Identifying the direct physiological targets of drugs and chemical probes remains challenging. Here we describe how resistance can be used to achieve ‘gold-standard’ validation of a chemical inhibitor’s direct target in human cells. This involves demonstrating that a silent mutation in the target that suppresses inhibitor activity in cell-based assays can also reduce inhibitor potency in biochemical assays. Further, phenotypes due to target inhibition can be identified as those observed in the inhibitor-sensitive cells, across a range of inhibitor concentrations, but not in genetically matched cells with a silent resistance-conferring mutation in the target. We propose that chemotype-specific resistance, which is generally considered to be a limitation of molecularly targeted agents, can be leveraged to deconvolve the mechanism of action of drugs and to properly use chemical probes.

Methylation of histones by lysine methyltransferases (KMTases) plays important roles in regulating chromatin function. It is also now clear that improper KMTases activity is linked to human diseases, such as cancer. We report an approach that employs drug-like ‘privileged’ scaffolds biased with motifs present in S-adenosyl methionine, the cofactor used by KMTases, to efficiently generate inhibitors for Set7, a biochemically well-characterized KMTase. Setin-1, the most potent inhibitor of Set7 we have developed also inhibits the KMTase G9a. Together these data suggest that these inhibitors should provide good starting points to generate useful probes for KMTase biology and guide the design of KMTase inhibitors with drug-like properties.

Microtubules are hollow tube-like biological polymers required for transport in diverse cellular contexts and are important drug targets. Microtubule function depends on interactions with associated proteins and post-translational modifications at specific sites located on its interior and exterior surfaces. However, we lack strategies to selectively perturb or probe these basic biochemical mechanisms. In this work, by combining amber suppression-mediated non-natural amino acid incorporation and tubulin overexpression in budding yeast, we demonstrate, for the first time, a general strategy for site-specific chemistry on microtubules. Probes and labels targeted to precise sites on the interior and exterior surfaces of microtubules will allow analysis and modulation of interactions with proteins and drugs, and elucidation of the functions of post-translational modifications.

Protein kinases may function more like variable rheostats rather than two-state switches. However, we lack approaches to properly analyze this aspect of kinase-dependent regulation. To address this, we develop a strategy in which a kinase inhibitor is identified using genetics-based screens, kinase mutations that confer resistance are characterized, and dose-dependent responses of isogenic drug-sensitive and resistant cells to inhibitor treatments are compared. This approach has the advantage that function of wild-type kinase, rather than mutants, is examined. To develop this approach, we focus on Ark1, the fission yeast member of the conserved Aurora kinase family. Applying this approach reveals that proper chromosome compaction in fission yeast needs high Ark1 activity, while other processes depend on significantly lower activity levels. Our strategy is general and can be used to examine the functions of other molecular rheostats.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (277 K)Download as PowerPoint slideHighlights► A genetics-based chemical screen leads to a fission yeast Aurora kinase inhibitor ► Inhibitor resistance conferring point mutation in the kinase gene is characterized ► Dose-dependent phenotypes in drug-resistant and sensitive cells are compared ► Key cell division processes depend of different levels of Aurora kinase activity

Post-translational modifications (PTMs) (e.g., acetylation, methylation, and phosphorylation) play crucial roles in regulating the diverse protein–protein interactions involved in essentially every cellular process. While significant progress has been made to detect PTMs, profiling protein–protein interactions mediated by these PTMs remains a challenge. Here, we report a method that combines a photo-cross-linking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein–protein interactions. To develop and apply this approach, we focused on trimethylated lysine-4 at the histone H3 N-terminus (H3K4Me3), a PTM linked to actively transcribed gene promoters. Our approach identified proteins previously known to recognize this modification and MORC3 as a new protein that binds H3M4Me3. This study indicates that our cross-linking-assisted and SILAC-based protein identification (CLASPI) approach can be used to profile protein–protein interactions mediated by PTMs, such as lysine methylation.

Chemical inhibitors can help analyze dynamic cellular processes, particularly when probes are active in genetically tractable model systems. Although fission yeast has served as an important model system, which shares more cellular processes (e.g., RNAi) with humans than budding yeast, its use for chemical biology has been limited by its multidrug resistance (MDR) response. Using genomics and genetics approaches, we identified the key transcription factors and drug-efflux transporters responsible for fission yeast MDR and designed strains sensitive to a wide-range of chemical inhibitors, including commonly used probes. We used this strain, along with acute chemical inhibition and high-resolution imaging, to examine metaphase spindle organization in a “closed” mitosis. Together, our findings suggest that our fission yeast strains will allow the use of several inhibitors as probes, discovery of new inhibitors, and analysis of drug action.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (121 K)Download as PowerPoint slideHighlights► Key factors responsible for multidrug resistance in fission yeast were identified ► A fission yeast strain sensitive to diverse chemical inhibitors was engineered ► Chemical inhibitors and these yeast strains can be used to probe dynamic mechanisms

It is difficult to determine a chemical inhibitor’s binding site in multiprotein mixtures, particularly when high-resolution structural studies are not straightforward. Building upon previous research involving photo-cross-linking and the use of mixtures of stable isotopes, we report a method, Stable Isotope Labeled Inhibitors for Cross-linking (SILIC), for mapping a small molecule inhibitor’s binding site in its target protein. In SILIC, structure–activity relationship data is used to design inhibitor analogues that incorporate a photo-cross-linking group along with either natural or ‘heavy’ stable isotopes. An equimolar mixture of these inhibitor analogues is cross-linked to the target protein to yield a robust signature for identifying inhibitor-modified peptide fragments in complex mass spectrometry data. As a proof of concept, we applied this approach to an ATP-competitive inhibitor of kinesin-5, a widely conserved motor protein required for cell division and an anticancer drug target. This analysis, along with mutagenesis studies, suggests that the inhibitor binds at an allosteric site in the motor protein.

The dynamic cellular reorganization needed for successful mitosis requires regulatory cues that vary across microns. The chromosomal passenger complex (CPC) is a conserved regulator involved in key mitotic events such as chromosome–microtubule attachment and spindle midzone formation. Recently, spatial phosphorylation gradients have been reported for CPC substrates, raising the possibility that CPC-dependent signaling establishes order on the micron-length scale in dividing cells. However, this hypothesis has not been tested, largely because of incomplete characterization of the CPC-dependent phosphorylation dynamics. Without these data it is difficult to evaluate perturbations of CPC signaling and select one that alters the spatial organization of substrate phosphorylation at a particular stage of mitosis, without changing overall phosphorylation levels. Here we examine the spatiotemporal dynamics of CPC-dependent phosphorylation along microtubules throughout mitosis using a Förster resonance energy transfer-based sensor. We find that a CPC substrate phosphorylation gradient, with highest phosphorylation levels between the two spindle poles, emerges when a cell enters mitosis. Interestingly, this gradient becomes undetectable at metaphase, but can be revealed by partially suppressing CPC activity, suggesting that high substrate phosphorylation levels can mask persistent CPC-dependent spatial patterning. After anaphase onset, the gradient emerges and persists until cell cleavage. Selective mislocalization of the CPC during anaphase suppresses gradient formation, but overall substrate phosphorylation levels remain unchanged. Under these conditions, the spindle midzone fails to organize and function properly. Our findings suggest a model in which the CPC establishes phosphorylation gradients to coordinate the spatiotemporal dynamics needed for error-free cell division.

Post-translational modifications (PTMs) of histones, proteins onto which DNA is packaged, are involved in many biological processes, including transcription, recombination, and chromosome segregation. As these PTMs can be dynamic, combinatorial, and mediators of weak interactions, the comprehensive profiling of all proteins that recognize histone PTMs is a daunting task. Here we describe an approach to design probes that can be used to identify proteins that directly interact with modified histones. Protein structure was used to guide the introduction of a photo-cross-linker in the probe, so as to convert weak interactions into covalent linkages. The probe also included an alkyne group to facilitate click chemistry-mediated conjugation of reporter tags for the rapid and sensitive detection (via rhodamine) and affinity enrichment (via biotin) of labeled proteins. In particular, we developed and validated a probe that can selectively capture proteins that recognize trimethyled lysine-4 of histone H3 (H3K4me3) in whole proteomes. A complete profiling of H3K4Me3 binding proteins should shed new light on cellular processes regulated by this PTM.

Accuracy in chromosome segregation depends on the assembly of a bipolar spindle. Unlike mitotic spindles, which have roughly equal amounts of kinetochore microtubules (kMTs) and nonkinetochore microtubules (non-kMTs), vertebrate meiotic spindles are predominantly comprised of non-kMTs, a large subset of which forms an antiparallel “barrel” array at the spindle equator. Though kMTs are needed to drive chromosome segregation, the contributions of non-kMTs are more mysterious. Here, we show that increasing the concentration of Op18/stathmin, a component of the chromosome-mediated microtubule formation pathway that directly controls microtubule dynamics, can be used to deplete non-kMTs in the vertebrate meiotic spindle assembled in Xenopus egg extracts. Under these conditions, kMTs and the spindle pole-associated non-kMT arrays persist in smaller spindles. In excess Op18, distances between sister kinetochores, an indicator of tension across centromeres, remain unchanged, even though kMTs flux poleward with a ≈30% slower velocity, and chromosomes oscillate more than in control metaphase spindles. Remarkably, kinesin-5, a conserved motor protein that can push microtubules apart and is required for the assembly and maintenance of bipolar meiotic spindles, is not needed to maintain spindle bipolarity in the presence of excess Op18. Our data suggest that non-kMTs in meiotic spindles contribute to normal kMT dynamics, stable chromosome positioning, and the establishment of proper spindle size. We propose that without non-kMTs, metaphase meiotic spindles are similar to mammalian mitotic spindles, which balance forces to maintain metaphase spindle organization in the absence of extensive antiparallel microtubule overlap at the spindle equator or a key mitotic kinesin.

Accurate segregation of chromosomes, essential for the stability of the genome, depends on ‘bi-orientation’—simultaneous attachment of each individual chromosome to both poles of the mitotic spindle1. On bi-oriented chromosomes, kinetochores (macromolecular complexes that attach the chromosome to the spindle) reside on the opposite sides of the chromosome’s centromere2. In contrast, sister kinetochores shift towards one side of the centromere on ‘syntelic’ chromosomes that erroneously attach to one spindle pole with both sister kinetochores. Syntelic attachments often arise during spindle assembly and must be corrected to prevent chromosome loss3. It is assumed that restoration of proper centromere architecture occurs automatically owing to elastic properties of the centromere1, 2. Here we test this assumption by combining laser microsurgery and chemical biology assays in cultured mammalian cells. We find that kinetochores of syntelic chromosomes remain juxtaposed on detachment from spindle microtubules. These findings reveal that correction of syntelic attachments involves an extra step that has previously been overlooked: external forces must be applied to move sister kinetochores to the opposite sides of the centromere. Furthermore, we demonstrate that the shape of the centromere is important for spindle assembly, because bipolar spindles do not form in cells lacking centrosomes when multiple chromosomes with juxtaposed kinetochores are present. Thus, proper architecture of the centromere makes an important contribution to achieving high fidelity of chromosome segregation.

Spindle checkpoint silencing is crucial for cell-cycle progression, but mechanisms underlying this process remain mysterious. Two papers, one in this issue of Developmental Cell (Meadows et al., 2011) and one in Current Biology (Rosenberg et al., 2011), begin to show how phosphatase PP1-gamma connects chromosome-microtubule attachment with anaphase entry.

Successful completion of diverse cellular functions, such as mitosis, positioning organelles, and assembling cilia, depends on the proper assembly of microtubule-based structures. While essentially all of the proteins needed to assemble these structures are now known, we cannot explain how even simple features such as size and shape are determined. As steps toward filling this knowledge gap, there have been several recent efforts toward reconstituting, with purified proteins, the basic structural motifs that recur in diverse cytoskeletal arrays. We discuss these studies and highlight how they shed light on the self-organized assembly of complex and dynamic cytoskeleton-based cellular structures.