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KSP (Eg5) Inhibion – Therapeuc Target for Mulple Diseases The Kinesin Spindle Protein (KSP; a.k.a. Eg5 or KIF11) is a plus end- directed 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 an-mitocs (can- cer) 1,2 , an-parasics (malaria) 3 , and an-fungals 4 . As a microtu- bule (MT) cross-linking enzyme, KSP plays a crical role in mitoc spindle pole separaon, and its inhibion results in the formaon of monoaster spindles which is thought to lead to mitoc catas- trophe and apoptosis (Fig. 1). The targeng of KSP as a treatment for cancer is well-documented 1,2,5,6 . The purpose of this newslet- ter is to briefly discuss KSP homologs as a therapeuc target for parasic and fungal diseases. KSP: A Treatment Target for Fungal and Parasic Diseases Drugs that target KSP are aracve because the prevalent treat- ments either produce deleterious side effects or the infecous organisms have developed resistance to currently ulized drugs. The human KSP protein has mulple druggable allosteric sites 3 , raising hopes that KSP homologs in parasites and fungi can be tar- geted for therapeuc intervenon. Biopharmaceucal companies focus on screening compounds from mulples sources looking for broad spectrum an-fungals that target KSP homologs. Model fungal cells include Saccharo- myces cerevisiae and Aspergillus nidulans which are used in con- juncon with mammalian cells to confirm species selecvity and lack of off-target effects 4 . Besides fungi, parasites are also a focus of KSP inhibitor research. L. Liu et al 3 screened compounds for their efficacy at targeng 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 consisng of a conserved region along with the L5 element which significantly varies in length and sequence across kinesins and/or KSP homo- logs (Table 1). These variaons confer inhibitor selecvity, making L5 an ideal target for allosteric inhibitors. Indeed, it helps form the surface pocket that the KSP inhibitor monastrol binds 8,9 . Thus, the L5 element offers excellent drug specificity and prevents cross-reacvity between different species 3 . Inhibitors were screened for their ability to modulate the basal and MT-smulated ATPase acvity of KSP homologs from Plasmo- dium falciparum and P. vivax as well as human KSP to confirm each inhibitor’s specificity for the parasic kinesin and lack of off- target effects 3 . Three different classes of Plasmodium KSP inhibi- tors were idenfied: 1). those selecve for Plasmodium kinesins; 2). those that inhibited both parasite and human KSP; and 3). those selecve for human KSP. Determinaon of the inhibitors’ binding site/sites on KSP is underway. Table 1. Sequence Alignment for Loop 5 of KSP Homologs 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. fumiga- tus (Cat. # EG02). We also offer compound screening assays with these kinesins or as part of a mul-motor protein screen. More- over, if the kinesin protein or assay is unavailable, we offer custom protein expression/purificaon and assay development services. In combinaon with our kinesin panel screen, we can also evalu- ate compounds for their effects on microtubule polymerizaon to idenfy the mechanism of acon for an-parasic and an- fungal 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 polymerizaon or species cross-reacvity. The Role of KSP (Eg5) in Cell Division Figure 1 Schemac representaon of the mitoc spindle and KSP (Eg5) cross- linking spindle microtubules. Adapted from ref. 2. References 1. Sarli V. and Giannis A. 2008. Targeng the kinesin spindle protein: Basic principles and clinical implicaons. 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 anmalarials tar- geng Plasmodium kinesin-5. J. Biol. Chem. 289, 16601-16614. 4. Nislow C.E. et al. “Anfungal assay”. US Patent 6,284,480. 4 September 2001. 5. Cytoskeleton 2014 Q2 CSD Newsleer. KSP/Eg5 Inhibion in Cancer: Theory and Therapy. www.cytoskeleton.com. 6. Cytoskeleton 2014 Q3 CSD Newsleer. Drugable site selecon for KSP inhibitors. www.cytoskeleton.com. 7. Waitzman J.S. et al. 2011. The loop 5 element structurally and kine- cally coordinates dimers of the kuman kinesin-5, Eg5. Biophys. J. 101, 2760-2769. 8. Yan Y. et al. 2004. Inhibion of a mitoc motor protein: Where, how, and conformaonal consequences. J. Mol. Biol. 335, 547–554. 9. Liu L. et al. 2011. Loop 5-directed compounds inhibit chimeric kine- sin-5 motors. Implicaons for conserved allosteric mechanisms. J. Biol. Chem. 286, 6201-6210. News Citaons Custom Modules www.cytoskeleton.com Future Topics Quarter 2, 2015 TBD Catalog Products Actin Proteins Activation Assays Antibodies ECM Proteins ELISA Kits G-LISA ® Kits Pull-down Assays Motor Proteins Small G-Proteins Tubulin & FtsZ Proteins Contact Us P: 1 (303) 322.2254 F: 1 (303) 322.2257 [email protected] www.cytoskeleton.com This Issue Contains KSP/Eg5 Inhibition in Parasites and Fungi Related Citations Custom Modules Q1 2015 + + + + + + + + + + + + + + + + + + Eg5 metaphase spindle chromosome
