This Issue Contains
Q1 2 0 1 5
Future Topics
Quarter 2, 2015
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Biopharmaceutical companies focus on screening compounds
from multiples sources looking for broad spectrum anti-fungals
that target KSP homologs. Model fungal cells include Saccharomyces cerevisiae and Aspergillus nidulans which are used in conjunction with mammalian cells to confirm species selectivity and
lack of off-target effects4.
Besides fungi, parasites are also a focus of KSP inhibitor research.
L. Liu et al3 screened compounds for their efficacy at targeting
KSP homologs in Plasmodium falciparum and P. vivax to evalulate
malaria treatments. The authors exploited the KSP allosteric site
characterized by Loop 5 (L5). L5 is an element in the a2 helix of
the KSP motor domain that defines an allosteric site consisting of
a conserved region along with the L5 element which significantly
varies in length and sequence across kinesins and/or KSP homologs (Table 1). These variations confer inhibitor selectivity, making
L5 an ideal target for allosteric inhibitors. Indeed, it helps form
the surface pocket that the KSP inhibitor monastrol binds8,9. Thus,
the L5 element offers excellent drug specificity and prevents
cross-reactivity between different species3.
Inhibitors were screened for their ability to modulate the basal
and MT-stimulated ATPase activity of KSP homologs from Plasmodium falciparum and P. vivax as well as human KSP to confirm
each inhibitor’s specificity for the parasitic kinesin and lack of offtarget effects3. Three different classes of Plasmodium KSP inhibitors were identified: 1). those selective for Plasmodium kinesins;
2). those that inhibited both parasite and human KSP; and 3).
those selective for human KSP. Determination of the inhibitors’
binding site/sites on KSP is underway.
HsKSP
MEGERSPNEEYT-------------------WE--EDPLAGI
PvKSP
MEGKILEHLKQYDNNKKVDLNESINSDISYCYELCENEDTGL
PfKSP
MEGRILEHLKHAEG-KKVDLSDSVNSDINYYYELCDSDDTGI
Hs: human; Pv: P. vivax; Pf: P. falciparum. Adapted from ref. 3.
Cytoskeleton Kinesin Motors and Custom Services
At Cytoskeleton, we have recombinant kinesin motor domains
that include the Eg5 homolog BimC motor domain proteins from
the filamentous fungi A. nidulans (Cat. # BM01) and A. fumigatus (Cat. # EG02). We also offer compound screening assays with
these kinesins or as part of a multi-motor protein screen. Moreover, if the kinesin protein or assay is unavailable, we offer custom
protein expression/purification and assay development services.
In combination with our kinesin panel screen, we can also evaluate compounds for their effects on microtubule polymerization
to identify the mechanism of action for anti-parasitic and antifungal compounds coming from phenotypic screens and/or as a
useful counterscreen for kinesin inhibitor drug discovery efforts
that desire to steer their SAR efforts away from compound effects
on tubulin polymerization or species cross-reactivity.
The Role of KSP (Eg5) in Cell Division
+
+
+ Eg5
+
+
+
+
+
+
+
+
+
+
+
+
+
metaphase
spindle
Figure 1 Schematic representation of the mitotic spindle and KSP (Eg5) crosslinking spindle microtubules. Adapted from ref. 2.
+
chromosome
+
References
1. Sarli V. and Giannis A. 2008. Targeting the kinesin spindle protein: Basic
principles and clinical implications. Clin. Cancer Res. 14, 7583-7587.
2. Wojcik E.J. et al. 2013. Kinesin-5: Cross-bridging mechanism to targeted
clinical therapy. Gene. 531, 133-149.
3. Liu L. et al. 2014. Small-molecule screen for candidate antimalarials targeting Plasmodium kinesin-5. J. Biol. Chem. 289, 16601-16614.
4. Nislow C.E. et al. “Antifungal assay”. US Patent 6,284,480. 4 September
2001.
5. Cytoskeleton 2014 Q2 CSD Newsletter. KSP/Eg5 Inhibition in Cancer:
Theory and Therapy. www.cytoskeleton.com.
6. Cytoskeleton 2014 Q3 CSD Newsletter. Drugable site selection for KSP
inhibitors. www.cytoskeleton.com.
7. Waitzman J.S. et al. 2011. The loop 5 element structurally and kinetically coordinates dimers of the kuman kinesin-5, Eg5. Biophys. J. 101,
2760-2769.
8. Yan Y. et al. 2004. Inhibition of a mitotic motor protein: Where, how,
and conformational consequences. J. Mol. Biol. 335, 547–554.
9. Liu L. et al. 2011. Loop 5-directed compounds inhibit chimeric kinesin-5 motors. Implications for conserved allosteric mechanisms. J. Biol.
Chem. 286, 6201-6210.
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KSP: A Treatment Target for Fungal and Parasitic Diseases
Drugs that target KSP are attractive because the prevalent treatments either produce deleterious side effects or the infectious
organisms have developed resistance to currently utilized drugs.
The human KSP protein has multiple druggable allosteric sites3,
raising hopes that KSP homologs in parasites and fungi can be targeted for therapeutic intervention.
Table 1. Sequence Alignment for Loop 5 of KSP Homologs
Citations
Motor Proteins
KSP (Eg5) Inhibition – Therapeutic Target for Multiple Diseases
The Kinesin Spindle Protein (KSP; a.k.a. Eg5 or KIF11) is a plus enddirected Kinesin-5 (a.k.a. BimC) subfamily member and has been
the focus of significant drug development efforts for decades.
Currently, KSP (or its homologs) is a target for anti-mitotics (cancer)1,2, anti-parasitics (malaria)3, and anti-fungals4. As a microtubule (MT) cross-linking enzyme, KSP plays a critical role in mitotic
spindle pole separation, and its inhibition results in the formation
of monoaster spindles which is thought to lead to mitotic catastrophe and apoptosis (Fig. 1). The targeting of KSP as a treatment
for cancer is well-documented1,2,5,6. The purpose of this newsletter is to briefly discuss KSP homologs as a therapeutic target for
parasitic and fungal diseases.
News
TBD
KSP/Eg5 Inhibition in Parasites and Fungi
Related Citations
Custom Modules
Custom Modules
Our recently expanded Custom Services Department provides additional resources for your research projects.
Cytoskeleton is leading the way to develop novel kinesin, dynein, and myosin based compound screens.
We are scientists dedicated to providing accurate data reported in a detailed and timely manner.
Clients Include:
About Custom Services
Like our regular product offerings, the Custom
Services Department emphasizes quality
products and services. We understand
that accuracy and timeliness are critical
elements for a successful project. Choose
from more than twenty defined modules
(for a full list, visit www.cytoskeleton.
com/custom-services), and then contact
Technical Support (tservice@cytoskeleton.
com) to guide you through the process.
•Merck & Co., Inc.
•Eli Lilly & Co.
•Amgen, Inc.
•Abbott Laboratories
•Pfizer, Inc.
•Astra-Zeneca plc
•GlaxoSmithKline plc
•Genentech, Inc.
•Johnson & Johnson
•Bristol-Meyers Squibb
Let’s get started, it’s as easy as 1,2,3 ...
1. Choose a module and ask for a quote (24h turn around time)
2. Review quote, specifications, and deliverables
3. Place order and receive regular updates until project is finished
Compound Screening Modules
Type
Format
Deliverable
Module # Timeline
(wks)
Eg5 Kinesin motor assay
Microtubule stimulated ATPase assay,
kinetic, absorbance at 360nm
96 assays, consisting of 40 duplicate single concentrations (or 5 x
IC50s), plus eight control wells. PDF Report with Executive Summary,
Introduction, Methods, Results and Data Analysis.
CDS050 or
CDS051
2
CDS056
2
CDS065
2
Ca2+/Sarcomere (thin filament)
Cardiac Myosin motor assay stimulated ATPase assay, kinetic,
absorbance at 360nm
Dynein motor assay
Tubulin polymerization
Same as CDS052.
Microtubule stimulated ATPase assay,
kinetic, absorbance at 360nm
Same as CDS052.
Tubulin (>99% pure) Polymerization
Assay, kinetic, fluorescence at
360nm/410nm
96 assays, with 40 duplicate single concentrations or 5 x IC50s, plus
CDS009 or
eight control wells (vinblastine, nocodazole or taxol). PDF Report with CDS010
Executive Summary, Introduction, Methods, Results and Data Analysis.
2
60 assays consisting of either 28 duplicate reactions plus 4 controls,
or 5 x IC50s plus 1 x control IC50. PDF report with Executive Summary,
Introduction, Methods, Results and Data Analysis.
2
GTP exchange factor plus Small
G-protein (e.g. Rho or Ras) with mantGEF/GTPase exchange assay GTP reporter. Kinetic, fluorescence at
360nm/450nm
CDS100
Gene Cloning and Protein Purification Modules
Type
Name
Recombinant Small Protein
Small protein or protein
Highly purified, His-tagged active protein lyophilized in 10 x 100 µg aliquots
domain (<30 kDa) with gene (or more depending on yield). Datasheet and assay method. Activity in line
provided by client
with published articles. E. coli expression.
Recombinant Small Protein
plus cloning
Small protein or protein
domain (<30 kDa) including
gene synthesis
Recombinant Kinesin Motor- Medium to large protein
Protein
or protein domain (30-100
kDa)
Recombinant Kinesin Motor edium to large protein or
Protein plus gene cloning
protein domain (30-100
kDa) with gene synthesis
Native or eukaryotic protein
Cited protein purification
expression & purification
Deliverable
Module #
Timeline (wks)
REC012
3
Same as above with gene synthesis.
REC022
6
Same as REC012.
REC032
3
Same as above with gene synthesis.
REC042
8
Same as above plus using a published procedure.
REC052
4-20
Nucleotide exchange (RFU)
Assay Development Modules
5500
Cdc42 + Dbs
Type
5000
4500
4000
3500
Cdc42 only
3000
2500
0
300
600
900
1200
Time (s)
1500
1800
Name
Deliverable
Module #
Timeline (wks)
GTP Exchange
G-protein GTP exchange
(fluor. kinetic, 360nm/460nm) assay using Mant-GTP
Report with optimized protocol, based on data from titrating four variables
([ionic], [MgCl2], [Mant-GTP] and temp.).
DEV026
4
GTPase assay
(abs, endpoint, 650nm)
GTP hydrolysis assay,
detecting phosphate
Same as above, except [Mant-GTP] is replaced by [G-protein] and if available [GAP protein].
DEV031
4
Motor ATPase
(abs, kinetic, 360nm)
ATP hydrolysis assay,
detecting phosphate
Report with optimized protocol, based on data from titrating five variables
([ionic], [MgCl2], [Motor], [microtubules] and temp.).
DEV034
4
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