Download Small molecule intervention in microtubule

Human Molecular Genetics, 2005, Vol. 14, Review Issue 2
doi:10.1093/hmg/ddi269
R291–R300
Small molecule intervention in microtubuleassociated human disease
Jantje M. Gerdes1 and Nicholas Katsanis1,2,*
1
McKusick-Nathans Institute of Genetic Medicine and 2Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD
21205, USA
Received June 30, 2005; Revised and Accepted July 20, 2005
Microtubules are essential for a number of cellular processes that include the transport of intracellular cargo
or organelles across long distances and the assembly of the mitotic spindle. The identification of numerous
microtubule-associated proteins and the progressive elucidation of the mechanisms of microtubule assembly and transport are beginning to have a profound impact on the study and treatment of human genetic
disease. A number of seemingly unrelated phenotypes have now been linked to microtubular dysfunction,
especially in systems dependent heavily on microtubule-based transport, such as neurons and ciliated
cells. In parallel, the association of microtubule transport defects with human genetic disease has led
to the realization that targeting various aspects of microtubular biology with small molecules might offer
new therapeutic paradigms, including the development of new therapeutic utility for seemingly old drugs.
In this review, we discuss the use of small molecules in the investigation of microtubule-associated processes and particularly the screens of chemical compound libraries for the identification of lead compounds
with potential utility in microtubule-associated disease processes.
MICROTUBULAR TRANSPORT
Microtubules play a central role in cellular transport, structural
integrity and cellular architecture (Fig. 1). As such, it is not
surprising that perturbations of microtubule dynamics and
transport can lead to a broad range of human phenotypes.
These defects are particularly prominent in structures and processes dependent heavily on the microtubule network, such as
the central nervous system, where proper neuronal function is
dependent on transport of cargo across long distances (up to
1 m in humans) and the microtubule-rich cilium of many
eukaryotic cell types (1 – 5).
Mammalian cells regulate their architecture and active
transport requirements by utilizing three major types of filaments: microtubules, microfilaments and intermediate filaments.
Intermediate filaments and microfilaments fulfill primarily
structural and mechanical functions, whereas microtubules are
critical for the intracellular transport of cargo, as well as the
assembly of the mitotic spindle. Like microfilaments, microtubules are polarized structures and assemble from heterodimers
of a- and b- tubulin in a GTP-dependent fashion. Microtubule
dynamics play an important role in mitosis and the cell cycle
(6), and interference with these dynamic processes can trigger
apoptosis (7). Transport along the microtubule network is accomplished by microtubular dynamics or by microtubule-associated
motor proteins and other microtubule-associated proteins
(MAPs), which either actively move cargo along the microtubules (motor proteins) or can serve as docking molecules to
bind cargo to motor proteins (8).
Microtubule-associated motor proteins are divided into two
major protein families, kinesins and dyneins, both of which
consist of globular motor domains that typically share high
sequence homology among the particular protein family; it
is the adjoining rod-like structure that is more diverse
(2,9,10). Members of the dynein family generally enable transport of cargo from the cell periphery to the center (9,11),
whereas the much larger kinesin superfamily is involved
primarily with transport towards the cell periphery, with the
exception of C-terminal kinesins which, though similar in
structure, facilitate transport towards the cell center (9,12).
MICROTUBULE-BINDING SMALL MOLECULES
Microtubules pose an interesting, albeit challenging, target for
specific remedial intervention. To date, all drugs or small
*To whom correspondence should be addressed at: McKusick-Nathans Institute of Genetic Medicine, 533 Broadway Research Building, 733 N. Broadway, Baltimore, MD 21205, USA. Tel: þ1 4105026660; Fax: þ1 4105020697; Email: [email protected]
# The Author 2005. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
R292
Human Molecular Genetics, 2005, Vol. 14, Review Issue 2
Figure 1. Schematic presentation of the role of microtubules in various cellular processes. The microtubules (blue) protrude from the microtubule-organizing
center (MTOC) which is located in proximity to the nucleus towards the cell periphery. Examples of small molecule effectors interfering with these microtubular
processes are shown in red.
molecules known to interact with microtubules disrupt microtubule dynamics by either stabilizing or destabilizing the
polymerized state. Disrupting microtubule dynamics affects
mainly rapidly dividing cells, such as cancer cells, which is
why these small molecules have been considered potent
agents for chemotherapy (Fig. 1) (6). Docetaxel (or taxotere)
(1) is a derivative of the natural product Paclitaxel (Taxolw)
(2), a known stabilizer of tubulin interaction. In contrast,
Podophyllotoxin (3), etoposide (4), vinblastine (5), vincristine
(6) and vinorelbine (7) are anticancer drugs that inhibit or
disrupt microtubules and microtubule assembly. In addition,
the antifungal and antimitotic FDA approved drug griseofulvin
(8) inhibits mitosis in metaphase and interacts with polymerized microtubules and MAPs. Currently, there are 16 microtubule interacting compounds listed in the chemical reference
database ChemBank, an electronic repository of structural
information and bioactivity data of chemical compounds
(http://chembank.broad.harvard.edu) (Table 1). In addition to
the aforementioned known drugs, there are more destabilizers
listed including the natural products cytochalasin A (9) and E
(10), TN-16 (11), myoseverin (12), nocodazole (13), vindesine
(14), the depsipetide Phomopsin A (15) and d-24851 (16). As
microtubule dynamics are of vital importance for all dividing
cells, disruption of this process will affect both cancerous and
normal cells alike; the increased rate of proliferation of cancer
cells renders antimotic drugs 1 –8 useful and potent inhibitors
of cancer growth, but also explains the drastic side effects of
cancer chemotherapy such as hair loss and a decrease in red
blood cells, because the proliferation of normal tissue is also
inhibited.
SPECIFIC INHIBITORS OF MAPS
Monastrol (17) was identified when screening for abnormal
mitotic behavior in cells treated with a library of small molecules (Fig. 2) (13). Treatment with monastrol results in
replacement of the bipolar mitotic spindle with a monastrol
microtubule array surrounded by a ring of chromosomes,
which in turn causes the cells to enter mitotic arrest. Subsequently, the human kinesin-like protein Eg5 was identified
as the target of monastrol (17) (13). In spite of considerable
sequence homology shared by all kinesin proteins, no effect
on conventional kinesin-associated microtubule motility was
observed. Similarly, there was no detectable alteration in the
localization and organization of the Golgi apparatus or
lysozymes. Therefore, monastrol is not considered a general
inhibitor of motor proteins but an example of a selective inhibitor of Eg5, thereby representing the first selective inhibitor of
a microtubule-associated motor protein. Until then, the nonhydrolyzable ATP analog AMP-PNP (18) and the marine
natural product adociasulfate-2 (19) (14) were the only kinesin
inhibitors known, both suffering drawbacks because of their
poor cell permeability and selectivity. Subsequent to the discovery of monastrol, the target of terpendole E (20), a natural
product with known antimitotic properties, was also identified
as kinesin Eg5 (15). Although the binding site for terpendole E
(20) is not known, the mechanism of action of adociasulfate-2
was established to be the area of the microtubule-binding
site of the kinesin heavy chains (14). The mechanism of
action for monastrol has been shown to be non-competitive
with respect to ATP or microtubule binding (16). However,
Human Molecular Genetics, 2005, Vol. 14, Review Issue 2
R293
Table 1. Bioactive compounds that target associated directly or indirectly with microtubules
Compound name
Structure
Bioactivity
Comments
1
Docetaxel
Microtubule stabilizer
Known drug—indications/usage:
anti-cancer, breast cancer,
non-small lung cancer
2
Taxol
Microtubule stabilizer, promotes and
stabilizes tubulin polymerization
Known drug—indications/usage:
antineoplastic
3
Podophyllotoxin
Tubulin binder, DNA topoisomerase
II inhibitor; inhibits microtubule
assembly, arrest cell cycle
Known drug—indications/usage:
cytostatic, antineoplastic
4
Etoposide
Topoisomerase II inhibitor; inhibits
microtubule assembly; arrests
cells in metaphase
Known drug—indications/usage:
anti-cancer, antineoplastic,
antimitotic
5
Vinblastine
Tubulin inhibitor; microtubule
disruptor
Known drug—indications/usage:
anti-cancer
6
Vincristine
Microtubule assembly inhibitor;
antibiotic
7
Vinorelbine
Microtubule inhibitor
Known drug—indications/usage:
anti-cancer, non-small cell
lung cancer
8
Griseofulvin
Interacts with polymerized
microtubules and associated
proteins; inhibits mitosis in
metaphase
Known drug–indications/usage:
antifungal, anti-mitotic
Continued
R294
Human Molecular Genetics, 2005, Vol. 14, Review Issue 2
Table 1. Continued
Compound name
Structure
Bioactivity
9
Cytocholasin A
Microtubule assembly inhibitor
10
Cytochalasin E
Microtubule assembly inhibitor
11
TN-16
Microtubule assembly inhibitor
12
Myoseverin
Microtubule disruptor
13
Nocodazole
Microtubule and mitosis inhibitor,
inhibits tubulin
14
Vindesine
Microtubule assembly inibitor;
antibiotic
15
Phomopsin A
Microtubule assembly inhibitor
16
d-24851
Microtubule stabilizer
17
Monastrol
Mitotic kinesin Eg5 inhibitor(13)
Comments
Continued
Human Molecular Genetics, 2005, Vol. 14, Review Issue 2
R295
Table 1. Continued
Compound name
Structure
Bioactivity
18
AMP-PNP
ATP- competitive inhibitor
19
Adociasulfate-2
Inhibitor of kinesin motors (14)
20
Terpendole-E
Arrests cells in metaphase; mitotic
kinesin Eg5 inhibitor (15)
21
Tubacin
Microtubule deacetylase (HDAC6)
inhibitor (58)
22
Scriptaid
Deacetylase inhibitor; HDAC
inhibitor (26)
23
DPD
Inhibits aggresome formation (26)
24
C2-8
Poly-Q aggregation inhibitor (38)
monastrol has been shown to bind to the motor domain of
Eg5 (16,17) and is thus an allosteric inhibitor of Eg5 activity.
Currently, several Eg5 inhibitors are undergoing clinical
evaluation (18,19).
In stable microtubules, there is an abundance of acetylated
a-tubulin, which is absent from more dynamic cellular structures such as neuronal growth cones or the leading edges of
Comments
fibroblasts. The role of a-tubulin acetylation remains unclear
and only recently an enzyme responsible for deacetylation
has been identified: histone deacetylase 6 (HDAC6) (20).
Unlike other histone deacetylases, HDAC6 is localized exclusively in the cytoplasm, in particular to punctate structures
concentrated perinuclearly, as well as the leading edge of
the cell (20). In this regard, it is reminiscent of p150Glued,
R296
Human Molecular Genetics, 2005, Vol. 14, Review Issue 2
Figure 2. (A) Monastrol causes monastrol spindles in mitotic cells (a-tubulin is shown in green and chromatin in blue). (a and b) Control BS-C-1 cells. (c and d)
BS-C-1 cells treated with 68 mM monastrol. No difference in the distribution of microtubule and chromatin in interphase cells was observed (b and d) (13). (B)
Hit compounds inhibit polyQ-aggresome formation in Saccharomyces cerevisiae (38). (C) Typical cellular tubulin acetylation phenotype induced by Taxol treatment. A549 cells were treated with vehicle (DMSO) as a control and 10 mM Taxol. (acetylated tubulin is shown in red and nuclei in blue) (59). (D) Axonal
degeneration and axonal microtubules in spinal ventral roots of Taxol-treated T44 tau TG mice. (a) L5ventral root of 12-month-wt mouse with regular large
and small myelinated axons evenly distributed within the nerve. (b and c) The ventral root axons from vehicle treated and untreated T44 tau TG mice are irregularly shaped and show prominent endoneurial space (especially untreated Tg mice). (d and e) The ventral roots in tau Tg mice treated with low and high doses
of Taxol (Paxceed) contain more regularly shaped axons with less endoneurial space than vehicle- and untreated Tg mice (55).
with which it colocalizes. HDAC6 shows deacetylase activity
with respect to polymerized microtubules; free a/b-tubulin
dimers are not deacetylated. In a cell motility assay, NIH
3T3 cells overexpressing wild-type HDAC6 move at least
3.5-fold faster than control cells in response to serum. This
apparent role in the regulation of cell motility by controlling
microtubule acetylation at the leading edge makes HDC6 an
attractive target in drug development for its implications in
metastasis and angiogenesis. Currently, four histone deacetylase
inhibitors are undergoing clinical trials for cancer treatment,
mainly in combination therapy (21). Although most histone
deacetylase inhibitors also affect microtubule acetylation,
there were no selective inhibitors for microtubule acetylation.
In a multidimensional, chemical genetic screen of over 7300
small molecules, tubacin (21) a selective and reversible inhibitor of HDAC6 related a-tubulin acetylation in mammalian
cells was discovered (22). Targeting of tubulin acetylation
does not affect cell cycle progression. Further investigation
revealed that deacetylation does not seem necessary for microtubule depolymerization. In neurodegenerative disorders,
hypoacetylation is frequently observed; increasing the levels
of acetylated a-tubulin by the inhibition of HDAC6 might
therefore not only be implicated in antimetastatic and anti-
angiogenic therapy but also in that of neurodegenerative
disorders.
Many neurodegenerative diseases are characterized by
inclusion bodies like Lewy bodies or aggresomes (23,24).
The hypothesis that aggresome formation is part of a cytoprotective response remains an issue of debate. A recent
study demonstrated the role of HDAC6 in the formation of
aggresomes (25). A ubiquitin-binding zinc finger domain in
HDAC6, as well as its affinity for dynein, suggest that
HDAC6 might represent a link between polyubiquitinated
misfolded proteins and dynein, thereby enabling the transport
along the microtubules towards the MTOC where aggresomes
develop. Aggresomes of misfolded proteins have a similar
appearance to Lewy bodies and accumulate upon the
inhibition of proteasome activity in cells. It has been shown
that both aggresomes and Lewy bodies contain HDAC6. As
cells are more sensitive to polyubiquitinated misfolded
protein stress without the presence of functional HDAC6
and hence in the absence of aggresome formation, it is
likely that aggresome formation is part of a cytoprotective
response.
A study designed to investigate the role and mechanism of
aggresome formation in mammalian cells employed a high
Human Molecular Genetics, 2005, Vol. 14, Review Issue 2
throughput assay to screen for small molecules that disrupt
aggresome formation in cultured cells (26). Among general
microtubule inhibitors and protein synthesis inhibitors, the
assay identified 12 candidate-specific aggresome inhibitors.
These can be sorted into two groups, one being cardiac glycosides and the other comprising two compounds: Scriptaid (22),
a previously known histone deacetylase inhibitor, and DPD
(23), a flavinoid which up to that point did not have any
associated biological activity (26). Upon treatment with
either compound, the misfolded protein forms small granular
structures throughout the cytoplasm, but assembly into aggresomes is almost completely suppressed. Other HDAC inhibitors show the same effect, and neither Scriptaid nor DPD
have a significant effect on MTOC formation or Golgi localization and assembly (26). A portion of the dynactin complex
precipitates with the misfolded proteins, suggesting that it
may bind to misfolded proteins themselves or their ubiquitin
labels. Interestingly, DPD does not inhibit deacetylase
activity, which might imply that the two inhibitors have a
different mechanism of action or that deacetylase activity
alone is not sufficient for aggresome formation.
A recent study hypothesized that the inhibition of both
proteasomal and aggresomal proteins degradation induces
the accumulation of polyubiquitinated proteins and significant
cell stress, thus triggering apoptosis (27). A combination of
bortezomib (a proteasome inhibitor) and tubacin (21) yielded
enhanced cytotoxicity even in plasma cells isolated from the
bone marrow of multiple myeloma patients. The cellular
stress response is mediated by both c-Jun N-terminal kinase
and caspase/PARP cleavage (27). Therefore, the combination
therapy of certain cancer types using proteasome and
aggresome inhibitors seems promising for clinical evaluation.
SMALL MOLECULE INTERVENTION IN
NEURODEGENERATIVE DISEASE
Huntington disease (HD) is a progressive neurodegenerative
disorder characterized by uncontrolled movements, changes
in personality and progressive dementia; patients die within
10 –20 years of onset. HD, as well as a number of other neurodegenerative disorders, have been linked to polyglutamine
expansion (28); in the case of HD, the causative gene Htt
has been shown to be expanded within the open reading
frame. At the cellular level, one major hallmark is the
formation of aggregates that contain polyQ-Htt protein in or
near the nuclei of affected neurons, both in brain tissue of
patients and HD transgenic mice (29 – 31). Histological
analyses have shown significantly dysfunctional neurons, as
well as loss of neurons, especially the medium spiny
neurons of the striatum (32). Motor and cognitive defects
have been observed in both patients and murine models of
HD before any neurodegeneration is detected (33). Yeast-2
hybrid screening and glutathione S-transferase-pulldown
experiments showed that wild-type Htt binds both HAP1 and
the p150Glued subunit of dynactin and seems to play a role
in microtubule-dependent axonal transport (34,35). Although
the role of inclusion body or aggresome remains unclear,
therapeutic strategies that aim to avoid polyQaggregation have shown some efficacy in vivo (36,37). A
R297
study aimed at identifying potential inhibitors of polyQdependent aggregation in HD neurons used initially a yeast
model of HD aggregation to screen 16 000 compounds
(Fig. 2) (38). The yeast strain was modified to express a
GFP labeled Htt homolog with an extended polyQ-trait
which formed aggregates and proved to be cytotoxic (39).
Overall, the modified yeast strain showed reduced growth
compared with wild-type yeast. The initial round of selection
tested for compounds that caused at least a 25% increase in
OD600 and/or EGFP fluorescence compared with controls
(38). Following rounds of screening included a visual assessment of aggresome formation in treated mammalian neuronal
cells (PC12) and filter trap assays for aggresome formation in
the presence of candidate compounds (both in vitro and
in vivo ). Final in vivo assessments of the candidate compounds
included the polyQ-aggregation in hippocampal slices derived
from HD transgenic mice and in a Drosophila HD model
(38). Of nine compounds able to rescue growth in the yeast
model, four did not block aggregation directly, suggesting
that they are probably not targeting soluble or aggregated
polyQ-Htt directly. Compound 24 inhibited aggresome formation both in vitro and in vivo and showed amelioration of
neurodegeneration in a dose-dependent manner in the HD
Drosophila model (38), thereby representing a strong lead
structure for human drug development. Moreover, the combination of initial cell-based high-throughput screening and subsequent evaluation in a diverse set of in vitro and in vivo
secondary assays that included animal models (brain slices
of HD transgenic mice and a HD Drosophila model) should
allow for screening of larger compound libraries and the application to other disease models.
The end-stage of Alzheimer disease (AD) is marked by
extracellular plaques of amyloid peptide Ab, neurofibrillary
tangles composed of hyperphosphorylated t-protein and the
decreased density of cholinergic neurons in the basal forebrain
(40). The major component of the extracellular amyloid
deposits is the 40 –42 amino acid b-amyloid (Ab), the
product of proteolytic cleavage of the much larger amyloid
b precursor protein APP (40). Both APP and the MAP t are
substrates of glycogen synthase kinase 3 (GSK3b), a protein
which plays a major role not only in neurodegenerative
disease but also in Wnt and Sonic hedgehog signaling (41).
Two different genes encode for GSK3a and GSK3b, a
splice variant of which, GSK3b2, is expressed in the brain
(41). GSK3s play a part in regulation of the cell-division
cycle, stem-cell renewal and differentiation, apoptosis, circadian rhythm, transcription and insulin action. This makes
them interesting drug targets for diseases as diverse as neurodegenerative disease, bipolar affective disorder and diabetes
(42). The GSK3 family is highly conserved throughout
evolution and specific inhibition of GSK3b, for example,
could potentially manipulate the kinesin-driven vesicle transport in neurons. Reduction of GSK3b activity levels by
siRNAs or known inhibitors such as lithium and kenpaullone
(25) decreased amyloid-b production in cultured cell lines
(43 –45). In Drosophila, GSK3b inhibition reverses vesicle
aggregation caused by overexpression of t that is associated
with loss of locomotor function (46). This finding not only
implicates GSK3b inhibitors as useful candidates for the
treatment of neurodegenerative diseases but also suggests
R298
Human Molecular Genetics, 2005, Vol. 14, Review Issue 2
Table 2. Representative examples of glycogen synthase kinase 3b inhibitors
Compound name
Structure
Kenpaullone (25)
Hymenialdisine
Flavopiridol
6-Bromoindirubin-30 -oxime
ARA014418
Staurosporine
CHIR98014
that the effects of t are dependent on its phosphorylation state.
To date, over 30 GSK3 inhibitors are known, most of which
bind in the ATP-binding pocket (42) (for representative
examples refer to Table 2). Almost all GSK3b inhibitors
share a low molecular weight (,600) and flat, hydrophobic
heterocycles comprising part of their structures. Binding is
mediated predominantly through hydrophobic interactions
with commonly two to three hydrogen bonds for spatial orientation. Most of these inhibitors compete with ATP in the ATPbinding site of the kinase. However, monovalent lithium ions
compete directly with divalent magnesium ions, impairing
kinase activity. Although ATP-binding sites of kinases are
remarkably similar, there are numerous examples of selective
inhibition of a specific kinase by interaction with the ATPbinding pocket (47). However, a major drawback is that
most of the current GSK3b inhibitors also exhibit an affinity
for other kinases, thereby reducing the specificity and their
potential as therapeutic compounds. Kenpaullone (25) is also
a known inhibitor for cyclin-dependent kinases and therefore
affects progression through the cell cycle. In addition, the
indirect reduction of GSK3 activity levels by the regulation
of the intracellular localization of GSK3 or interference with
the interaction of GSK3 with scaffold proteins or downstream
targets could represent viable routes for drug discovery as well
(48). Although it has been difficult to develop inhibitors of
protein –protein interactions in the past, recent work on the
inhibition of the interaction between MDM2 and p53 has
demonstrated that this strategy is feasible, at least in some
cases (49).
Both Ab and kinesin light chains, as well as t, are substrates
of GSK3b. Thus, increased GSK3b activity does not only lead
to increased levels of Ab and phosphorylated kinesin light
chains, it can also cause hyperphosphorylation of t which is
expressed predominantly in neurons of the central nervous
system. t is thought to play a role in the assembly and stabilization of neuronal microtubules, as well as in organelle
transport along axons and dendrites. Overall, t seems to be implicated in several neurodegenerative diseases, especially those
including parkinsonism or dementia (50–52). The sequestrations
of hyperphosphorylated t-protein are key features of human
tauopathies. In addition, tauopathies are marked by filamentous
t-inclusions, reduced numbers of microtubules, impaired fast
axonal transport (FAT), neurodegeneration and motor weakness.
These hallmarks are recapitulated in a transgenic PrP T44 mouse
model which expresses the shortest human t-isoform T44 under
the control of the mouse prion protein promoter PrP (53,54). A
recent study tested the hypothesis that microtubule-stabilizing
drugs might find a therapeutic application in human tauopathies
by attenuating the loss of normal t functions that results from
t-sequestration into tangles (55). Previous in vitro studies had
shown already that microtubule-binding substances such as
Taxol mitigate Ab-induced neuronal death and t-phosphorylation (56). Indeed, Paclitaxel (2), a microtubule stabilizing
compound administered commonly in cancer chemotherapy,
was able to correct the FAT deficit in the PrP T44 Tg mouse
model (55). Moreover, an increased number of microtubules
and amelioration of motor impairments in mice could be
observed. These results provide a mechanistic link of tauopathies and impairment of microtubule stability. Markedly, no
changes in the abundance of spinal chord t-spheroids have
been observed. Consequently, the motor and FAT impairment
related to human tauopathies do not seem to depend on the
attenuation of aggresome formation. The mitigation of the
FAT phenotype in PrP T44 mice demonstrates a form of in
vivo therapy that corrects loss-of-function rather than correcting the gain-of-function of disease proteins. However,
because of the central role of microtubule in a wide variety of
disease processes, therapeutic microtubules stabilization will
almost certainly have to be combined with more specific
emerging therapies for Alzheimer or other human tauopathies.
CONCLUDING REMARKS
The central role of microtubules and microtubular transport
makes them an appealing target in selective drug development.
Human Molecular Genetics, 2005, Vol. 14, Review Issue 2
All currently marketed drugs that affect microtubules disrupt
microtubular dynamics without any specificity for diseased
cells. Interference with microtubular dynamics will trigger
apoptosis and thus cause cell death mainly in rapidly dividing
cells such as cancer cells. Considering the serious and
sometimes life-threatening side effects, only rare (or perhaps
desperate) circumstances would justify similarly harsh
therapeutic regimens. Future drugs will have to offer higher
selectivity and specificity, which translates into fewer side
effects and better patient compliance. Moreover, defects of
microtubular or intraflagellar transport will have to target
specific parts of the transport machinery to avoid interference
with other pathways. Specific inhibitors of GSK3b have such
potential with implications in neurodegenerative diseases,
mood disorders and diabetes (42).
The mechanism of action of monastrol (17) differs from
known cancer treatments and may provide advantages in
the treatment of multi-drug resistant cancer cell lines. At
present, there are several derivatives undergoing clinical
trials. Monastrol (17) represents the first selective inhibition
of a specific motor protein (13). The inhibition of Eg5 specifically will prove useful for further investigations of its role in
cellular processes. It also demonstrated that despite considerable sequence homology between members of the kinesin
protein family, it is possible to find and develop selective
inhibitors for a single motor protein. Similarly, the selective
inhibition of the a-tubulin deacetylase activity of histone
deacetylases will allow further investigation of the role of
a-tubulin acetylation in cellular processes (22). Finally, the
interplay between chemistry and cell biology, suggesting
that HDAC6 is the link between polyubiquitinated misfolded
proteins and dynein transport, exemplifies the future research
at the interface between these two disciplines. The combination of methods of both fields has driven investigation of
incompletely understood cellular processes, such as aggresome formation, and has facilitated further investigations of
the role of HDAC6 in the cell. In addition, closer collaboration
at the interface of biology and chemistry facilitates and accelerates the identification of bioactive compounds. By combining both in vitro and in vivo assays, the problem of
bioavailability is factored into the process of lead identification (38). Moreover, cell- or even tissue-based assays can
be employed for both lead and target identifications.
Although a great demand for more selective small molecule
effectors targeting specific pathological processes remains,
new combination therapies might fill the need for some
well-characterized diseases. The use of classical cancer
drugs in lower doses for the treatment of Alzheimer disease
attenuates one aspect of the disease pathology, the impairment of FAT (55). Combination with kinase inhibitors, for
example, to mitigate the hyperphosphorylation phenotype
could serve as a new therapeutic regimen for AD patients.
Similarly, many known drugs or bioactive molecules in combination could prove effective in entirely different clinical
implications by targeting similar pathological mechanisms.
So, while lacking a specific and singular target (e.g. for
Alzheimer disease), the oligo-factorial approach utilizing
bioactive molecules targeting several aspects of processes
known to be involved in pathogenesis is potentially able to
fill the need for treatment.
R299
Ciliary dysfunction has been linked to several human diseases including Retinitis pigmentosa (RP), polycystic kidney
disease (PKD), as well as the human obesity syndrome
Bardet–Biedl syndrome (5,57). The close connection between
microtubules and ciliary function indicates that small molecule
effectors targeting microtubules might also be implicated in the
treatment of such diverse diseases as RP or obesity.
With the emerging role of microtubule-associated processes
in human disease, the synthesis and discovery of small
molecules modulating these processes will evolve rapidly.
Improved selectivity will necessitate a more focused mechanism of function than that of hitherto known microtubule
effectors. In the light of the recent successful inhibition of
the protein –protein interaction between MDM2 and p53
(49), the directed manipulation of such interactions between
microtubules and their associated proteins has become possible. Further investigations in this field of interdisciplinary
research will certainly address this question.
Conflict of Interest statement. None declared.
REFERENCES
1. Gunawardena, S. and Goldstein, L.S.B. (2003) Cargo-carrying motor
vehicles on the neuronal highway: transport pathways and
neurodegenerative disease. J. Neurobiol., 58, 258– 271.
2. Mandelkow, E. and Mandelkow, E.-M. (2002) Kinesin motors and
disease. Trends Cell Biol., 12, 585–591.
3. Pazour, G.J. and Rosenbaum, J.L. (2002) Intraflagellar transport and
cilia-dependent diseases. Trends Cell Biol., 12, 551 –555.
4. Badano, J.L., Teslovich, T.M. and Katsanis, N. (2005) The centrosome in
human genetic disease. Nature Rev. Genet., 6, 194 –205.
5. Gerdes, J.M. and Katsanis, N. (2005) Microtubule transport defects in
neurological and ciliary disease. Cell Mol. Life Sci., 62, 1556–1570.
6. Kirschner, M.W. and Mitchison, T. (1986) Microtubule dynamics. Nature,
324, 621–621.
7. Mollinedo, F. and Gajate, C. (2003) Microtubules, microtubule-interfering
agents and apoptosis. Apoptosis, 8, 413–450.
8. Su, Q.N., Cai, Q., Gerwin, C., Smith, C.L. and Sheng, Z.H. (2004)
Syntabulin is a microtubule-associated protein implicated in syntaxin
transport in neurons. Nature Cell Biol., 6, 941–953.
9. Hirokawa, N. (1998) Kinesin and dynein superfamily proteins and the
mechanism of organelle transport. Science, 279, 519 –526.
10. Vallee, R.B., Williams, J.C., Varma, D. and Barnhart, L.E. (2003) Dynein:
an ancient motor protein involved in multiple modes of transport.
J. Neurobiol., 58, 189–200.
11. Milisav, I. (1998) Dynein and dynein-related genes. Cell Mot. Cytoskel.,
39, 261–272.
12. Miki, H., Setou, M., Kaneshiro, K. and Hirokawa, N. (2001) All kinesin
superfamily protein, KIF, genes in mouse and human. Proc. Natl Acad.
Sci. USA, 98, 7004–7011.
13. Mayer, T.U., Kapoor, T.M., Haggarty, S.J., King, R.W., Schreiber, S.L.
and Mitchison, T.J. (1999) Small molecule inhibitor of mitotic spindle
bipolarity identified in a phenotype-based screen. Science, 286, 971–974.
14. Sakowicz, R., Berdelis, M.S., Ray, K., Blackburn, C.L., Hopmann, C.,
Faulkner, D.J. and Goldstein, L.B. (1998) A marine natural product
inhibitor of kinesin motors. Science, 298, 292 –295.
15. Nakazawa, J., Yajima, J., Usui, T., Ueki, M., Takatsuki, A., Imoto, M.,
Toyoshima, Y.Y. and Osada, H. (2003) A novel action of terpendole E on
the motor activity of mitotic kinesin Eg5. Chem. Biol., 10, 131 –137.
16. Maliga, Z., Kapoor, T.M. and Mitchison, T.J. (2002) Evidence that
monastrol is an allosteric inhibitor of the mitotic kinesin Eg5. Chem. Biol.,
9, 989–996.
17. DeBonis, S., Simorre, J.P., Crevel, I., Lebeau, L., Skoufias, D.A.,
Blangy, A., Ebel, C., Gans, P., Cross, R., Hackney, D.D. et al. (2003)
Interaction of the mitotic inhibitor monastrol with human kinesin Eg5.
Biochemistry, 42, 338 –349.
R300
Human Molecular Genetics, 2005, Vol. 14, Review Issue 2
18. Marcus, A.I., Peters, U., Thomas, S.L., Garrett, S., Zelnak, A.,
Kapoor, T.M. and Giannakakou, P. (2005) Mitotic kinesin inhibitors
induce mitotic arrest and cell death in Taxol-resistant and -sensitive
cancer cells. J. Biol. Chem., 280, 11569–11577.
19. Bergnes, G., Brejc, K. and Belmont, L. (2005) Mitotic kinesins: prospects
for antimitotic drug discovery. Curr. Topics Med. Chem., 5, 127– 145.
20. Hubbert, C., Guardiola, A., Shao, R., Kawaguchi, Y., Ito, A., Nixon, A.,
Yoshida, M., Wang, X.-F. and Yao, T.-P. (2002) HDAC6 is a
microtubule-associated deacetylase. Nature, 417, 455 –458.
21. Lesney, M.S. (2004) Enlisting apoptosis. Modern Drug Disc., 7, 55.
22. Haggarty, S.J., Koeller, K.M., Wong, J.C., Grozinger, C.M. and
Schreiber, S.L. (2003) Domain-selective small-molecule inhibitor of
histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc.
Natl Acad. Sci. USA, 100, 4389–4394.
23. Ross, C.A. and Poirier, M.A. (2004) Protein aggregation and
neurodegenerative disease. Nature Med., 10, S10–S17.
24. Goedert, M. (2001) The significance of tau and alpha-synuclein inclusions
in neurodegenerative diseases. Curr. Opin. Genet. Develop., 11, 343–351.
25. Kawaguchi, Y., Kovacs, J.A., McLaurin, A., Vance, J.M., Ito, A. and
Yao, T.-P. (2003) The deacetylase HDAC6 regulates aggresome
formation and cell viability in response to misfolded protein stress.
Cell, 115, 727–738.
26. Corcoran, L.J., Mitchison, T.J. and Liu, Q. (2004) A novel action of
histone deacetylase inhibitors in a protein aggresome disease model.
Curr. Biol., 14, 488–492.
27. Hideshima, T., Bradner, J.E., Wong, J., Chauhan, D., Richardson, P.,
Schreiber, S.L. and Anderson, K. (2005) Small-molecule inhibition of
proteasome and aggresome function induces synergistic antitumor activity
in multiple myeloma. Proc. Natl Acad. Sci. USA, 102, 8567–8572.
28. Ross, C.A. (2002) Polyglutamine pathogenesis: emergence of unifying
mechanisms for Huntington’s disease and related disorders. Neuron, 35,
819 –822.
29. Davies, S.W., Turmaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H.,
Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L. and Bates, G.P.
(1997) Formation of neuronal intranuclear inclusions underlies the
neurological dysfunction in mice transgenic for the HD mutation. Cell, 90,
537 –548.
30. DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P.,
Vonsattel, J.P. and Aronin, N. (1997) Aggregation of huntingtin in
neuronal intranuclear inclusions and dystrophic neurites in brain.
Science, 277, 1990–1993.
31. Yamamoto, A., Lucas, J.J. and Hen, R. (2000) Reversal of neuropathology
and motor dysfunction in a conditional model of Huntington’s disease.
Cell, 101, 57–66.
32. Vonsattel, J.P., Myers, R.H., Stevens, T.J., Ferrante, R.J. and Bird, E.D.
(1985) Neuropathological classification of Huntington’s disease. J.
Neuropath. Exp. Neurol., 44, 559 –577.
33. Gauthier, L.R., Charrin, B.C., Borrell-Pages, M., Dompierre, J.P.,
Rangone, H., Cordelieres, F.P., De Mey, J., MacDonald, M.E.,
Lessmann, V., Humbert, S. et al. (2004) Huntingtin controls neurotrophic
support and survival of neurons by enhancing BDNF vesicular transport
along microtubules. Cell, 118, 127 –138.
34. Li, X.-J., Li, S.H., Sharp, A.H., Nucifora, F.C., Schilling, G., Lanahan, A.,
Worley, P., Snyder, S.H. and Ross, C.A. (1995) A huntingtin-associated
protein enriched in brain with implication for pathology. Nature, 378,
398 –402.
35. Engelender, S., Sharp, A.H., Colomer, V., Tokito, M.K., Lanahan, A.,
Worley, P., Holzbaur, E.L. and Ross, C.A. (1997) Huntingtin-associated
protein1 interacts with the p150Glued subunit of dynactin. Hum. Mol.
Genet., 6, 2205–2212.
36. Kazantsev, A., Walker, H.A., Slepko, N., Bear, J.E., Preisinger, E.,
Steffan, J.S., Zhu, Y.Z., Gertler, F.B., Housman, D.E., Marsh, J.L. et al.
(2002) A bivalent Huntingtin binding peptide suppresses polyglutamine
aggregation and pathogenesis in Drosophila. Nat. Genet., 30, 367– 376.
37. Sanchez, I., Mahlke, C. and Yuan, J.Y. (2003) Pivotal role of
oligomerization in expanded polyglutamine neurodegenerative disorders.
Nature, 421, 373 –379.
38. Zhang, X., Smith, D.L., Merin, A.B., Engemann, S., Russel, D.E.,
Roark, M., Washington, S.L., Maxwell, M.M., Marsh, J.L.,
Thompson, L.M. et al. (2005) A potent small molecule inhibits
polyglutamine aggregation in Huntington’s disease neurons and
suppresses neurodegeneration in vivo. Proc. Natl Acad. Sci. USA, 102,
892 –897.
39. Meriin, A.B., Zhang, X.Q., He, X.W., Newnam, G.P., Chernoff, Y.O. and
Sherman, M.Y. (2002) Huntingtin toxicity in yeast model depends on
polyglutamine aggregation mediated by a prion-like protein Rnq1. J. Cell
Biol., 157, 997–1004.
40. Phinney, A.L., Horne, P., Yang, J., Janus, C., Bergeron, C. and
Westaway, D. (2003) Mouse models of Alzheimer’s disease: the long and
filamentous road. Neurol. Res., 25, 590–600.
41. Cohen, P. and Frame, S. (2001) The renaissance of GSK3. Nat. Rev. Mol.
Cell Biol., 2, 769–776.
42. Meijer, L., Flajolet, M. and Greengard, P. (2004) Pharmacological
inhibitors of glycogen synthase kinase 3. Trends Pharmacol. Sci., 25,
471–480.
43. Ryder, J., Su, Y.A., Liu, F., Li, B.L., Zhou, Y. and Ni, B.H. (2003)
Divergent roles of GSK3 and CDK5 in APP processing. Biochem.
Biophys. Res. Comm., 312, 922 –929.
44. Phiel, C.J., Wilson, C.A., Lee, V.M.Y. and Klein, P.S. (2003) GSK-3
alpha regulates production of Alzheimer’s disease amyloid-beta peptides.
Nature, 423, 435–439.
45. Sun, X., Sato, S., Murayama, O., Murayama, M., Park, J.M.,
Yamaguchi, H. and Takashima, A. (2002) Lithium inhibits amyloid
secretion in COS7 cells transfected with amyloid precursor protein C100.
Neurosci. Lett., 321, 61 –64.
46. Mudher, A., Shepherd, D., Newman, T.A., Mildren, P., Jukes, J.P.,
Squire, A., Mears, A., Berg, S., MacKay, D., Asuni, A.A. et al. (2004)
GSK-3 beta inhibition reverses axonal transport defects and behavioural
phenotypes in Drosophila. Mol. Psych., 9, 522 –530.
47. Noble, M.E.M., Endicott, J.A. and Johnson, L.N. (2004) Protein kinase
inhibitors: insights into drug design from structure. Science, 303,
1800–1805.
48. Phelps, M.A., Foraker, A.B. and Swaan, P.W. (2003) Cytoskeletal motors
and cargo in membrane trafficking: opportunities for high specificity in
drug intervention. Drug Disc. Today, 8, 494–502.
49. Vassilev, L.T., Vu, B.T., Graves, B., Carvajal, D., Podlaski, F.,
Filipovic, Z., Kong, N., Kammlott, U., Lukacs, C., Klein, C. et al. (2004)
In vivo activation of the p53 pathway by small-molecule antagonists of
MDM2. Science, 303, 844 –848.
50. Rademakers, R., Cruts, M. and van Broeckhoven, C. (2004) The role of
tau (MAPT) in frontotemporal dementia and related tauopathies. Human
Mut., 24, 277–295.
51. Geschwind, D.H. (2003) Tau phosphorylation, tangles, and
neurodegeneration: the chicken or the egg? Neuron, 40, 457–460.
52. Higuchi, M., Lee, V.M.Y. and Trojanowski, J.Q. (2002) Tau and
axonopathy in neurodegenerative disorders. Neuromol. Med., 2, 131–150.
53. Ishihara, T., Zhang, B., Higuchi, M., Yoshiyama, Y., Trojanowski, J.Q.
and Lee, V.M.Y. (2001) Age-dependent induction of congophilic
neurofibrillary tau inclusions in tau transgenic mice. Am. J. Pathol., 158,
555–562.
54. Ishihara, T., Hong, M., Zhang, B., Nakagawa, Y., Lee, M.K.,
Trojanowski, J.Q. and Lee, V.M.Y. (1999) Age-dependent emergence and
progression of a tauopathy in transgenic mice overexpressing the shortest
human tau isoform. Neuron, 24, 751 –762.
55. Zhang, B., Maiti, A., Shively, S., Lakhani, F., McDonald-Jones, G.,
Bruce, J., Lee, E.B., Xie, S.X., Joyce, S., Li, C. et al. (2005)
Microtubule-binding drugs offset tau sequestration by stabilizing
microtubules and reversing fast axonal transport deficits in a tauopathy
model. Proc. Natl Acad. Sci. USA, 102, 227–231.
56. Michaelis, M.L., Ansar, S., Chen, Y., Reiff, E.R., Seyb, K.I., Himes, R.H.,
Audus, K.L. and Georg, G.I. (2005) Beta-amyloid-induced
neurodegeneration and protection by structurally diverse microtubulestabilizing agents. J. Pharmacol. Experim. Therap., 312, 659 –668.
57. Ansley, S.J., Badano, J.L., Blacque, O.E., Hill, J., Hoskins, B.E.,
Leitch, C.C., Kim, J.C., Ross, A.J., Eichers, E.R., Teslovich, T.M. et al.
(2003) Basal body dysfunction is a likely cause of pleiotropic
Bardet–Biedl syndrome. Nature, 425, 628 –633.
58. Haggarty, S.J., Koeller, K.M., Wong, J.C., Butcher, R.A. and
Schreiber, S.L. (2003) Multidimensional chemical genetic analysis of
diversity-oriented synthesis-derived deacetylase inhibitors using
cell-based assays. Chem. Biol., 10, 383 –396.
59. Koeller, K.M., Haggarty, S.J., Perkins, B.D., Leykin, I., Wong, J.C.,
Kao, M.C.J. and Schreiber, S.L. (2003) Chemical genetic modifier
screens: small molecule trichostatin suppressors as probes of intracellular
histone and tubulin acetylation. Chem. Biol., 10, 397–410.