We directly resolved discrete exocytic fusion events mediating insertion of AMPA-type glutamate receptors (AMPARs) to the somatodendritic surface of rat hippocampal pyramidal neurons, in slice and dissociated cultures, using protein tagging with a pH-sensitive GFP (green fluorescent protein) variant and rapid (10 frames/s) fluorescence microscopy. AMPAR-containing exocytic events occurred under basal culture conditions in both the cell body and dendrites; potentiating chemical stimuli produced an NMDA receptor-dependent increase in the frequency of individual exocytic events. The number of AMPARs inserted per exocytic event, estimated using singlemolecule analysis, was quite uniform but individual events differed significantly in kinetic properties affecting the subsequent surface distribution of receptors. "Transient" events, from which AMPARs dispersed laterally immediately after surface insertion, generated a pronounced but short-lived (dissipating within ϳ1 s) increase in surface AMPAR fluorescence extending locally (2-5 m) from the site of exocytosis. "Persistent" events, from which inserted AMPARs dispersed slowly (typically over 5-10 s), affected local surface receptor concentration to a much smaller degree. Both modes of exocytic insertion occurred throughout the dendritic shaft, but remarkably, neither mode of insertion was observed directly into synaptic spines. AMPARs entered spines preferentially from transient events occurring in the adjoining dendritic shaft, driven apparently by mass action and short-range lateral diffusion, and locally delivered AMPARs remained mostly in the mobile fraction. These results suggest a highly dynamic mechanism for both constitutive and activitydependent surface delivery of AMPARs, mediated by kinetically distinct exocytic modes that differ in propensity to drive lateral entry of receptors to nearby synapses.

Key Points Cold agglutinin–driven classical pathway activity terminates prior to the initiation of the terminal cascade in CAD patient blood. By inhibiting cold agglutinin–mediated complement deposition on the cellular membrane, TNT003 prevents RBCs from being phagocytosed.

Background and purpose: Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain-related diseases. The current work describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist. Experimental approach: The antagonistic potencies (pIC50) of AF-353 for rat and human P2X3 and human P2X2/3 receptors were determined using methods of radioligand binding, intracellular calcium flux and whole cell voltage-clamp electrophysiology. Key results: The pIC50 estimates for these receptors ranged from 7.3 to 8.5, while concentrations 300-fold higher had little or no effect on other P2X channels or on an assortment of receptors, enzymes and transporter proteins. In contrast to A-317491 and TNP-ATP, competition binding and intracellular calcium flux experiments suggested that AF-353 inhibits activation by ATP in a non-competitive fashion. Favourable pharmacokinetic parameters were observed in rat, with good oral bioavailability (%F = 32.9), reasonable half-life (t1/2 = 1.63 h) and plasma-free fraction (98.2% protein bound). Conclusions and implications:The combination of a favourable pharmacokinetic profile with the antagonist potency and selectivity for P2X3 and P2X2/3 receptors suggests that AF-353 is an excellent in vivo tool compound for study of these channels in animal models and demonstrates the feasibility of identifying and optimizing molecules into potential clinical candidates, and, ultimately, into a novel class of therapeutics for the treatment of pain-related disorders.

Cold agglutinin disease is a difficult-to-treat autoimmune hemolytic anemia in which immunoglobulin M antibodies bind to erythrocytes and fix complement, resulting in predominantly extravascular hemolysis. This trial tested the hypothesis that the anti-C1s antibody sutimlimab would ameliorate hemolytic anemia. Ten patients with cold agglutinin disease participated in the phase 1b component of a first-in-human trial. Patients received a test dose of 10-mg/kg sutimlimab followed by a full dose of 60 mg/kg 1 to 4 days later and 3 additional weekly doses of 60 mg/kg. All infusions were well tolerated without premedication. No drug-related serious adverse events were observed. Seven of 10 patients with cold agglutinin disease responded with a hemoglobin increase >2 g/dL. Sutimlimab rapidly increased hemoglobin levels by a median of 1.6 g/dL within the first week, and by a median of 3.9 g/dL (interquartile range, 1.3-4.5 g/dL; 95% confidence interval, 2.1-4.5) within 6 weeks (P = .005). Sutimlimab rapidly abrogated extravascular hemolysis, normalizing bilirubin levels within 24 hours in most patients and normalizing haptoglobin levels in 4 patients within 1 week. Hemolytic anemia recurred when drug levels were cleared from the circulation 3 to 4 weeks after the last dose of sutimlimab. Reexposure to sutimlimab in a named patient program recapitulated the control of hemolytic anemia. All 6 previously transfused patients became transfusion-free during treatment. Sutimlimab was safe, well tolerated, and rapidly stopped C1s complement–mediated hemolysis in patients with cold agglutinin disease, significantly increasing hemoglobin levels and precluding the need for transfusions. This trial was registered at www.clinicaltrials.gov as #NCT02502903.

Building on prototype 1, which achieves 120 degrees of phosgene-powered unidirectional rotation to rotamer 6 (see Figure 5 in the full article), 7 was designed to accomplish repeated unidirectional rotation (see Scheme 7). Compound 7 contains an amino group on each blade of the triptycene and a 4-(dimethylamino)pyridine (DMAP) unit to selectively deliver phosgene (or its equivalent) to the amine in the "firing position". The synthesis of 7 is described: the key constructive steps are a benzyne addition to an anthracene to generate the triptycene, a stilbene photocyclization to construct the helicene, and a Stille coupling to incorporate the DMAP unit. The DMAP unit was shown to regioselectively relay 1,1'-carbonyldiimidazole (but not phosgene) to the proximal amino group, as designed, but rotation of the triptycene does not occur. Extensive attempts to troubleshoot the problem led to the conclusion that the requisite intramolecular urethane formation, as demonstrated in the prototype (1 --> 4), does not occur with 7 (to give 85) or 97 (to give 100). We speculate that either (i) hydrogen bonding between the hydroxypropyl group and functionality present in 7 but absent from 1 or (ii) a Bürgi-Dunitz (or similar) interaction involving the DMAP (see 106) prevents achievement of a conformation conducive to intramolecular urethane formation.

Serotonin-gated ion channels (5-HT3) are members of the ligand-gated channel family, which includes channels that are opened directly by the neurotransmitter acetylcholine, GABA, glycine, or glutamate. Although there is general agreement that the second transmembrane domain (M2) lines the pore, the position of the gate in the M2 is less certain. Here, we used substituted cysteine accessibility method (SCAM) to provide new evidence for a centrally located gate that moves during channel activation. In the closed state, three cysteine substitutions, located on the extracellular side of M2, were modified by methanethiosulfonate (MTS) reagents. In contrast, 13 cysteine substitutions were modified in the open state with MTS reagents. The pattern of inhibition (every three to four substitutions) was consistent with an alpha helical structure for the middle and cytoplasmic segments of the M2 transmembrane domain. Unexpectedly, open-state modification of two amino acids in the center of M2 with three different MTS reagents prevented channels from fully closing in the absence of neurotransmitter. Our results are consistent with a model in which the central region of the M2 transmembrane domain is inaccessible in the closed state and moves during channel activation.

No abstract

The classical pathway (CP) of complement may contribute to the pathogenesis of antibody-mediated rejection (ABMR). Selective CP blockade may be a promising strategy to counteract rejection. The objective of this first-in-patient phase 1b trial was to evaluate the safety/tolerability and CP-blocking potential of 4 weekly doses (60 mg/kg) of the anti-C1s antibody BIVV009 in complement-mediated disorders. Here we describe the results in a cohort of 10 stable kidney transplant recipients (median of 4.3 years posttransplantation) with late active ABMR and features of CP activation, such as capillary C4d or complement-fixing donor-specific antibodies (DSA). During 7 weeks follow-up, no severe adverse events were reported, and BIVV009 profoundly inhibited overall and DSA-triggered CP activation in serum. Five of 8 C4d-positive recipients turned C4d-negative in 5-week follow-up biopsies, while another 2 recipients showed a substantial decrease in C4d scores. There was, however, no change in microcirculation inflammation, gene expression patterns, DSA levels, or kidney function. In conclusion, we demonstrate that BIVV009 effectively blocks alloantibody-triggered CP activation, even though short-course treatment had no effect on indices of activity in late ABMR. This initial trial provides a valuable basis for future studies designed to clarify the therapeutic value of CP blockade in transplantation. ClinicalTrials.gov NCT#02502903.