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The hydration shell of a protein molecule influences its functional important dynamics while the protein molecule influences the hydration shell. Neutron scattering experiments have been used to separate both effects. Neutron crystallography is used to determine the positions and the mean square displacements, 〈 x 2 〉, of the protons in met-myoglobin. Mean square displacements obtained by incoherent neutron scattering stem from motions occurring on a time scale faster than 100 ps. The combination of the two experimental results allows to separate three types of protein protons. The 〈 x 2 〉-values of lysine protons stem from motions faster than 100 ps. Half of the 〈 x 2 〉-values of methyl protons is caused by motions faster than 100 ps, the other halve comes from slower motions. The backbone protons move slower than 100 ps. Incoherent neutron scattering on a perdeuterated myoglobin with a 1 H 2 O hydration shell allowed the study of the diffusion in this shell.

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Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

Axonal branching is essential for neurons to establish contacts to different targets. It therefore provides the physical basis for the integration and distribution of information within the nervous system. During embryonic and early postnatal development, several axonal branching modes may be distinguished that might be regulated by activities of the growth cone or by the axon shaft. The various forms of axonal branching are dependent on intrinsic components and are regulated by extrinsic factors that activate specific signaling systems. This article focuses on components implicated in cyclic guanosine monophosphate signaling that regulate axon bifurcation – a specific form of branching – within the spinal cord in animal models. This cascade is composed of the ligand CNP, the guanylyl cyclase Npr2 and the cyclic guanosine monophosphate-dependent kinase I. In the absence of one of these components, axons of dorsal root ganglion neurons do not form T-shaped branches when entering the spinal cord, while collateral (interstitial) branching, another branching mode of the same type of the neuron, is not affected. It will be important to analyze human patients with mutations in the corresponding genes to get insights into the pathophysiological effects of impaired sensory axon branching in the spinal cord.

Clinical intuition and resent research (Senju et al., 2009) suggests that adults with high-functioning autism (HFA) are able to use explicit verbal information but fail to react upon subtle nonverbal cues in order to understand others and navigate social encounters. In order to investigate the relative influence of different domains of socially relevant information in HFA as compared to matched controls, we used verbal and nonverbal stimuli as a basis for an interpersonal impression formation task. Results demonstrated a reduced sensitivity to nonverbal cues in individuals with HFA when consideration of conflicting information from the different domains was required, although HFA participants could well evaluate nonverbal stimuli in isolation. Thus, in a more complex experimental setting nonverbal information had a comparably weak impact on the impression formation confirming that social processing is preferentially based on verbal information in HFA.

cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase Npr2 (also known as guanylyl cyclase B) by the ligand CNP controls sensory axon bifurcation of DRG and cranial sensory ganglion (CSG) neurons entering the spinal cord or hindbrain, respectively. Previous studies have shown that Npr2 is phosphorylated on serine and threonine residues in its kinase homology domain (KHD). However, it is unknown whether phosphorylation of Npr2 is essential for axon bifurcation. Here, we generated a knock-in mouse line in which the seven regulatory serine and threonine residues in the KHD of Npr2 were substituted by alanine (Npr2-7A), resulting in a nonphosphorylatable enzyme. Real-time imaging of cGMP in DRG neurons with a genetically encoded fluorescent cGMP sensor or biochemical analysis of guanylyl cyclase activity in brain or lung tissue revealed the absence of CNP-induced cGMP generation in theNpr27A/7Amutant. Consequently, bifurcation of axons, but not collateral formation, from DRG or CSG in this mouse mutant was perturbed at embryonic and mature stages. In contrast, axon branching was normal in a mouse mutant in which constitutive phosphorylation of Npr2 is mimicked by a replacement of all of the seven serine and threonine sites by glutamic acid (Npr2–7E). Furthermore, we demonstrate that theNpr27A/7Amutation causes dwarfism as described for global Npr2 mutants. In conclusion, ourin vivostudies provide strong evidence that phosphorylation of the seven serine and threonine residues in the KHD of Npr2 is an important regulatory element of Npr2-mediated cGMP signaling which affects physiological processes, such as axon bifurcation and bone growth.SIGNIFICANCE STATEMENTThe branching of axons is a morphological hallmark of virtually all neurons. It allows an individual neuron to innervate different targets and to communicate with neurons located in different regions of the nervous system. The natriuretic peptide receptor 2 (Npr2), a transmembrane guanylyl cyclase, is essential for the initiation of bifurcation of sensory axons when entering the spinal cord or the hindbrain. By using two genetically engineered mouse lines, we show that phosphorylation of specific serine and threonine residues in juxtamembrane regions of Npr2 are required for its enzymatic activity and for axon bifurcation. These investigations might help to understand the regulation of Npr2 and its integration in intracellular signaling systems.

Identification of the molecular mechanisms underlying axonal branching in vivo has begun in several neuronal systems, notably the projections formed by dorsal root ganglion (DRG) neurons or retinal ganglion cells (RGC). cGMP signalling is essential for sensory axon bifurcation at the spinal cord, whereas brain-derived neurotrophic factor (BDNF) and ephrinA signalling establish position-dependent branching of RGC axons. In the latter system, the degradation of specific signalling components, via the ubiquitin-proteasome system, may provide an additional mechanism involved in axon branching of RGC. The process of arborisation is essential for neurons to innervate multiple targets and to build topographic maps. The various forms of branching found in different types of neurons are regulated by distinct signalling pathways activated by multiple extracellular cues in addition to axonal guidance factors. These signalling cascades, together with transcriptional programs, most likely interact and trigger the polymerisation or depolymerisation of the actin and tubulin cytoskeleton to regulate branching.

While most often forbidden by university policy, homophobic attitudes and intolerance of gay/lesbian athletes may still exist within intercollegiate athletic departments. Against this backdrop, this study examines attitudes towards sexual orientation from a sample of Division I and III male university athletes (n=397) from four universities in the south-eastern United States. The study's primary research questions are: a) utilizing ethnicity as the independent variable, what are the sampled male athletes' attitudes towards sexual orientations? and b) is there a relationship between male college athletes' ethnicity and their sexual-orientation attitudes? Using the frameworks of social script and critical race theories, this article discusses the results' significance for university athletic administrators, faculty and college athletes.

The goal of the study was to investigate the size of the corpus callosum (CC) and its subsegments in relation to total brain volume (TBV) as an empirical indicator of impaired connectivity in autism with special respect to gender. In MRI data sets of 29 adults with high-functioning autism (HFA) and 29 age-, gender- and IQ-matched control subjects, the TBV was measured and the CC was analyzed as a whole and in subsegments employing two different manual segmentation procedures. With respect to diagnosis, there were no significant differences in the dependent variables (CC, CC subsegments, and TBV). With respect to gender, only TBV was significantly increased in males compared with females, resulting in a significantly decreased CC/TBV ratio in males. This finding, however, was independent from gender and can be fully attributed to brain size. Our findings do not support the following hypotheses: (1) a hypothesis of impaired CC in HFA adults as a subgroup of patients with autism spectrum disorders, and (2) the sexual dimorphism hypothesis of the CC.

The dynamic and reversible acetylation of proteins catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) is a major epigenetic regulatory mechanism of gene transcription 1 associated with multiple diseases. While HDAC inhibitors are approved to treat certain cancers, progress on the development of drug-like HAT inhibitors has lagged 2. The HAT paralogs p300 and CBP (p300/CBP) are key transcriptional co-activators essential for a multitude of cellular processes and also implicated in human pathological conditions, including cancer 3. Current p300/CBP HAT domain inhibitors including natural products, 4 bi-substrate analogs (Lys-CoA) 5 and the widely utilized C646 6, 7 lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like p300/CBP catalytic inhibitor. We show the first high resolution (1.95Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 is acetyl-CoA competitive. A-485 selectively inhibited proliferation across lineage-specific tumor types, including several hematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen sensitive and castrate resistant prostate cancer and inhibited tumor growth in a castration resistant xenograft model. These results demonstrate the feasibility of selectively targeting the catalytic activity of histone acetyltransferases.

The coxsackievirus–adenovirus receptor (CAR) is a member of the Ig superfamily strongly expressed in the developing nervous system. Our histological investigations during development reveal an initial uniform distribution of CAR on all neural cells with a concentration on membranes that face the margins of the nervous system (e.g., the basal laminae and the ventricular side). At more advanced stages, CAR becomes downregulated and restricted to specific regions including areas rich in axonal and dendritic surfaces.To study the function of CAR on neural cells, we used the fiber knob of the adenovirus, extracellular CAR domains, blocking antibodies to CAR, as well as CAR-deficient neural cells. Blocking antibodies were found to inhibit neurite extension in retina organ and retinal explant cultures, whereas the application of the recombinant fiber knob of the adenovirus subtype Ad2 or extracellular CAR domains promoted neurite extension and adhesion to extracellular matrices.We observed a promiscuous interaction of CAR with extracellular matrix glycoproteins, which was deduced from analytical ultracentrifugation experiments, affinity chromatography, and adhesion assays. The membrane proximal Ig domain of CAR, termed D2, was found to bind to a fibronectin fragment, including the heparin-binding domain 2, which promotes neurite extension of wild type, but not of CAR-deficient neural cells. In contrast to heterophilic interactions, homophilic association of CAR involves both Ig domains, as was revealed by ultracentrifugation, chemical cross-linking, and adhesion studies. The results of these functional and binding studies are correlated to a U-shaped homodimer of the complete extracellular domains of CAR detected by x-ray crystallography.

The dynamic and reversible acetylation of proteins catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) is a major epigenetic regulatory mechanism of gene transcription associated with multiple diseases. While HDAC inhibitors are approved to treat certain cancers, progress on the development of drug-like HAT inhibitors has lagged. The HAT paralogs p300 and CBP (p300/CBP) are key transcriptional co-activators essential for a multitude of cellular processes and also implicated in human pathological conditions, including cancer. Current p300/CBP HAT domain inhibitors including natural products, bi-substrate analogs (Lys-CoA) and the widely utilized C646 lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like p300/CBP catalytic inhibitor. We show the first high resolution (1.95Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 is acetyl-CoA competitive. A-485 selectively inhibited proliferation across lineage-specific tumor types, including several hematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen sensitive and castrate resistant prostate cancer and inhibited tumor growth in a castration resistant xenograft model. These results demonstrate the feasibility of selectively targeting the catalytic activity of histone acetyltransferases. Citation Format: Kenneth D. Bromberg, Loren M. Lasko, Clarissa G. Jakob, Wei Qiu, Debra Montgomery, Enrico L. Digiammarino, Todd M. Hansen, Roberto M. Risi, Robin R. Frey, Vlasios Manaves, Bailin Shaw, Mikkel Algire, Paul Hessler, Lloyd T. Lam, Tamar Uziel, Emily Favire, Debra Ferguson, Fritz G. Buchanan, Ruth L. Martin, Maricel Torrent, Saul H. Rosenberg, Michael R. Michaelides, Albert Lai. Discovery of a potent catalytic p300/CBP inhibitor that targets lineage-specific tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A23.

ProbodyTM therapeutics are antibody prodrugs designed to remain largely inactive until proteolytically activated in the tumor microenvironment (TME), potentially enabling the safer targeting of antigens that are highly expressed in both tumor and normal tissue. CD71 (transferrin receptor) is an example of an ideal Probody Drug Conjugate (PDC) target, not only because it efficiently internalizes and can deliver a cytotoxic payload intracellularly, but also because it is expressed at high levels both in many different tumor types as well as in dividing normal cells. We have previously demonstrated that while an anti-CD71 antibody drug conjugate (ADC) is highly toxic, a CD71-targeting PDC is both efficacious in mouse tumor models and well tolerated in nonhuman primates. Two key components of a Probody therapeutic prodomain that reduce its binding to normal tissue and allow for its tumor-specific activation are 1) a mask that reduces the ability of the antibody binding site to interact with target antigen and 2) a protease-activatable substrate that is cleaved in the TME, resulting in removal of the mask. Here, we demonstrate how modulating mask strength and substrate cleavability can optimize efficacy and safety of a CD71-targeting PDC in preclinical models. Through this process, we have selected a lead molecule, CX-2029, for further development. CX-2029 is a CD71-targeting PDC conjugated to vcMMAE with a Drug to Probody Ratio (DPR) of 2, achieved by purification. At dose levels consistent with those expected in humans, a more strongly masked PDC, CX-2018, was less efficacious in a mouse xenograft tumor model compared to PDC CX-2016, which has a weaker mask, demonstrating that mask strength affects antitumor activity. Further, PDC CX-2019, which has the same mask but a less cleavable substrate than CX-2016, was similarly efficacious in a mouse xenograft tumor model, demonstrating that both substrates are sufficiently cleaved in the TME to activate the PDCs. However, CX-2019 was better tolerated in NHP at 6 mg/kg than CX-2016, suggesting that the less cleavable substrate in CX-2019 leads to a better therapeutic index. Using a LC/MS/MS method, we showed lower levels of circulating activated CX-2019 compared with circulating activated CX-2016, which is consistent with CX-2019’s improved tolerability. Lead CX-2029 contains the same mask and substrate as CX-2019 but differs in having a DPR of 2 versus ~3 for CX-2019. Up to 6 mg/kg of CX-2029 as a single dose produced complete regressions and durable responses in mouse xenograft tumor models encompassing multiple indications, and was tolerated in monkeys at doses of up to 12 mg/kg. These data demonstrate that, in preclinical models, tuning of mask strength and substrate cleavability can optimize the efficacy and tolerability of Probody Therapeutics and have the potential to enable the safe and effective targeting of highly expressed tumor antigens like CD-71. CX-2029 is currently under development, with an IND filing expected in 2018. PROBODY is a trademark of CytomX Therapeutics, Inc. Citation Format: Shweta Singh, Laura Serwer, Niharika Chauhan, Amy DuPage, Michael Krimm, Ken Wong, Yuanhui Huang, Andrew Jang, Eric Ureno, Adam Miller, Sarah Patrick, Shanti Duvur, Fritz Buchanan, Matthew M. Ravn, Rob Leanna, Ilaria Badagnani, Tracy Henriques, Shouchun Liu, Claus Krebber, Sridhar Viswanathan, Jennifer Richardson, Susan Morgan-Lappe, Michael Kavanaugh. Optimizing a CD71-targeting Probody drug conjugate (PDC) for activity in multiple solid tumor and lymphoma models and for tolerability in nonhuman primates [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B116.