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1.  Activity of the Kinesin Spindle Protein Inhibitor Ispinesib (SB-715992) in Models of Breast Cancer 
Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein, a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have shown a 9% response rate in patients with locally advanced or metastatic breast cancer and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities, or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer, we explored the activity of ispinesib alone and in combination with several therapies approved for the treatment of breast cancer.
Experimental Design
We measured the ispinesib sensitivity and pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo and tested the ability of ispinesib to enhance the antitumor activity of approved therapies.
In vitro, ispinesib displayed broad antiproliferative activity against a panel of 53 breast cell lines. In vivo, ispinesib produced regressions in each of five breast cancer models and tumor-free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the antitumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine and exhibited activity comparable with paclitaxel and ixabepilone.
These findings support further clinical exploration of kinesin spindle protein inhibitors for the treatment of breast cancer.
PMCID: PMC2844774  PMID: 20068098
2.  Initial Testing (Stage 1) of the Kinesin Spindle Protein Inhibitor Ispinesib by the Pediatric Preclinical Testing Program 
Pediatric blood & cancer  2009;53(7):1255-1263.
Ispinesib is a highly specific inhibitor of kinesin spindle protein (KSP, HsEg5), a mitotic kinesin required for separation of the spindle poles. Here we report the activity of ispinesib against the in vitro and in vivo panels of the Pediatric Preclinical Testing Program (PPTP).
Ispinesib was tested against the PPTP in vitro panel cell lines at concentrations from 0.1 nM to 1 μM and against the in vivo tumor panel xenografts by intraperitoneal administration (5 or 10 mg/kg) every 4 days for 3 doses repeated at day 21.
Ispinesib was highly potent against the PPTP’s in vitro cell lines with a median IC50 of 4.1 nM. Ispinesib (10 mg/kg) induced unexplained toxicity in mice bearing osteosarcoma xenografts and exceeded the MTD in 12 of 40 non-osteosarcoma xenografts. Ispinesib induced significant tumor growth delay in 88% (23/26) of evaluable xenografts. Using a time to event measure of efficacy, ispinesib had intermediate and high levels of activity against 4 (21%) and 5 (26%) of the 19 evaluable solid tumor xenografts, respectively. Ispinesib induced maintained complete responses (CR) in a rhabdoid tumor, a Wilms tumor and a Ewing sarcoma xenograft. Ispinesib (5 mg/kg) produced 2 complete and 2 partial responses among 6 evaluable xenografts in the ALL panel. The in vivo pattern of activity was distinctive from that previously reported for vincristine.
Ispinesib demonstrated broad in vivo antitumor activity, including maintained complete responses for several xenografts, although with high toxicity rates at the doses studied.
PMCID: PMC2763980  PMID: 19554570
Preclinical Testing; Developmental Therapeutics; Ispinesib
3.  KIF11 inhibition for glioblastoma treatment: reason to hope or a struggle with the brain? 
BMC Cancer  2009;9:196.
Glioblastomas (GBM) are typically comprised of morphologically diverse cells. Despite current advances in therapy, including surgical resection followed by radiation and chemotherapy, the prognosis for patients with GBM remains poor. Unfortunately, most patients die within 2 years of diagnosis of their disease. Molecular abnormalities vary among individual patients and also within each tumor. Indeed, one of the distinguishing features of GBM is its marked genetic heterogeneity. Due to the brain location of the tumor, the potential target inhibition for anticancer therapy must exhibit a manageable neurotoxicity profile in the concentration range in which the compounds show anti-proliferative activity.
Kinesin KIF11 inhibition by small molecules such as Monastrol or Ispinesib is currently under investigation in the field of malignant tumors. In the current study we have assessed the relevance of the anti-mitotic Kinesin-like protein KIF11 in human GBM cell-lines.
In this study the target was validated using a set of well characterised and potentially specific small molecule inhibitors of KIF11: an ispinesib analog, Monastrol, a Merck compound and 3 simplified derivatives of the Merck compound. Following an in silico selection, those compounds predicted to bear a favorable BBB permeation profile were assessed for their phenotypic effect on cell lines derived both from primary (U87MG) as well as treated (DBTRG-05-MG) glioblastomas. For some compounds, these data could be compared to their effect on normal human astrocytes, as well as their neurotoxicity on primary rat cortical neurons. The ispinesib analogue 1 showed an anti-proliferative effect on GBM cell lines by blocking them in the G2/M phase in a concentration range which was shown to be harmless to primary rat cortical neurons. Furthermore, ispinesib analog increased caspase 3/7-induced apoptosis in U87MG cells.
In the area of cell cycle inhibition, KIF11 is critical for proper spindle assembly and represents an attractive anticancer target. Our results suggest that KIF11 inhibitors, when able to permeate the blood-brain-barrier, could represent an interesting class of anticancer drugs with low neurotoxic effects in the treatment of brain tumors.
PMCID: PMC2711111  PMID: 19545421
4.  Triphenylbutanamines: Kinesin Spindle Protein Inhibitors with in Vivo Antitumor Activity† 
Journal of Medicinal Chemistry  2012;55(4):1511-1525.
The human mitotic kinesin Eg5 represents a novel mitotic spindle target for cancer chemotherapy. We previously identified S-trityl-l-cysteine (STLC) and related analogues as selective potent inhibitors of Eg5. We herein report on the development of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from the STLC scaffold. This new generation systematically improves on potency: the most potent C-trityl analogues exhibit Kiapp ≤ 10 nM and GI50 ≈ 50 nM, comparable to results from the phase II clinical benchmark ispinesib. Crystallographic studies reveal that they adopt the same overall binding configuration as S-trityl analogues at an allosteric site formed by loop L5 of Eg5. Evaluation of their druglike properties reveals favorable profiles for future development and, in the clinical candidate ispinesib, moderate hERG and CYP inhibition. One triphenylbutanamine analogue and ispinesib possess very good bioavailability (51% and 45%, respectively), with the former showing in vivo antitumor growth activity in nude mice xenograft studies.
PMCID: PMC3428805  PMID: 22248262
5.  A Pediatric Phase 1 Trial and Pharmacokinetic Study of Ispinesib: A Children's Oncology Group Phase I Consortium Study 
Pediatric blood & cancer  2010;55(7):1323-1328.
To determine the maximum-tolerated dose, dose-limiting toxicities and pharmacokinetics of the kinesin spindle protein inhibitor ispinesib in pediatric patients with recurrent or refractory solid tumors.
Subjects and Methods
Ispinesib was administered as 1-hour intravenous infusion weekly × 3, every 28 days. Cohorts of 3-6 patients were enrolled at 5, 7, 9 and 12 mg/m2/dose. Serial plasma samples for pharmacokinetic analyses were obtained after the first dose.
Twenty-four (13 females) patients with a median (range) age of 10 years (1-19) were enrolled on the study. At the 12 mg/m2 dose level dose-limiting neutropenia occurred in 2/6 patients and hyperbilirubinemia in 1/6 patients, while at the 9 mg/m2 dose level 1/6 patients had dose-limiting neutropenia. There were no objective responses, but 3 patients (diagnoses of anaplastic astrocytoma, alveolar soft part sarcoma and ependymoblastoma) had stable disease for 4 to 7 courses. There was substantial inter-patient variation in drug disposition. The median (range) terminal elimination half-life was 16 hours (8-44) and the plasma drug clearance was 5 L/hr/m2 (1-14).
The maximum tolerated and recommended phase II dose for ispinesib administered weekly × 3 every 28 days for children with solid tumors is 9 mg/m2/dose. Plans for a phase II trial in select pediatric solid tumors are in development.
PMCID: PMC3053384  PMID: 20712019
Ispinesib; pediatric cancer; solid tumors; Children Oncology Group; phase 1 trials
6.  A phase I trial of ispinesib, a kinesin spindle protein inhibitor, with docetaxel in patients with advanced solid tumours 
British Journal of Cancer  2008;98(5):894-899.
The aim of this study is to define the maximum tolerated dose (MTD), safety, pharmacokinetics (PKs) and efficacy of ispinesib (SB-715992) in combination with docetaxel. Patients with advanced solid tumours were treated with ispinesib (6–12 mg m−2) and docetaxel (50–75 mg m−2). Docetaxel was administered over 1 h followed by a 1-h infusion of ispinesib on day 1 of a 21-day schedule. At least three patients were treated at each dose level. Blood samples were collected during cycle 1 for PK analysis. Clinical response assessments were performed every two cycles using RECIST guidelines. Twenty-four patients were treated at four dose levels. Prolonged neutropaenia and febrile neutropaenia were dose limiting in six and two patients, respectively. The MTD was ispinesib 10 mg m−2 with docetaxel 60 mg m−2. Pharmacokinetic assessment demonstrated concentrations of ispinesib and docetaxel, consistent with published data from single agent studies of the drugs. Seven patients (six hormone refractory prostate cancer (HRPC), one renal cancer) had a best response of stable disease (⩾18 weeks). One patient with HRPC had a confirmed >50% prostatic-specific antigen decrease. The MTD for ispinesib and docetaxel was defined and the combination demonstrated an acceptable toxicity profile. Preliminary PK data suggest no interaction between ispinesib and docetaxel.
PMCID: PMC2266864  PMID: 18319713
docetaxel; kinesin spindle protein; mitotic kinesin; phase I; SB-715992
7.  Optimized S-Trityl-l-cysteine-Based Inhibitors of Kinesin Spindle Protein with Potent in Vivo Antitumor Activity in Lung Cancer Xenograft Models 
Journal of Medicinal Chemistry  2013;56(5):1878-1893.
The mitotic kinesin Eg5 is critical for the assembly of the mitotic spindle and is a promising chemotherapy target. Previously, we identified S-trityl-l-cysteine as a selective inhibitor of Eg5 and developed triphenylbutanamine analogues with improved potency, favorable drug-like properties, but moderate in vivo activity. We report here their further optimization to produce extremely potent inhibitors of Eg5 (Kiapp < 10 nM) with broad-spectrum activity against cancer cell lines comparable to the Phase II drug candidates ispinesib and SB-743921. They have good oral bioavailability and pharmacokinetics and induced complete tumor regression in nude mice explanted with lung cancer patient xenografts. Furthermore, they display fewer liabilities with CYP-metabolizing enzymes and hERG compared with ispinesib and SB-743921, which is important given the likely application of Eg5 inhibitors in combination therapies. We present the case for this preclinical series to be investigated in single and combination chemotherapies, especially targeting hematological malignancies.
PMCID: PMC3759169  PMID: 23394180
8.  Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference 
PLoS ONE  2013;8(4):e60334.
ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [125I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC50 value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.
PMCID: PMC3622673  PMID: 23593196
9.  Pseudo-merohedral twinning and noncrystallographic symmetry in orthorhombic crystals of SIVmac239 Nef core domain bound to different-length TCRζ fragments 
P212121 crystals of SIV Nef core domain bound to a peptide fragment of the T-cell receptor ζ subunit exhibited noncrystallographic symmetry and nearly perfect pseudo-merohedral twinning simulating tetragonal symmetry. For a different peptide fragment, nontwinned tetragonal crystals were observed but diffracted to lower resolution. The structure was determined after assignment of the top molecular-replacement solutions to various twin or NCS domains followed by refinement under the appropriate twin law.
HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling ζ subunit of the T-­cell receptor (TCRζ). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nefcore) in complex with two different TCRζ fragments are described. The structure of SIVmac239 Nefcore bound to the longer TCRζ polypeptide (Leu51–Asp93) was determined to 3.7 Å resolution (R work = 28.7%) in the tetragonal space group P43212. The structure of SIVmac239 Nefcore in complex with the shorter TCRζ polypeptide (Ala63–Arg80) was determined to 2.05 Å resolution (R work = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P212121. The reduction in crystal space-group symmetry induced by the truncated TCRζ polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, −l) and a and b unit-cell parameters that were nearly identical predisposed the P212121 crystal form to pseudo-merohedral twinning.
PMCID: PMC2815668  PMID: 20124696
pseudo-merohedral twinning; noncrystallographic symmetry; pseudosymmetry; human immunodeficiency virus; Nef; T-cell receptor
10.  DrugTargetSeqR: a genomics- and CRISPR/Cas9-based method to analyze drug targets 
Nature chemical biology  2014;10(8):626-628.
To identify the physiological targets of drugs and bioactive small molecules we have developed an approach, named DrugTargetSeqR, which combines high-throughput sequencing, computational mutation discovery and CRISPR/Cas9-based genome editing. We apply this approach to ispinesib and YM155, drugs that have undergone clinical trials as anti-cancer agents, and demonstrate target identification and uncover genetic and epigenetic mechanisms likely to cause drug resistance in human cancer cells.
PMCID: PMC4123312  PMID: 24929528
11.  The Kinesin-12 Kif15 is a processive track-switching tetramer 
eLife  2014;3:e01724.
Kinesin-12 motors are a little studied branch of the kinesin superfamily with the human protein (Kif15) implicated in spindle mechanics and chromosome movement. In this study, we reconstitute full-length hKif15 and its microtubule-targeting factor hTpx2 in vitro to gain insight into the motors mode of operation. We reveal that hKif15 is a plus-end-directed processive homotetramer that can step against loads of up to 3.5 pN. We further show that hKif15 is the first kinesin that effectively switches microtubule tracks at intersections, enabling it to navigate microtubule networks, such as the spindle. hKif15 tetramers are also capable of cross-linking microtubules, but unexpectedly, this does not depend on hTpx2. Instead, we find that hTpx2 inhibits hKif15 stepping when microtubule-bound. Our data reveal that hKif15 is a second tetrameric spindle motor in addition to the kinesin-5 Eg5 and provides insight into the mechanisms by which hKif15 and its inhibitor hTpx2 modulate spindle microtubule architecture.
eLife digest
Before a cell can divide, it produces an extra copy of all its chromosomes, and it must then ensure that each daughter cell ends up with one copy of each chromosome. During the division process, a structure called the spindle forms in the cell. This spindle is made of thread-like extensions called microtubules that grow from two poles at opposite ends of the cell. These microtubules are responsible for getting the chromosomes to line up in the middle of the cell, and then pulling half of the chromosomes to one end of the cell, and half to the other end. The cell then divides into two daughter cells.
Two motor proteins—so-called because they consume chemical energy to ‘walk’ along the microtubules—have important roles in this process: Kif11 motor proteins mainly drive the formation of the spindle and thus division of the chromosomes. A cell that does not contain Kif11 can only divide if it contains extra copies of a second motor protein called Kif15: this suggests that Kif15 can serve as some sort of back up for Kif11.
Normal cells only divide when new cells are needed for growth or to replace old cells that have died. Cancer cells, on the other hand, divide in a way that is not controlled. Drugs that interfere with Kif11 have been developed in the hope that they will stop cancer cells dividing, but these drugs have not been very effective in clinical tests, possibly due to the Kif15 back up. Scientists hope, therefore, that a better understanding of the role of Kif15 may lead to improved cancer treatments.
Drechsler et al. have isolated individual Kif15 motor proteins and used advanced microscopy techniques to study them in action. These experiments showed that Kif15 motor proteins can travel long distances along a single microtubule, and can also switch to a different microtubule at intersections. This movement of Kif15 is stopped when they bump into Tpx2 proteins, which are sitting on the microtubules. Together, these proteins can also form links between microtubules that can withstand high forces. These properties provide a starting point to understand how Kif15 can act as a back up for Kif11 in cells. In the future, it will be important to work out how Kif11 and Kif15 motor proteins work together in teams to build the spindle.
PMCID: PMC3965211  PMID: 24668168
kinesin-12; mitotic spindle; mitosis; human
12.  Mapping the Structural and Dynamical Features of Kinesin Motor Domains 
PLoS Computational Biology  2013;9(11):e1003329.
Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes that mediate directed movement along microtubules. Characterizing these distinct conformations and their interconversion mechanism is essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of conventional and accelerated molecular dynamics simulations that together map the structural dynamics of the kinesin motor domain. All experimental structures were found to reside in one of three distinct conformational clusters (ATP-like, ADP-like and Eg5 inhibitor-bound). These groups differ in the orientation of key functional elements, most notably the microtubule binding α4–α5, loop8 subdomain and α2b-β4-β6-β7 motor domain tip. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. However, groupings were coincident with distinct neck-linker orientations. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicate that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were also evident. In multiple ATP bound simulations, the neck-linker, loop8 and the α4–α5 subdomain display correlated motions that are absent in ADP bound simulations. Further dissection of these couplings provides evidence for a network of dynamic communication between the active site, microtubule-binding interface and neck-linker via loop7 and loop13. Additional simulations indicate that the mutations G325A and G326A in loop13 reduce the flexibility of these regions and disrupt their couplings. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state and support a model where neck-linker docking leads to a tighter coupling of the microtubule and nucleotide binding regions. Furthermore, simulations highlight sites critical for large-scale conformational changes and the allosteric coupling between distal functional sites.
Author Summary
Kinesin motor proteins transport cargo along microtubule tracks to support essential cellular functions including cell growth, axonal signaling and the separation of chromosomes during cell division. All kinesins contain one or more conserved motor domains that modulate binding and movement along microtubules via cycles of ATP hydrolysis. Important conformational transitions occurring during this cycle have been characterized with extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in conformational change and microtubule interaction modulation remain unclear. Here we describe a comprehensive principal component analysis of available motor domain crystallographic structures supplemented with extensive unbiased conventional and accelerated molecular dynamics simulations that together characterize the response of kinesin motor domains to ATP binding and hydrolysis. Our studies reveal atomic details of conformational transitions, as well as novel nucleotide-dependent dynamical couplings, of distal regions and residues potentially important for the allosteric link between nucleotide and microtubule binding sites.
PMCID: PMC3820509  PMID: 24244137
13.  Probing Spindle Assembly Mechanisms with Monastrol, a Small Molecule Inhibitor of the Mitotic Kinesin, Eg5 
The Journal of Cell Biology  2000;150(5):975-988.
Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest–deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.
PMCID: PMC2175262  PMID: 10973989
monastrol; Eg5; kinesin; MAD2; kinetochore
14.  The Origin of Minus-end Directionality and Mechanochemistry of Ncd Motors 
PLoS Computational Biology  2012;8(11):e1002783.
Adaptation of molecular structure to the ligand chemistry and interaction with the cytoskeletal filament are key to understanding the mechanochemistry of molecular motors. Despite the striking structural similarity with kinesin-1, which moves towards plus-end, Ncd motors exhibit minus-end directionality on microtubules (MTs). Here, by employing a structure-based model of protein folding, we show that a simple repositioning of the neck-helix makes the dynamics of Ncd non-processive and minus-end directed as opposed to kinesin-1. Our computational model shows that Ncd in solution can have both symmetric and asymmetric conformations with disparate ADP binding affinity, also revealing that there is a strong correlation between distortion of motor head and decrease in ADP binding affinity in the asymmetric state. The nucleotide (NT) free-ADP (φ-ADP) state bound to MTs favors the symmetric conformation whose coiled-coil stalk points to the plus-end. Upon ATP binding, an enhanced flexibility near the head-neck junction region, which we have identified as the important structural element for directional motility, leads to reorienting the coiled-coil stalk towards the minus-end by stabilizing the asymmetric conformation. The minus-end directionality of the Ncd motor is a remarkable example that demonstrates how motor proteins in the kinesin superfamily diversify their functions by simply rearranging the structural elements peripheral to the catalytic motor head domain.
Author Summary
Proteins belonging to the kinesin superfamily are responsible for vesicle or organelle transport, spindle morphogenesis, and chromosome sorting during cell division. Interestingly, while most proteins in kinesin superfamily that share the common catalytic motor head domain have plus-end directionality along microtubules, kinesin-14 (Ncd) exhibits minus-end directionality. Despite the several circumstantial evidences on the determining factors for the motor directionality in the last decade, a comprehensive understanding of the mechanism governing the Ncd minus-end directionality is still missing. Our studies provide a clear explanation for this minus-end directionality and the associated mechanochemical cycle. Here, we modeled an Ncd motor by employing structural details available in the literature to simulate its conformational dynamics. Simulations using our structure-based model of Ncd assert that the dynamics due to a simple rearrangement of structural elements, peripheral to the catalytic motor domain, is the key player in determining both the directionality and mechanochemistry unique to Ncd motors. Although Ncd has a different directionality, it uses a similar strategy to kinesin-1 of structural adaptation of the catalytic motor domain. Therefore using the same physical principle of protein folding and very similar structural elements, motors in the kinesin superfamily are able to achieve a variety of biological function.
PMCID: PMC3499263  PMID: 23166486
15.  The crystal structure and biochemical characterization of Kif15: a bifunctional molecular motor involved in bipolar spindle formation and neuronal development 
The structural and biochemical study of Kif15 provides insight into this potential drug target and allows comparison with Eg5, a kinesin that partially shares the functions of Kif15.
Kinesins constitute a superfamily of microtubule-based motor proteins with important cellular functions ranging from intracellular transport to cell division. Some kinesin family members function during the mitotic phase of the eukaryotic cell cycle and are crucial for the successful progression of cell division. In the early stages of mitosis, during prometaphase, certain kinesins are required for the formation of the bipolar spindle, such as Eg5 and Kif15, which seem to possess partially overlapping functions. Because kinesins transform the chemical energy from ATP hydrolysis into mechanical work, inhibition of their function is a tractable approach for drug development. Drugs targeting Eg5 have shown promise as anticancer agents. Kif15 has recently come to the fore because it can substitute the functions of Eg5, and may itself have potential as a prospective drug target. Here, the initial biochemical, kinetic and structural characterization of Kif15 is reported and it is compared with the functionally related motor Eg5. Although Kif15 contains ADP in the catalytic site, its motor-domain structure was captured in the ‘ATP-like’ configuration, with the neck linker docked to the catalytic core. The interaction of Kif15 with microtubules was also investigated and structural differences between these two motors were elucidated which indicate profound differences in their mode of action, in agreement with current models of microtubule cross-linking and sliding.
PMCID: PMC3919264  PMID: 24419385
human kinesins; mitosis; bipolar spindle formation; Kif15; Eg5
16.  Mitotic Spindle Assembly around RCC1-Coated Beads in Xenopus Egg Extracts 
PLoS Biology  2011;9(12):e1001225.
Beads coated with the guanine nucleotide exchange factor RCC1 and a kinesin motor protein are sufficient to induce mitotic spindle assembly in Xenopus egg cytoplasm.
During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 µm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.
Author Summary
The mitotic spindle is a bipolar structure that is responsible for separating the two sets of duplicated chromosomes in a dividing cell, thereby delivering one set to each of the two daughter cells. It is built from dynamic filaments called microtubules, as well as hundreds of other components that contribute to the organization and dynamics of the microtubules and to chromosome movement. To understand which proteins are essential for spindle formation and function, we would like to be able to build it from purified components. As a step towards this goal, we coupled individual proteins to inert glass beads (as a substitute for chromosomes), to determine what combination of proteins can induce spindle assembly in a complex cytoplasm derived from frog eggs. We found that a single enzyme called RCC1 is sufficient to activate a pathway that stabilizes and organizes microtubules into a bipolar structure around the bead, but that this bead then oscillated back and forth between the poles of the spindle. By coupling a microtubule-based motor protein together with RCC1 on the bead, we were able to balance the bead in the center of the spindle. Thus, two proteins immobilized on a bead can substitute for a chromosome and induce stable spindle formation.
PMCID: PMC3246454  PMID: 22215983
17.  B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast 
PLoS Genetics  2010;6(5):e1000935.
Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.
Author Summary
The assembly of a bipolar mitotic spindle is essential for the accurate segregation of sister chromatids during mitosis and, hence, for successful cell division. Spindle assembly depends on the successful duplication of the spindle poles, followed by their separation to opposing ends of the cell. Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle, the relevant CDK targets have not been identified. Motor proteins of the kinesin-5 family generate movement on the microtubules that make up the spindle and are believed to power spindle pole separation. By employing the budding yeast Saccharomyces cerevisiae as a model, we have found evidence that cyclin/CDKs control spindle assembly by phosphorylating the kinesins-5 Kip1 and Cin8. When phosphorylation at a conserved CDK site in the motor domain of Kip1 is blocked, spindle pole separation is greatly diminished but neither protein abundance nor localization is affected. We have also obtained direct evidence by mass spectrometry that Kip1 and Cin8 are phosphorylated in vivo at consensus CDK sites in their tail domains. Our findings suggest that B-cyclin/CDKs regulate spindle assembly by regulating kinesin-5 motor activity.
PMCID: PMC2865516  PMID: 20463882
18.  A seesaw model for intermolecular gating in the kinesin motor protein 
Biophysical Reviews  2011;3(2):85-100.
Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine. Kinesin-1, similar to many kinesin family members, assembles to form homodimers that use alternating ATPase cycles of the catalytic motor domains, or “heads”, to proceed unidirectionally along its partner filament (the microtubule) via a hand-over-hand mechanism. Cryo-electron microscopy has now revealed 8-Å resolution, 3D reconstructions of kinesin-1•microtubule complexes for all three of this motor’s principal nucleotide-state intermediates (ADP-bound, no-nucleotide, and ATP analog), the first time filament co-complexes of any cytoskeletal motor have been visualized at this level of detail. These reconstructions comprehensively describe nucleotide-dependent changes in a monomeric head domain at the secondary structure level, and this information has been combined with atomic-resolution crystallography data to synthesize an atomic-level "seesaw" mechanism describing how microtubules activate kinesin’s ATP-sensing machinery. The new structural information revises or replaces key details of earlier models of kinesin’s ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin–microtubule complex is essential for understanding the structural basis of the cycle. I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement.
PMCID: PMC3117274  PMID: 21765878
Kinesin; Microtubule; Motor protein; ATPase; Cryo-electron microscopy; Structure
Journal of theoretical biology  2011;289:107-115.
Loop 5 (L5) is a conserved loop that projects from the α2-helix adjacent to the nucleotide site of all kinesin-family motors. L5 is critical to the function of the mitotic kinesin-5 family motors and is the binding site for several kinesin-5 inhibitors that are currently in clinical trials. Its conformational dynamics and its role in motor function are not fully understood. Our previous work using EPR spectroscopy suggested that L5 alters the nucleotide pocket conformation of the kinesin-5 motor Eg5 [1]. EPR spectra of a spin-labeled nucleotide analog bound at the nucleotide site of Eg5 display a highly immobilized component that is absent if L5 is shortened or if the inhibitor STLC is added [1], which X-ray structures suggest stabilizes a L5 conformation pointing away from the nucleotide site. These data, coupled with the proximity of L5 to the nucleotide site suggest L5 could interact with a bound nucleotide, modulating function. Here we use molecular dynamics (MD) simulations of Eg5 to explore the interaction of L5 with the nucleotide site in greater detail. We performed MD simulations in which the L5-domain of the Eg5•ADP X-ray structure was manually deformed via backbone bond rotations. The L5-domain of Eg5 was sufficiently lengthy that portions of L5 could be located in proximity to bound ADP. The MD simulations evolved to thermodynamically stable structures at 300K showing that L5 can interact directly with bound nucleotide with significant impingement on the ribose hydroxyls, consistent with the EPR spectroscopy results. Taken together, these data provide support for the hypothesis that L5 modulates Eg5 function via interaction with the nucleotide-binding site.
PMCID: PMC3191284  PMID: 21872609
kinesin-5; nucleotide-analog spin probes; EPR spectroscopy; molecular dynamics simulation
20.  Structure of a monoclinic polymorph of human carbonic anhydrase II with a doubled a axis 
The crystal structure of human carbonic anhydrase II with a doubled a axis from that of the usually observed monoclinic cell has been determined and refined to 1.4 Å resolution. The two molecules comprising the asymmetric unit are related by a noncrystallographic translation of ½ along a, but one of the molecules has two alternate orientations related by a rotation of approximately 2°.
The crystal structure of human carbonic anhydrase II with a doubled a axis from that of the usually observed monoclinic unit cell has been determined and refined to 1.4 Å resolution. The diffraction data with h = 2n + 1 were systematically weaker than those with h = 2n. Consequently, the scaling of the data, structure solution and refinement were challenging. The two molecules comprising the asymmetric unit are related by a noncrystallographic translation of ½ along a, but one of the molecules has two alternate positions related by a rotation of approximately 2°. This rotation axis is located near the edge of the central β-sheet, causing a maximum distance disparity of 1.7 Å between equivalent atoms on the diametrically opposite side of the molecule. The crystal-packing contacts are similar to two sequential combined unit cells along a of the previously determined monoclinic unit cell. Abnormally high final R cryst and R free values (20.2% and 23.7%, respectively) are not unusual for structures containing pseudo-translational symmetry and probably result from poor signal to noise in the weak h-odd data.
PMCID: PMC2865368  PMID: 20445238
doubled axis; systematically weak data; crystal contacts; translational symmetry; rotational disorder
21.  Common Mechanistic Themes for the Powerstroke of Kinesin-14 motors 
Journal of structural biology  2013;184(2):10.1016/j.jsb.2013.09.020.
Kar3Cik1 is a heterodimeric kinesin-14 from Saccharomyces cerevisiae involved in spindle formation during mitosis and karyogamy in mating cells. Kar3 represents a canonical kinesin motor domain that interacts with microtubules under the control of ATP-hydrolysis. In vivo, the localization and function of Kar3 is differentially regulated by its interacting stoichiometrically with either Cik1 or Vik1, two closely related motor homology domains that lack the nucleotide-binding site. Indeed, Vik1 structurally resembles the core of a kinesin head. Despite being closely related, Kar3Cik1 and Kar3Vik1 are each responsible for a distinct set of functions in vivo and also display different biochemical behavior in vitro. To determine a structural basis for their distinct functional abilities, we used cryo-electron microscopy and helical reconstruction to investigate the 3-D structure of Kar3Cik1 complexed to microtubules in various nucleotide states and compared our 3-D data of Kar3Cik1 with that of Kar3Vik1 and the homodimeric kinesin-14 Ncd from Drosophila melanogaster. Due to the lack of an X-ray crystal structure of the Cik1 motor homology domain, we predicted the structure of this Cik1 domain based on sequence similarity to its relatives Vik1, Kar3 and Ncd. By molecular docking into our 3-D maps, we produced a detailed near-atomic model of Kar3Cik1 complexed to microtubules in two distinct nucleotide states, a nucleotide-free state and an ATP-bound state. Our data show that despite their functional differences, heterodimeric Kar3Cik1 and Kar3Vik1 and homodimeric Ncd, all share striking structural similarities at distinct nucleotide states indicating a common mechanistic theme within the kinesin-14 family.
PMCID: PMC3851574  PMID: 24099757
Kinesin-14; Kar3Cik1; Microtubules; Helical 3-D analysis; Molecular docking; Cryo-electron microscopy
22.  Localization and function of Kinesin-5-like proteins during assembly and maintenance of mitotic spindles in Silvetia compressa 
BMC Research Notes  2009;2:106.
Kinesin-5 (Eg-5) motor proteins are essential for maintenance of spindle bipolarity in animals. The roles of Kinesin-5 proteins in other systems, such as Arabidopsis, Dictyostelium, and sea urchin are more varied. We are studying Kinesin-5-like proteins during early development in the brown alga Silvetia compressa. Previously, this motor was shown to be needed to assemble a bipolar spindle, similar to animals. This report builds on those findings by investigating the localization of the motor and probing its function in spindle maintenance.
Anti-Eg5 antibodies were used to investigate localization of Kinesin-5-like proteins in brown algal zygotes. In interphase zygotes, localization was predominantly within the nucleus. As zygotes entered mitosis, these motor proteins strongly associated with spindle poles and, to a lesser degree, with the polar microtubule arrays and the spindle midzone. In order to address whether Kinesin-5-like proteins are required to maintain spindle bipolarity, we applied monastrol to synchronized zygotes containing bipolar spindles. Monastrol is a cell-permeable chemical inhibitor of the Kinesin-5 class of molecular motors. We found that inhibition of motor function in pre-formed spindles induced the formation of multipolar spindles and short bipolar spindles.
Based upon these localization and inhibitor studies, we conclude that Kinesin-5-like motors in brown algae are more similar to the motors of animals than those of plants or protists. However, Kinesin-5-like proteins in S. compressa serve novel roles in spindle formation and maintenance not observed in animals.
PMCID: PMC2706839  PMID: 19527496
23.  NSC 622124 Inhibits Human Eg5 and Other Kinesins Via Interaction with the Conserved Microtubule-Binding Site 
Biochemistry  2009;48(8):1754-1762.
Kinesin-5 proteins are essential for formation of a bipolar mitotic spindle in most, and perhaps all, eukaryotic cells. Several Kinesin-5 proteins, notably the human version, HsEg5, are targets of a constantly expanding group of small-molecule inhibitors, which hold promise both as tools to probe mechanochemical transduction and as anti-cancer agents. Although most such compounds are selective for HsEg5 and closely related Kinesin-5 proteins, some, such as NSC 622124, exhibit activity against at least one kinesin from outside the Kinesin-5 family. Here we show NSC 622124, despite identification in a screen that yielded inhibitors now known to target the HsEg5 monastrol-binding site, does not compete with 14C-monastrol for binding to HsEg5, and is able to inhibit the basal and microtubule-stimulated ATPase activity of the monastrol-insensitive Kinesin-5, KLP61F. NSC 622124 competes with microtubules, but not ATP, for interaction with HsEg5, and disrupts the microtubule binding of HsEg5, KLP61F and Kinesin-1. Proteolytic degradation of an HsEg5•NSC622124 complex revealed that segments of the α3 and α5 helices map to the inhibitor-binding site. Overall, our results demonstrate that NSC 622124 targets the conserved microtubule-binding site of kinesin proteins. Further, unlike compounds previously reported to target the kinesin microtubule-binding site, NSC 622124 does not produce any enhancement of basal ATPase activity, and thus acts solely as a negative regulator through interaction with a site traditionally viewed as a binding region for positive regulators (i.e., microtubules). Our work emphasizes the concept that microtubule-dependent motor proteins may be controlled at multiple sites by both positive and negative effectors.
PMCID: PMC3244877  PMID: 19236100
24.  Computational Studies of the Farnesyltransferase Ternary Complex Part I: Substrate Binding† 
Biochemistry  2005;44(50):16513-16523.
Farnesyltransferase (FTase) catalyzes the transfer of farnesyl from farnesyldiphosphate (FPP) to cysteine residues at or near the C-terminal of protein acceptors with a CaaX motif (a, aliphatic; X, Met). Farnesylation is a critical modification to many switch proteins involved in extracellular signal transduction pathway, which facilitates their fixation on the cell membrane where the extracellular signal is processed. The malfunction caused by mutations in these proteins often causes uncontrolled cell reproduction and leads to tumor formation. FTase inhibitors have been extensively studied as potential anticancer agents in recent years, several of which have advanced to different phases of clinical trials. However, the mechanism of this biologically important enzyme has not been firmly established. Understanding how FTase recruits the FPP substrate is the first and foremost step towards further mechanistic investigations and the design of effective FTase inhibitors. Molecular dynamic simulations were carried out on the ternary structure of FTase complexed with the FPP substrate and an acetyl-capped tetrapeptide (acetyl-CVIM), which revealed that the FPP substrate maintains an inactive conformation and the binding of the diphosphate group can be largely attributed to residues R291β, K164α, K294β, and H248β. The FPP substrate assumes an extended conformation in the binding site with restricted rotation of the backbone dihedral angles; however, it does not have a well defined conformation when unbound in solution. This is evident from multinanosecond MD simulations of the FPP substrate in vacuum and solution. Our conclusion is further supported by theoretical j-coupling calculations. Our results on the FPP binding are in good agreement with previous experimental kinetic studies on FTase mutants. The hypothetical conformational activation of the FPP substrate is currently under investigation.
PMCID: PMC2566548  PMID: 16342942
25.  Effective killing of the human pathogen Candida albicans by a specific inhibitor of non-essential mitotic kinesin Kip1p 
Molecular Microbiology  2007;65(2):347-362.
Kinesins from the bipolar (Kinesin-5) family are conserved in eukaryotic organisms and play critical roles during the earliest stages of mitosis to mediate spindle pole body separation and formation of a bipolar mitotic spindle. To date, genes encoding bipolar kinesins have been reported to be essential in all organisms studied. We report the characterization of CaKip1p, the sole member of this family in the human pathogenic yeast Candida albicans. C. albicans Kip1p appears to localize to the mitotic spindle and loss of CaKip1p function interferes with normal progression through mitosis. Inducible excision of CaKIP1 revealed phenotypes unique to C. albicans, including viable homozygous Cakip1 mutants and an aberrant spindle morphology in which multiple spindle poles accumulate in close proximity to each other. Expression of the C. albicans Kip1 motor domain in Escherichia coli produced a protein with microtubule-stimulated ATPase activity that was inhibited by an aminobenzothiazole (ABT) compound in an ATP-competitive fashion. This inhibition results in ‘rigor-like’, tight association with microtubules in vitro. Upon treatment of C. albicans cells with the ABT compound, cells were killed, and terminal phenotype analysis revealed an aberrant spindle morphology similar to that induced by loss of the CaKIP1 gene. The ABT compound discovered is the first example of a fungal spindle inhibitor targeted to a mitotic kinesin. Our results also show that the non-essential nature and implementation of the bipolar motor in C. albicans differs from that seen in other organisms, and suggest that inhibitors of a non-essential mitotic kinesin may offer promise as cidal agents for antifungal drug discovery.
PMCID: PMC1976386  PMID: 17573815

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