Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.

Cells integrate mechanical properties of their surroundings to form multicellular, three-dimensional tissues of appropriate size and spatial organisation. Actin cytoskeleton-linked proteins such as talin, vinculin and filamin function as mechanosensors in cells, but it has yet to be tested whether the mechanosensitivity is important for their function in intact tissues. Here we tested, how filamin mechanosensing contributes to oogenesis in Drosophila. Mutations that require more or less force to open the mechanosensor region demonstrate that filamin mechanosensitivity is important for the maturation of actin-rich ring canals that are essential for Drosophila egg development. The open mutant was more tightly bound to the ring canal structure while the closed mutant dissociated more frequently. Thus, our results show that an appropriate level of mechanical sensitivity is required for filamins’ function and dynamics during Drosophila egg growth and support the structure-based model in which the opening and closing of the mechanosensor region regulates filamin binding to cellular components.

Though used widely in cancer therapy, paclitaxel only elicits a response in a fraction of patients. A strong determinant of paclitaxel tumor response is the state of microtubule dynamic instability. However, whether the manipulation of this physiological process can be controlled to enhance paclitaxel response has not been tested. Here, we show a previously unrecognized role of the microtubule-associated protein CRMP2 in inducing microtubule bundling through its carboxy terminus. This activity is significantly decreased when the FER tyrosine kinase phosphorylates CRMP2 at Y479 and Y499. The crystal structures of wild-type CRMP2 and CRMP2-Y479E reveal how mimicking phosphorylation prevents tetramerization of CRMP2. Depletion of FER or reducing its catalytic activity using sub-therapeutic doses of inhibitors increases paclitaxel-induced microtubule stability and cytotoxicity in ovarian cancer cells and in vivo. This work provides a rationale for inhibiting FER-mediated CRMP2 phosphorylation to enhance paclitaxel on-target activity for cancer therapy.

Filamins are multi-domain, actin cross-linking, and scaffolding proteins. In addition to the actin cross-linking function, filamins have a role in mechanosensor signaling. The mechanosensor function is mediated by domain-domain interaction in the C-terminal region of filamins. Recently, we have shown that there is a three-domain interaction module in the N-terminal region of filamins, where the neighboring domains stabilize the structure of the middle domain and thereby regulate its interaction with ligands. In this study, we have used small-angle X-ray scattering as a tool to screen for potential domain-domain interactions in the N-terminal region. We found evidence of four domain-domain interactions with varying flexibility. These results confirm our previous study showing that domains 3, 4, and 5 exist as a compact three domain module. In addition, we report interactions between domains 11–12 and 14–15, which are thus new candidate sites for mechanical regulation.

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Covalent probes can display unmatched potency, selectivity and duration of action, however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening. Such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge, and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found selective hits for most targets. Combination with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2, and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile fragment screening as a practical and efficient tool for covalent ligand discovery.Fragment based screening, which focuses on very low molecular-weight compounds, is a successful hit discovery approach for reversible inhibitors 28,29 , that has led to several drugs and chemical probes 29,30 . Compared to traditional HTS, fragment-based screening offers better coverage of chemical space and higher probability of binding due to lower complexity 31,32 . The major limitation in fragment-based screening is the weak binding affinity of fragment hits, which not only necessitates very sensitive biophysical detection methods, coupled with elaborate validation cascades to eliminate attendant artefacts, but additionally makes progressing hits to potency difficult and expensive. In particular, it requires large compound series with typically ambiguous structure-activity relationships, because no method to date can reliably rationalize which are the dominant interactions of the original fragment. Screening covalent fragments addresses both problems: covalent binders are easy to detect by mass spectrometry; and because the dominant interaction is unambiguous, namely the covalent bond, designing followup series is simplified, and the primary hits are already potent.A prominent covalent fragment screening approach is disulfide tethering 33,34 , which entails incubating a library of disulfide-containing fragments with the target. Disulfide exchange with the target cysteine selects for fragments that are reversibly stabilized in its vicinity. Disulfide tethering was successfully applied to a variety of targets containing both native and introduced cysteine residues 35 . Recently it led to the discovery of a promising K-Ras G12C inhibitor 36 . Disulfides are not, however, suitable as cellular probes, and replacing them with a suitable electrophile is in general no less challenging than starting from a reversible ligand.A potential solution is to directly screen mild electrophile fragments. Electrophile...

Synaptojanin1 (Synj1) is a phosphoinositide phosphatase, important in clathrin uncoating during endocytosis of presynaptic vesicles. It was identified as a potential drug target for Alzheimer’s disease, Down syndrome, and TBC1D24-associated epilepsy, while also loss-of-function mutations in Synj1 are associated with epilepsy and Parkinson’s disease. Despite its involvement in a range of disorders, structural, and detailed mechanistic information regarding the enzyme is lacking. Here, we report the crystal structure of the 5-phosphatase domain of Synj1. Moreover, we also present a structure of this domain bound to the substrate diC8-PI(3,4,5)P3, providing the first image of a 5-phosphatase with a trapped substrate in its active site. Together with an analysis of the contribution of the different inositide phosphate groups to catalysis, these structures provide new insights in the Synj1 mechanism. Finally, we analysed the effect of three clinical missense mutations (Y793C, R800C, Y849C) on catalysis, unveiling the molecular mechanisms underlying Synj1-associated disease.

The chemotherapeutic agent paclitaxel increases cancer cell microtubule stability, induces mitotic arrest and cancer cell death and improves survival of cancer patients. However, in ovarian and other cancers, it only elicits a response in less than half of patients who receive it. We have previously shown that enhancing microtubule stability in cells prior to paclitaxel treatment, further increases paclitaxel-induced microtubule stability and cancer cell death (Ahmed et al., Cancer Cell, 2007;12:514 and Ahmed et al., Cancer Research 2011;71:5806). We now report that the microtubule-associated protein CRMP2, and FER kinase are expressed in almost half of high-grade serous ovarian cancers and that inhibiting the phosphorylation of CRMP2 by FER is sufficient for enhancing paclitaxel-induced microtubule stability and cytotoxicity in multiple ovarian cancer cell lines. We are also able propose a detailed mechanism underlying these observations. A combination of biochemistry, super-resolution fluorescence microscopy, total internal reflection fluorescence (TIRF) microscopy and electron microscopy reveals that CRMP2 induces microtubule nucleation, elongation, bundling and stability. Moreover, FER phosphorylates CRMP2 at 6 tyrosine residues, two of which (Tyr479 and Tyr499) are critical for modulating CRMP2 function; this agrees with site-directed mutagenesis in cancer cells which points to Tyr479 as a critical site for association between CRMP2 and microtubules. A comparison of the crystal structures of wild-type CRMP2 and Tyr479Glu/Tyr499Glu phosphomimetic mutants indicate that phosphorylation of these sites induces significant conformational changes that prevent CRMP2 from forming tetramers that are known to be critical for its microtubule bundling activity. This then leads us to propose a molecular model of how CRMP2 associates with microtubules, explaining how targeting CRMP2 phosphorylation can be exploited for enhancing the therapeutic efficacy of paclitaxel. Citation Format: Yiyan Zheng, Ritika Sethi, Frank von Delft, Ahmed Ashour Ahmed. The atomic basis for paclitaxel sensitization following loss of CRMP2 phosphorylation in ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3856.