Multi-parameter optimization: Development of a morpholin-3-one derivative with an improved kinetic profile for imaging monoacylglycerol lipase in the brain.

Monoacylglycerol lipase (MAGL) is a gatekeeper in regulating endocannabinoid signaling and has gained substantial attention as a therapeutic target for neurological disorders. We recently discovered a morpholin-3-one derivative as a novel scaffold for imaging MAGL via positron emission tomography (PET). However, its slow kinetics in vivo hampered the application. In this study, structural optimization was conducted and eleven novel MAGL inhibitors were designed and synthesized. Based on the results from MAGL inhibitory potency, in vitro metabolic stability and surface plasmon resonance assays, we identified compound 7 as a potential MAGL PET tracer candidate. [11C]7 was synthesized via direct 11CO2 fixation method and successfully mapped MAGL distribution patterns on rodent brains in in vitro autoradiography. PET studies in mice using [11C]7 demonstrated its improved kinetic profile compared to the lead structure. Its high specificity in vivo was proved by using MAGL KO mice. Although further studies confirmed that [11C]7 is a P-glycoprotein (P-gp) substrate in mice, its low P-gp efflux ratio on cells transfected with human protein suggests that it should not be an issue for the clinical translation of [11C]7 as a novel reversible MAGL PET tracer in human subjects. Overall, [11C]7 ([11C]RO7284390) showed promising results warranting further clinical evaluation.

Chemoprobe-based assays of histone lysine demethylase 1A target occupation enable in vivo pharmacokinetics and pharmacodynamics studies of KDM1A inhibitors.

Screening of cellular activity for inhibitors of histone lysine modifiers is most frequently performed indirectly by analyzing changes in the total levels of histone marks targeted by lysine methylases/demethylases. However, inhibition of histone lysine modifiers often leads to local rather than total changes in histone marks. Also, because histone modifications can be modulated by more than one cellular enzyme, it is not always clear whether changes in histone marks are a direct or indirect consequence of the inhibitor treatment applied. Direct assessment of target occupation can provide a useful tool to quantify the fraction of an epigenetic modifier that is bound to a pharmacological inhibitor (target engagement). Here, we developed and used a novel chemoprobe-based immunoassay to quantify target engagement in cells. Quantification of the fraction of free KDM1A was made possible, in an immune-based assay, by coupling a biotinylated chemoprobe to a warhead capable of selectively and irreversibly binding to the free active form of KDM1A. The results obtained confirmed that this approach is able to determine the degree of target engagement in a dose-dependent manner. Furthermore, the assay can be also used on tissue extracts to analyze the in vivo pharmacokinetics and pharmacodynamics relationship of KDM1A inhibitors, as has been exemplified with ORY-1001 (iadademstat), a potent and irreversible inhibitor of KDM1A. The principle of this assay may be applied to other targets, and the KDM1A probe may be employed in chemoproteomic analyses.

Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly.

Centrioles are microtubule-based organelles that organize the microtubule network and seed the formation of cilia and flagella. New centrioles assemble through a stepwise process dependent notably on the centriolar protein SAS-5 in Caenorhabditis elegans SAS-5 and its functional homologues in other species form oligomers that bind the centriolar proteins SAS-6 and SAS-4, thereby forming an evolutionarily conserved structural core at the onset of organelle assembly. Here, we report a novel interaction of SAS-5 with microtubules. Microtubule binding requires SAS-5 oligomerization and a disordered protein segment that overlaps with the SAS-4 binding site. Combined in vitro and in vivo analysis of select mutants reveals that the SAS-5-microtubule interaction facilitates centriole assembly in C. elegans embryos. Our findings lead us to propose that the interdependence of SAS-5 oligomerization and microtubule binding reflects an avidity mechanism, which also strengthens SAS-5 associations with other centriole components and, thus, promotes organelle assembly.

Novel peptide probes to assess the tensional state of fibronectin fibers in cancer.

Transformations of extracellular matrix (ECM) accompany pathological tissue changes, yet how cell-ECM crosstalk drives these processes remains unknown as adequate tools to probe forces or mechanical strains in tissues are lacking. Here, we introduce a new nanoprobe to assess the mechanical strain of fibronectin (Fn) fibers in tissue, based on the bacterial Fn-binding peptide FnBPA5. FnBPA5 exhibits nM binding affinity to relaxed, but not stretched Fn fibers and is shown to exhibit strain-sensitive ECM binding in cell culture in a comparison with an established Fn-FRET probe. Staining of tumor tissue cryosections shows large regions of relaxed Fn fibers and injection of radiolabeled 111In-FnBPA5 in a prostate cancer mouse model reveals specific accumulation of 111In-FnBPA5 in tumor with prolonged retention compared to other organs. The herein presented approach enables to investigate how Fn fiber strain at the tissue level impacts cell signaling and pathological progression in different diseases.

Radiolabeled 111In-FGF-2 Is Suitable for In Vitro/Ex Vivo Evaluations and In Vivo Imaging.

Fibroblast growth factor-2 (FGF-2) is a potent modulator of cell growth and regulation, with improper FGF-2 signaling being involved in impaired responses to injury or even cancer. Therefore, the exploitation of FGF-2 as a therapeutic drives the prerequisite for effective insight into drug disposition kinetics. In this article, we present an 111In-radiolabeled FGF-2 derivative for noninvasive imaging in small animals deploying single photon emission tomography (SPECT). 111In-FGF-2 is equally well suitable for in vitro and ex vivo investigations as 125I-FGF-2. Furthermore, 111In-FGF-2 permits the performance of in vivo imaging, for example for the analysis of FGF-2 containing pharmaceutical formulations in developmental or preclinical stages. 111In-FGF-2 had affinity for the low-molecular-weight heparin enoxaparin identical to that of unlabeled FGF-2 (Kd: 0.6 ± 0.07 µM and 0.33 ± 0.03 µM, respectively) as assessed by isothermal titration calorimetry. The binding of 111In-FGF-2 to heparan sulfate proteoglycans (HPSGs) and the biological activity were comparable to those of unlabeled FGF-2, with EC50 values of 12 ± 2 pM and 25 ± 6 pM, respectively. In vivo biodistribution in healthy nude mice indicated a predominant accumulation of 111In-FGF-2 in filtering organs and minor uptake in the retina and the salivary and pituitary glands, which was confirmed by SPECT imaging. Therefore, 111In-FGF-2 is a valid tracer for future noninvasive animal imaging of FGF-2 in pharmaceutical development.

The Human Centriolar Protein CEP135 Contains a Two-Stranded Coiled-Coil Domain Critical for Microtubule Binding.

Centrioles are microtubule-based structures that play important roles notably in cell division and cilium biogenesis. CEP135/Bld10p family members are evolutionarily conserved microtubule-binding proteins important for centriole formation. Here, we analyzed in detail the microtubule-binding activity of human CEP135 (HsCEP135). X-ray crystallography and small-angle X-ray scattering in combination with molecular modeling revealed that the 158 N-terminal residues of HsCEP135 (HsCEP135-N) form a parallel two-stranded coiled-coil structure. Biochemical, cryo-electron, and fluorescence microscopy analyses revealed that in vitro HsCEP135-N interacts with tubulin, protofilaments, and microtubules and induces the formation of microtubule bundles. We further identified a 13 amino acid segment spanning residues 96-108, which represents a major microtubule-binding site in HsCEP135-N. Within this segment, we identified a cluster of three lysine residues that contribute to the microtubule bundling activity of HsCEP135-N. Our results provide the first structural information on CEP135/Bld10p proteins and offer insights into their microtubule-binding mechanism.

Termination of Protofilament Elongation by Eribulin Induces Lattice Defects that Promote Microtubule Catastrophes.

Microtubules are dynamic polymers built of tubulin dimers that attach in a head-to-tail fashion to form protofilaments, which further associate laterally to form a tube. Asynchronous elongation of individual protofilaments can potentially lead to an altered microtubule-end structure that promotes sudden depolymerization, termed catastrophe [1-4]. However, how the dynamics of individual protofilaments relates to overall growth persistence has remained unclear. Here, we used the microtubule targeting anti-cancer drug Eribulin [5-7] to explore the consequences of stalled protofilament elongation on microtubule growth. Using X-ray crystallography, we first revealed that Eribulin binds to a site on ß-tubulin that is required for protofilament plus-end elongation. Based on the structural information, we engineered a fluorescent Eribulin molecule. We demonstrate that single Eribulin molecules specifically interact with microtubule plus ends and are sufficient to either trigger a catastrophe or induce slow and erratic microtubule growth in the presence of EB3. Interestingly, we found that Eribulin increases the frequency of EB3 comet "splitting," transient events where a slow and erratically progressing comet is followed by a faster comet. This observation possibly reflects the "healing" of a microtubule lattice. Because EB3 comet splitting was also observed in control microtubules in the absence of any drugs, we propose that Eribulin amplifies a natural pathway toward catastrophe by promoting the arrest of protofilament elongation.

SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture.

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.

Kinesin-Binding Protein Controls Microtubule Dynamics and Cargo Trafficking by Regulating Kinesin Motor Activity.

Kinesin motor proteins play a fundamental role for normal neuronal development by controlling intracellular cargo transport and microtubule (MT) cytoskeleton organization. Regulating kinesin activity is important to ensure their proper functioning, and their misregulation often leads to severe human neurological disorders. Homozygous nonsense mutations in kinesin-binding protein (KBP)/KIAA1279 cause the neurological disorder Goldberg-Shprintzen syndrome (GOSHS), which is characterized by intellectual disability, microcephaly, and axonal neuropathy. Here, we show that KBP regulates kinesin activity by interacting with the motor domains of a specific subset of kinesins to prevent their association with the MT cytoskeleton. The KBP-interacting kinesins include cargo-transporting motors such as kinesin-3/KIF1A and MT-depolymerizing motor kinesin-8/KIF18A. We found that KBP blocks KIF1A/UNC-104-mediated synaptic vesicle transport in cultured hippocampal neurons and in C. elegans PVD sensory neurons. In contrast, depletion of KBP results in the accumulation of KIF1A motors and synaptic vesicles in the axonal growth cone. We also show that KBP regulates neuronal MT dynamics by controlling KIF18A activity. Our data suggest that KBP functions as a kinesin inhibitor that modulates MT-based cargo motility and depolymerizing activity of a subset of kinesin motors. We propose that misregulation of KBP-controlled kinesin motors may represent the underlying molecular mechanism that contributes to the neuropathological defects observed in GOSHS patients.

Structural insights on cholesterol endosynthesis: Binding of squalene and 2,3-oxidosqualene to supernatant protein factor.

We present the crystal structures of the SEC14-like domain of supernatant protein factor (SPF) in complex with squalene and 2,3-oxidosqualene. The structures were resolved at 1.75Å (complex with squalene) and 1.6Å resolution (complex with 2,3-oxidosqualene), leading in both cases to clear images of the protein/substrate interactions. Ligand binding is facilitated by removal of the Golgi-dynamics (GOLD) C-terminal domain of SPF, which, as shown in previous structures of the apo-protein, blocked the opening of the binding pocket to the exterior. Both substrates bind into a large hydrophobic cavity, typical of such lipid-transporter family. Our structures report no specific recognition mode for the epoxide group. In fact, for both molecules, ligand affinity is dominated by hydrophobic interactions, and independent investigations by computational models or differential scanning micro-calorimetry reveal similar binding affinities for both ligands. Our findings elucidate the molecular bases of the role of SPF in sterol endo-synthesis, supporting the original hypothesis that SPF is a facilitator of substrate flow within the sterol synthetic pathway. Moreover, our results suggest that the GOLD domain acts as a regulator, as its conformational displacement must occur to favor ligand binding and release during the different synthetic steps.

Localizing chemical groups while imaging single native proteins by high-resolution atomic force microscopy.

Simultaneous high-resolution imaging and localization of chemical interaction sites on single native proteins is a pertinent biophysical, biochemical, and nanotechnological challenge. Such structural mapping and characterization of binding sites is of importance in understanding how proteins interact with their environment and in manipulating such interactions in a plethora of biotechnological applications. Thus far, this challenge remains to be tackled. Here, we introduce force-distance curve-based atomic force microscopy (FD-based AFM) for the high-resolution imaging of SAS-6, a protein that self-assembles into cartwheel-like structures. Using functionalized AFM tips bearing Ni(2+)-N-nitrilotriacetate groups, we locate specific interaction sites on SAS-6 at nanometer resolution and quantify the binding strength of the Ni(2+)-NTA groups to histidine residues. The FD-based AFM approach can readily be applied to image any other native protein and to locate and structurally map histidine residues. Moreover, the surface chemistry used to functionalize the AFM tip can be modified to map other chemical interaction sites.

Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A.

Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral ß-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open ß-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry.

Centrioles are evolutionary conserved organelles that give rise to cilia and flagella as well as centrosomes. Centrioles display a characteristic ninefold symmetry imposed by the spindle assembly abnormal protein 6 (SAS-6) family. SAS-6 from Chlamydomonas reinhardtii and Danio rerio was shown to form ninefold symmetric, ring-shaped oligomers in vitro that were similar to the cartwheels observed in vivo during early steps of centriole assembly in most species. Here, we report crystallographic and EM analyses showing that, instead, Caenorhabotis elegans SAS-6 self-assembles into a spiral arrangement. Remarkably, we find that this spiral arrangement is also consistent with ninefold symmetry, suggesting that two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Sequence analysis suggests that SAS-6 spirals are restricted to specific nematodes. This oligomeric arrangement may provide a structural basis for the presence of a central tube instead of a cartwheel during centriole assembly in these species.

Structural basis of the 9-fold symmetry of centrioles.

The centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles.

eIF4G stimulates the activity of the DEAD box protein eIF4A by a conformational guidance mechanism.

The activity of eIF4A, a key player in translation initiation, is regulated by other translation factors through currently unknown mechanisms. Here, we provide the necessary framework to understand the mechanism of eIF4A's regulation by eIF4G. In solution, eIF4A adopts a defined conformation that is different from the crystal structure. Binding of eIF4G induces a 'half-open' conformation by interactions with both domains, such that the helicase motifs are pre-aligned for activation. A primary interface acts as an anchor for complex formation. We show here that formation of the secondary interface is essential for imposing the 'half-open' conformation on eIF4A, and it is critical for the functional interaction of eIF4G with eIF4A. Via this bipartite interaction, eIF4G guides the transition of eIF4A between the 'half-open' and closed conformations, and stimulates its activity by accelerating the rate-limiting step of phosphate release. Subtle changes induced by eIF4G may be amplified by input signals from other translation factors, leading to an efficient regulation of translation initiation.

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins.

DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

The DNA-gate of Bacillus subtilis gyrase is predominantly in the closed conformation during the DNA supercoiling reaction.

Gyrase is the only type II topoisomerase that introduces negative supercoils into DNA. Supercoiling is catalyzed via a strand-passage mechanism, in which the gate DNA (gDNA) is transiently cleaved, and a second DNA segment, the transfer DNA (tDNA), is passed through the gap before the gDNA is religated. Strand passage requires an opening of the so-called DNA-gate by approximately 2 nm. A single-molecule FRET study reported equal populations of open and closed DNA-gate in topoisomerase II. We present here single-molecule FRET experiments that monitor the conformation of DNA bound to the DNA-gate of Bacillus subtilis gyrase and the conformation of the DNA-gate itself. DNA bound to gyrase adopts two different conformations, one slightly, one severely distorted. DNA distortion requires cleavage, but neither ATP nor the presence of a tDNA. At the same time, the DNA-gate of gyrase is predominantly in the closed conformation. In agreement with the single molecule data and with the danger of dsDNA breaks for genome integrity, <5% of cleavage complexes are detected in equilibrium. Quinolone inhibitors favor DNA cleavage by B. subtilis gyrase, but disfavor DNA distortion, and the DNA-gate remains in the closed conformation. Our results demonstrate that DNA binding, distortion and cleavage, and gate-opening are mechanistically distinct events. During the relaxation and supercoiling reactions, gyrase with an open DNA-gate is not significantly populated, consistent with gate-opening as a very rare event that only occurs briefly to allow for strand passage.