The depolymerase activity of MCAK shows a graded response to Aurora B kinase phosphorylation through allosteric regulation.

Kinesin-13 motors regulate precise microtubule dynamics and limit microtubule length throughout metazoans by depolymerizing microtubule ends. Recently, the kinesin-13 motor family member MCAK (also known Kif2C) has been proposed to undergo large conformational changes during its catalytic cycle, as it switches from being in solution to being bound to microtubules. Here, we reveal that MCAK has a compact conformation in solution through crosslinking and electron microscopy experiments. When MCAK is bound to the microtubule ends, it adopts an extended conformation with the N-terminus and neck region of MCAK interacting with the microtubule. Interestingly, the region of MCAK that interacts with the microtubule is the region phosphorylated by Aurora B and contains an end binding (EB) protein-binding motif. The level of phosphorylation of the N-terminus results in a graded microtubule depolymerase activity. Here, we show that the N-terminus of MCAK forms a platform to integrate Aurora B kinase downstream signals and in response fine-tunes its depolymerase activity during mitosis. We propose that this allosteric control mechanism allows decoupling of the N-terminus from the motor domain of MCAK to allow MCAK depolymerase activity at kinetochores.

Modified Peptide Inhibitors of the Keap1-Nrf2 Protein-Protein Interaction Incorporating Unnatural Amino Acids.

Noncovalent inhibitors of the Keap1-Nrf2 protein-protein interaction (PPI) have therapeutic potential in a range of disease states including neurodegenerative diseases (Parkinson's and Alzheimer's diseases), chronic obstructive pulmonary disease and various inflammatory conditions. By stalling Keap1-mediated ubiquitination of Nrf2, such compounds can enhance Nrf2 transcriptional activity and activate the expression of a range of genes with antioxidant response elements in their promoter regions. Keap1 inhibitors based on peptide and small-molecule templates have been identified. In this paper we develop the structure-activity relationships of the peptide series and identify a group of ligands incorporating unnatural amino acids that demonstrate improved binding affinity in fluorescence polarisation, differential scanning fluorimetry and isothermal titration calorimetry assays. These modified peptides have the potential for further development into peptidomimetic chemical probes to explore the role of Nrf2 in disease and as potential lead structures for drug development.

New MKLP-2 inhibitors in the paprotrain series: Design, synthesis and biological evaluations.

Members of the kinesin superfamily are involved in key functions during intracellular transport and cell division. Their involvement in cell division makes certain kinesins potential targets for drug development in cancer chemotherapy. The two most advanced kinesin targets are Eg5 and CENP-E with inhibitors in clinical trials. Other mitotic kinesins are also being investigated for their potential as prospective drug targets. One recently identified novel potential cancer therapeutic target is the Mitotic kinesin-like protein 2 (MKLP-2), a member of the kinesin-6 family, which plays an essential role during cytokinesis. Previous studies have shown that inhibition of MKLP-2 leads to binucleated cells due to failure of cytokinesis. We have previously identified compound 1 (paprotrain) as the first selective inhibitor of MKLP-2. Herein we describe the synthesis and biological evaluation of new analogs of 1. Our structure-activity relationship (SAR) study reveals the key chemical elements in the paprotrain family necessary for MKLP-2 inhibition. We have successfully identified one MKLP-2 inhibitor 9a that is more potent than paprotrain. In addition, in vitro analysis of a panel of kinesins revealed that this compound is selective for MKLP-2 compared to other kinesins tested and also does not have an effect on microtubule dynamics. Upon testing in different cancer cell lines, we find that the more potent paprotrain analog is also more active than paprotrain in 10 different cancer cell lines. Increased selectivity and higher potency is therefore a step forward toward establishing MKLP-2 as a potential cancer drug target.

Depsidones from Lichens as Natural Product Inhibitors of M-Phase Phosphoprotein 1, a Human Kinesin Required for Cytokinesis.

M-Phase Phosphoprotein 1 (MPP1), a microtubule plus end directed kinesin, is required for the completion of cytokinesis. Previous studies have shown that MPP1 is upregulated in various types of bladder cancer. This article describes inhibitor screening leading to the identification of a new class of natural product inhibitors of MPP1. Two compounds with structural similarity, norlobaridone (1) and physodic acid (2), were found to inhibit MPP1. Physodic acid is not competitive with ATP, indicating the presence of an allosteric inhibitor-binding pocket. Initial drug-like property screening indicates that physodic acid is more soluble than norlobaridone and has more favorable lipophilicity. However, both suffer from high clearance in human microsomal stability assays mediated by the lability of the lactone ring as well as hydroxylation of the alkyl chains as shown by metabolite identification studies. In cell-based assays physodic acid is a weak inhibitor with EC50 values of about 30 µM in a range of tumor cell lines. The two depsidones identified and characterized here could be used for future improvement of their activity against MPP1 and will be useful chemical probes for studying this unique molecular motor in more depth.

Analysis of the human cofilin 1 structure reveals conformational changes required for actin binding.

The actin cytoskeleton is the chassis that gives a cell its shape and structure, and supplies the power for numerous dynamic processes including motility, endocytosis, intracellular transport and division. To perform these activities, the cytoskeleton undergoes constant remodelling and reorganization. One of the major actin-remodelling families are the cofilin proteins, made up of cofilin 1, cofilin 2 and actin-depolymerizing factor (ADF), which sever aged ADP-associated actin filaments to reduce filament length and provide new potential nucleation sites. Despite the significant interest in cofilin as a central node in actin-cytoskeleton dynamics, to date the only forms of cofilin for which crystal structures have been solved are from the yeast, Chromalveolata and plant kingdoms; none have previously been reported for an animal cofilin protein. Two distinct regions in animal cofilin are significantly larger than in the forms previously crystallized, suggesting that they would be uniquely organized. Therefore, it was sought to determine the structure of human cofilin 1 by X-ray crystallography to elucidate how it could interact with and regulate dynamic actin-cytoskeletal structures. Although wild-type human cofilin 1 proved to be recalcitrant, a C147A point mutant yielded crystals that diffracted to 2.8 Šresolution. These studies revealed how the actin-binding helix undergoes a conformational change that increases the number of potential hydrogen bonds available for substrate binding.

Structural insights into a unique inhibitor binding pocket in kinesin spindle protein.

Human kinesin Eg5 is a target for drug development in cancer chemotherapy with compounds in phase II clinical trials. These agents bind to a well-characterized allosteric pocket involving the loop L5 region, a structural element in kinesin-5 family members thought to provide inhibitor specificity. Using X-ray crystallography, kinetic, and biophysical methods, we have identified and characterized a distinct allosteric pocket in Eg5 able to bind inhibitors with nanomolar K(d). This pocket is formed by key structural elements thought to be pivotal for force generation in kinesins and may represent a novel site for therapeutic intervention in this increasingly well-validated drug target.

Development and validation of RdRp Screen, a crystallization screen for viral RNA-dependent RNA polymerases.

Members of the Flaviviridae family constitute a severe risk to human health. Whilst effective drugs have been developed against the hepacivirus HCV, no antiviral therapy is currently available for any other viruses, including the flaviviruses dengue (DENV), West Nile and Zika viruses. The RNA-dependent RNA polymerase (RdRp) is responsible for viral replication and represents an excellent therapeutic target with no homologue found in mammals. The identification of compounds targeting the RdRp of other flaviviruses is an active area of research. One of the main factors hampering further developments in the field is the difficulty in obtaining high-quality crystal information that could aid a structure-based drug discovery approach. To address this, we have developed a convenient and economical 96-well screening platform. We validated the screen by successfully obtaining crystals of both native DENV serotype 2 and 3 RdRps under several conditions included in the screen. In addition, we have obtained crystal structures of RdRp3 in complex with a previously identified fragment using both soaking and co-crystallization techniques. This work will streamline and accelerate the generation of crystal structures of viral RdRps and provide the community with a valuable tool to aid the development of structure-based antiviral design.

Is the Fate of Clinical Candidate Arry-520 Already Sealed? Predicting Resistance in Eg5-Inhibitor Complexes.

Arry-520 is an advanced drug candidate from the Eg5 inhibitor class undergoing clinical evaluation in patients with relapsed or refractory multiple myeloma. Here, we show by structural analysis that Arry-520 binds stoichiometrically to the motor domain of Eg5 in the conventional allosteric loop L5 pocket in a complex that suggests the same structural mechanism as other Eg5 inhibitors. We have previously shown that acquired resistance through mutations in the allosteric-binding site located at loop L5 in the Eg5 structure appears to be independent of the inhibitors' scaffold, which suggests that Arry-520 will ultimately have the same fate. When Arry-520 was assessed in two cell lines selected for the expression of either Eg5(D130A) or Eg5(L214A) STLC-resistant alleles, mutations previously shown to convey resistance to this class of inhibitors, it was inactive in both. Surprisingly, when the cells were challenged with ispinesib, another Eg5 inhibitor, the Eg5(D130A) cells were resistant, but those expressing Eg5(L214A) were strikingly sensitive. Molecular dynamics simulations suggest that subtle differences in ligand binding and flexibility in both compound and protein may alter allosteric transmission from the loop L5 site that do not necessarily result in reduced inhibitory activity in mutated Eg5 structures. Although we predict that cells challenged with Arry-520 in the clinical setting are likely to acquire resistance through point mutations in the Eg5-binding site, the data for ispinesib suggest that this resistance mechanism is not scaffold independent as previously thought, and new inhibitors can be designed that retain inhibitory activity in these resistant cells.

The structure of the binary methyltransferase-SAH complex from Zika virus reveals a novel conformation for the mechanism of mRNA capping.

Zika virus, a flavivirus like Dengue and West Nile viruses, poses a significant risk as a pathogen in the category of emerging infectious diseases. Zika infections typically cause nonspecific, mild symptoms, but can also manifest as a neurological disorder like Guillain-Barré syndrome. Infection in pregnant women is linked to microcephaly in newborn infants. The methyltransferase domain of the non-structural protein 5 is responsible for two sequential methylations of the 5'-RNA cap. This is crucial for genome stability, efficient translation, and escape from the host immune response. Here we present the crystal structures of the Zika methyltransferase domain in complex with the methyl-donor SAM and its by-product SAH. The methyltransferase-SAH binary complex presents a new conformation of a "closed" or "obstructed" state that would restrict the binding of new RNA for capping. The combination and comparison of our new structures with recently published Zika methyltransferase structures provide a first glimpse into the structural mechanism of Zika virus mRNA capping.

Crystal structure of the Eg5 - K858 complex and implications for structure-based design of thiadiazole-containing inhibitors.

The thiadiazole scaffold is an important core moiety in a variety of clinical drug candidates targeting a range of diseases. For example, the 2,4,5-substituted 1,3,4-thiadiazole scaffold is present in a lead compound and at least two clinical candidates targeting the human motor protein Eg5, against neoplastic diseases. An inhibitor named K858 has in vivo activity in various mouse xenografts whereas the clinical candidates (S)-ARRY-520 and (R)-Litronesib have entered clinical trials with the former one in phase III clinical trials either alone or in combination with a proteasome inhibitor against relapsed/refractory multiple myeloma. Astonishingly, structural data are lacking for all thiadiazole-containing Eg5 inhibitors. Here we report the structure determination of two crystal forms of the ternary Eg5-ADP-K858 complex, locking the motor in the so-called final inhibitor bound state, thus blocking ADP release, a crucial stage for Eg5 activity. K858 acts at the established allosteric inhibitor-binding pocket formed of helix α2, loop L5 and helix α3. The structure of the complex has far reaching consequences for thiadiazole containing Eg5 inhibitors. For example, we could rationalise the structure-activity relationship in the crucial 5-position of the thiadiazole scaffold and the complex will serve in the future as a basis for strucutre-based drug design.

The C-terminal region of the motor protein MCAK controls its structure and activity through a conformational switch.

The precise regulation of microtubule dynamics is essential during cell division. The kinesin-13 motor protein MCAK is a potent microtubule depolymerase. The divergent non-motor regions flanking the ATPase domain are critical in regulating its targeting and activity. However, the molecular basis for the function of the non-motor regions within the context of full-length MCAK is unknown. Here, we determine the structure of MCAK motor domain bound to its regulatory C-terminus. Our analysis reveals that the MCAK C-terminus binds to two motor domains in solution and is displaced allosterically upon microtubule binding, which allows its robust accumulation at microtubule ends. These results demonstrate that MCAK undergoes long-range conformational changes involving its C-terminus during the soluble to microtubule-bound transition and that the C-terminus-motor interaction represents a structural intermediate in the MCAK catalytic cycle. Together, our work reveals intrinsic molecular mechanisms underlying the regulation of kinesin-13 activity.

Mitotic kinesin Eg5 overcomes inhibition to the phase I/II clinical candidate SB743921 by an allosteric resistance mechanism.

Development of drug resistance during cancer chemotherapy is one of the major causes of chemotherapeutic failure for the majority of clinical agents. The aim of this study was to investigate the underlying molecular mechanism of resistance developed by the mitotic kinesin Eg5 against the potent second-generation ispinesib analogue SB743921 (1), a phase I/II clinical candidate. Biochemical and biophysical data demonstrate that point mutations in the inhibitor-binding pocket decrease the efficacy of 1 by several 1000-fold. Surprisingly, the structures of wild-type and mutant Eg5 in complex with 1 display no apparent structural changes in the binding configuration of the drug candidate. Furthermore, ITC and modeling approaches reveal that resistance to 1 is not through conventional steric effects at the binding site but through reduced flexibility and changes in energy fluctuation pathways through the protein that influence its function. This is a phenomenon we have called "resistance by allostery".

Optimized S-trityl-L-cysteine-based inhibitors of kinesin spindle protein with potent in vivo antitumor activity in lung cancer xenograft models.

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 (K(i)(app) < 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.

Indolobenzazepin-7-ones and 6-, 8-, and 9-membered ring derivatives as tubulin polymerization inhibitors: synthesis and structure--activity relationship studies.

Several small weight indole derivatives (D-64131, D-24851, BPR0L075, BLF 61-3, and ATI derivatives) are potent tubulin polymerization inhibitors and show nanomolar antiproliferative activity. Among them, indolobenzazepin-7-ones were recently disclosed as potent antimitotic agents. In an effort to improve this structure, we prepared new derivatives in order to evaluate their antiproliferative activity. 5,6,7,9-Tetrahydro-8H-indolo[2,3-e][3]benzazocin-8-one (1m) was found to be the most potent derivative inhibiting the cell growth of several cancer cell lines in the lower nanomolar range.