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Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts, no new antibiotic class with activity against Gram-negative bacteria has been approved in over 50 years. LepB inhibitors (LepBi) based on the arylomycin class of natural products are a novel class of antibiotics and function by inhibiting the bacterial type I signal peptidase (SPase) in Gram-negative bacteria. One critical aspect of LepBi development involves optimization of the membrane-anchored lipophilic portion of the molecule. We therefore developed an approach that assesses the effect of this portion on the complicated equilibria of plasma protein binding, crossing the outer membrane of Gram-negative bacteria and anchoring in the bacterial inner membrane to facilitate SPase binding. Our findings provide important insights into the development of antibacterial agents where the target is associated with the inner membrane of Gram-negative bacteria., Competing Interests: The authors declare the following competing financial interest(s): M.F.T.K.Y-C.C., Yuan C., J.J.C. and M.R.D. are employees and shareholders of Genentech, a member of the Roche group. R.I.H., T.C.R. and P.A.S. are shareholders of RQx Pharmaceuticals, Inc., (© 2023 American Chemical Society.)

Amino-quinazoline BRaf kinase inhibitor 2 was identified from a library screen as a modest inhibitor of the unfolded protein response (UPR) regulating potential anticancer target IRE1α. A combination of crystallographic and conformational considerations were used to guide structure-based attenuation of BRaf activity and optimization of IRE1α potency. Quinazoline 6-position modifications were found to provide up to 100-fold improvement in IRE1α cellular potency but were ineffective at reducing BRaf activity. A salt bridge contact with Glu651 in IRE1α was then targeted to build in selectivity over BRaf which instead possesses a histidine in this position (His539). Torsional angle analysis revealed that the quinazoline hinge binder core was ill-suited to accommodate the required conformation to effectively reach Glu651, prompting a change to the thienopyrimidine hinge binder. Resulting analogues such as 25 demonstrated good IRE1α cellular potency and imparted more than 1000-fold decrease in BRaf activity., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)

NMDA receptors (NMDARs) play subunit-specific roles in synaptic function and are implicated in neuropsychiatric and neurodegenerative disorders. However, the in vivo consequences and therapeutic potential of pharmacologically enhancing NMDAR function via allosteric modulation are largely unknown. We examine the in vivo effects of GNE-0723, a positive allosteric modulator of GluN2A-subunit-containing NMDARs, on brain network and cognitive functions in mouse models of Dravet syndrome (DS) and Alzheimer's disease (AD). GNE-0723 use dependently potentiates synaptic NMDA receptor currents and reduces brain oscillation power with a predominant effect on low-frequency (12-20 Hz) oscillations. Interestingly, DS and AD mouse models display aberrant low-frequency oscillatory power that is tightly correlated with network hypersynchrony. GNE-0723 treatment reduces aberrant low-frequency oscillations and epileptiform discharges and improves cognitive functions in DS and AD mouse models. GluN2A-subunit-containing NMDAR enhancers may have therapeutic benefits in brain disorders with network hypersynchrony and cognitive impairments., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.

The N -methyl-d-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, gated by the endogenous coagonists glutamate and glycine, permeable to Ca 2+ and Na + . NMDAR dysfunction is associated with numerous neurological and psychiatric disorders, including schizophrenia, depression, and Alzheimer's disease. Recently, we have disclosed GNE-0723 ( 1 ), a GluN2A subunit-selective and brain-penetrant positive allosteric modulator (PAM) of NMDARs. This work highlights the discovery of a related pyridopyrimidinone core with distinct structure-activity relationships, despite the structural similarity to GNE-0723. GNE-5729 ( 13 ), a pyridopyrimidinone-based NMDAR PAM, was identified with both an improved pharmacokinetic profile and increased selectivity against AMPARs. We also include X-ray structure analysis and modeling to propose hypotheses for the activity and selectivity differences.

To enhance physiological function of NMDA receptors (NMDARs), we identified positive allosteric modulators (PAMs) of NMDARs with selectivity for GluN2A subunit-containing receptors. X-ray crystallography revealed a binding site at the GluN1-GluN2A dimer interface of the extracellular ligand-binding domains (LBDs). Despite the similarity between the LBDs of NMDARs and AMPA receptors (AMPARs), GluN2A PAMs with good selectivity against AMPARs were identified. Potentiation was observed with recombinant triheteromeric GluN1/GluN2A/GluN2B NMDARs and with synaptically activated NMDARs in brain slices from wild-type (WT), but not GluN2A knockout (KO), mice. Individual GluN2A PAMs exhibited variable degrees of glutamate (Glu) dependence, impact on NMDAR Glu EC50, and slowing of channel deactivation. These distinct PAMs also exhibited differential impacts during synaptic plasticity induction. The identification of a new NMDAR modulatory site and characterization of GluN2A-selective PAMs provide powerful molecular tools to dissect NMDAR function and demonstrate the feasibility of a therapeutically desirable type of NMDAR enhancement., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Angiographic indexes of restenosis after coronary stent placement in patients show a bimodal pattern suggesting the existence of two populations with different risk of restenosis. This is reflected in the arterial remodeling response of inbred mouse strains arguing for a genetic control of the mechanisms leading to lumen narrowing. As bone marrow-derived cells (BMCs) contribute to vascular healing after arterial injury, we investigated the role of BMCs in the genetic control of restenosis., Methods and Results: 129X1/SvJ mice developed significantly more neointima and late lumen loss compared to C57BL/6 mice. Gene expression analysis of intimal tissue revealed major differences in the expression of inflammatory and hematopoietic stem and progenitor cell-associated genes in response to arterial injury. In 129X1/SvJ mice stronger mobilization of lin(-)sca-1(+)CXCR4(+) cells was observed after vascular injury. Bone marrow transplantation identified the extent of neointima formation as clearly dependent on the genetic background of BMCs (ie, mice with 129X1/SvJ BMCs developed more intimal hyperplasia). The inflammatory response and the recruitment of BMCs to the site of arterial injury were significantly increased in mice with 129X1/SvJ BMCs., Conclusions: The genetically controlled mechanisms leading to lumen narrowing in vascular remodeling are dependent on mobilization and recruitment capacities of particular BMCs.

Atherosclerosis, restenosis, and posttransplant graft atherosclerosis are characterized by endothelial damage, infiltration of inflammatory cells, and proliferation of smooth muscle cells. The CXCR3-activating chemokines interferon-gamma inducible protein 10 (IP10) and MIG (monokine induced by interferon-gamma) have been implicated in vascular repair and remodeling. The underlying molecular mechanisms, however, remain elusive. Here, we show that wire-mediated arterial injury induced local and systemic expression of IP10 and MIG, resulting in enhanced recruitment of CXCR3(+) leukocytes and hematopoietic progenitor cells. This was accompanied by profound activation of mammalian target of rapamycin complex (mTORC)1, increased reactive oxygen species production, apoptosis, and intimal hyperplasia. Genetic and pharmacological inactivation of CXCR3 signaling not only suppressed recruitment of inflammatory cells but also abolished mTORC1 activation, reduced reactive oxygen species generation, and blocked apoptosis of vascular cells, resulting in significant reduction of intimal hyperplasia in vivo. In vitro, stimulation of T cells with IP10 directly activated mTORC1 and induced generation of reactive oxygen species and apoptosis in an mTORC1-dependent manner. These results strongly indicate that CXCR3-dependent activation of mTORC1 directly links stimulation of the Th1 immune system with the proliferative response of intimal cells in vascular remodeling.

Aims: Neointima formation after vascular injury is strongly associated with inflammation. Rapamycin inhibits human neointima formation and reduces expression of the proinflammatory cytokine endothelial-monocyte activating peptide II (EMAP-II) in vitro. Here we investigated the interplay between EMAP-II and rapamycin after vascular injury in vivo., Methods and Results: In a mouse model of vascular injury, mice were either not treated, given everolimus, a rapamycin derivate, or subjected to simultaneous challenge with everolimus and EMAP-II. EMAP-II expression was measured in coronary artery smooth muscle cells (CASMC) and monocytic cells in vitro and in patients after percutaneous coronary intervention (PCI). After vascular injury, rapamycin reduced neointima formation and adventitial thickening. Immunohistochemistry revealed reduced EMAP-II protein expression and suppressed recruitment of inflammatory cells. Simultaneous challenge with EMAP-II counteracted these effects of rapamycin. Expression of EMAP-II and its inhibition by rapamycin was confirmed in CASMC and monocytic cells. In patients, EMAP-II upregulation was confined to PCI of distal coronary artery segments and profoundly suppressed by oral rapamycin treatment., Conclusion: These data suggest important yet unrecognized roles of EMAP-II and adventitial inflammation in neointima formation: Through inhibition of EMAP-II, rapamycin reduces the recruitment of inflammatory cells to the adventitia and supports an early and bland healing.

Cell-surface mucin glycoproteins are altered with the onset of oncogenesis. Knowledge of mucin structure could be used in vaccine strategies that target tumor-associated mucin motifs. Thus far, however, mucins have resisted detailed molecular analysis. Reported herein is the solution conformation of a highly complex segment of the mucin CD43. The elongated secondary structure of the isolated mucin strand approaches the stability of motifs found in folded proteins. The features required for the mucin motif to emerge are also described. Immunocharacterization of related constructs strongly suggests that the observed epitopes represent distinguishing features of tumor cell-surface architecture.