumor-bearing mice with vehicle, 15 mg/kg tanespimycin, or 90 mg/kg tanespimycin, 15 mg/kg
of 1, or 30 mg/kg 1 daily for 5 days with a 2 day holiday for a total of 13 doses (n ) 10 mice per group) is shown.
mycin (Supporting Information Table 2). Taken together, these
results indicate that the activity of the non-benzoquinone
ansamycins created in this study is independent of reductive
activation by NQO1.

1520 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Letters
Analysis of effects on different Hsp90 client proteins includ-
ing ErbB2, Raf-1, and pAkt corroborates Hsp90 as the target
of the non-benzoquinone ansamycins. Treatment of SKBr3 cells
with the potent cytostatic analogues results in the rapid loss of
the Hsp90 client ErbB2 and induction of Hsp70, whereas
treatment with the less active analogues shows less effect on
these markers of Hsp90 inhibition24-26 (Supporting Information
Figure 2). The response of the client proteins in these cells to
treatment by the potent non-benzoquinone ansamycins thus
mirrors that observed following treatment with tanespimycin
or alvespimycin.
Acknowledgment. We acknowledge financial support from
the National Institutes of Health SBIR Grant 5R44 CA096262-
03.
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clinical evaluation, higher antiproliferative activity against
NQO1 deficient cell lines, potent and long-lasting effects on
Hsp90 client proteins in vivo, and robust, superior efficacy in
mouse xenograft models.
Supporting Information Available: Experimental details, NMR
and LC/MS/MS data for isolated compounds, panel of AHBA
analogues used for preparation of compounds, effect of compounds
on client proteins, impact of treatment on body weight, effect of
dicumarol on cytotocity of isolated compounds. This material is
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JM900012A