Discovery of nanobodies against SARS-CoV-2 and an uncommon neutralizing mechanism

SARS-CoV-2 and its variants continue to threaten public health. The virus recognizes the host cell by attaching its Spike receptor-binding domain (RBD) to the host receptor ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here we report the isolation, and biological and structural characterization of two single-chain antibodies (nanobodies, DL4 and DL28) from RBD-immunized alpaca. Both nanobodies bind Spike with affinities that exceeded the detection limit (picomolar) of the biolayer interferometry assay and neutralize the original SARS-CoV- 2 strain with IC50 of 0.086 g mL-1 (DL4) and 0.385 g mL-1 (DL28). DL4 and a more potent, rationally designed mutant, neutralizes the Alpha variant as potently as the original strain but only displays marginal activity against the Beta variant. By contrast, the neutralizing activity of DL28, when in the Fc-fused divalent form, was less affected by the mutations in the Beta variant (IC50 of 0.414 g mL-1 for Alpha, 1.060 g mL-1 for Beta). Crystal structure studies reveal that DL4 blocks ACE2-binding by direct competition, while DL28 neutralizes SARS-CoV-2 by an uncommon mechanism through which DL28 distorts the receptor-binding motif in RBD and hence prevents ACE2-binding. Our work provides two neutralizing nanobodies for potential therapeutic development and reveals an uncommon mechanism to design and screen novel neutralizing antibodies against SARS-CoV-2.

Antidepressant effect of total flavonoids extracted from Xiaobuxin-Tang in forced swimming tests and learned helplessness in rats and mice / 中国药理学与毒理学杂志

AIM Xiaobuxin-Tang (XBXT) is a traditional Chinese herbal decoction which is composed of Haematitum, Flos Inulae, Folium Phyllostachydis Henonis and Semen Sojae Preparatum. The present study was to investigate if the total flavonoids extracted from XBXT (XBXT-2) had antidepressant effect. METHODS Forced swimming tests in mice and rats, and learned helplessness (LH) model of rats were adopted to affirm the antidepressant effect of XBXT-2 with the test on spontaneous motor activity. Plasma corticosterone level in the LH rats was measured with ELISA. RESULTS Single administraton of XBXT-2 at the doses of 50 and 100 mg·kg-1 (ig) significantly decreased the duration of immobility time in the forced swimming tests in mice and rats. Researches on LH model of rats indicated that XBXT-2 at doses of 50 and 25 mg·kg-1 markedly reduced the number of escape failure in shuttle box. Meanwhile, the plasma corticosterone level of the LH rats was significantly decreased. XBXT-2 50-200 mg·kg-1 had no effects on spontaneous motor activity in mice. CONCLUSION XBXT-2 possesses significant antidepressant-like effect. The mechanism may involve the inhibition of the hyperaction of the hypothalamic-pituitary-adrenal axis.

Effect of inulin-type hexasaccharide on forced swimming tests in mice and rats and DRL 72 s in rats / 中国药理学通报

AIM　To determine whether O-β -D-fructofuranosyl-〔(2→1 )-O-β-D-fructofuransyl〕4α-D-glucopyranoside (inulin-type hexasaccharide, IHS), a monomer extracted from the roots of Morinda of ficinalis How, has antidepressant action. METHODS Fo rced swimming tests in mice and rats and differential-reinforcement-of-low-r ate 72 second schedule (DRL 72 s) in rats were used. RESULTS In the forced swimming test in mice, IHS (80 mg*kg-1, po), like the effe ct of clinically effective antidepressant desipramine (10 mg*kg-1, ip)， produced significant decrease in immobility time. IHS (20 mg*kg-1，po ) also elicited significant decrease in immobility time in forced swimming test in rats, which was comparable to the effect of desipramine (40 mg*kg-1, po). Moreover, in the DRL 72 s in rats, IHS (5～10 mg*kg-1, ip), s imilar to desipramine (5 mg*kg-1, ip), elicited significant increase in reinforcers. CONCLUSION These findings demonstrate that IHS has antidepressant action and is an effective component extracted from the root s of Morinda officinalis How.

Interactions of nicotinic and muscarinic acetylcholine receptors / 中国药理学通报

Nicotinic and muscarinic acetylcholine receptors have the same endogenous ligand ACh and are distributed together in many tissues, so it is reasonable to believe that there must be some interactions existed between them. The functions of muscarinic receptors in the tissues innervated by the parasympathetic cholinergic postfibers, can be modulated by the ganglionic nicotinic receptors through stimulating ACh release. In ganglia, the postsynaptic nicotinic receptor activities can be modulated by the presynaptic muscarinic and nicotinic autoreceptors through regulating ACh release. Moreover, The functions of muscarinic receptors can be changed by nicotinic receptor desensitization or blockade. The two types of receptor act on each other and keep in a varied homeostasis of cholinergic nervous system.

Effect of antidepressants on the hippocampal neurogenesis in chronically stressed mice / 中国药理学通报

AIM To explore possible action mechanism of antidepressants. METHODS Using flow cytometry, the cell proliferation was detected. The proliferation of hippocampal progenitor cells and level of brain derived neurotrophic factor (BDNF) were measured by immunohistochemistry. RESULTS Treatment with N methyl D aspartate (NMDA) 600 ?mol?L -1 for 3 d significantly decreased the percentage of S phase in PC12 cells, while in the presence of classical antidepressants, desipramine (DIM) or fluoxetine (FLU) 1, 5 ?mol?L -1 , the percentage of S phase increased. Furthermore, the proliferation of hippocampal progenitor cells, as well as the BDNF level in dentate gyrus (subgranular zone) significantly decreased in chronically stressed mice for 24 d, while chronic administration with DIM or FLU 10 mg?kg -1 (ip) normalized it. Meanwhile, the BDNF level in dentate gyrus also elevated after DIM or FLU treatments. CONCLUSION Up regulation of the hippocampal neurogenesis is the common action mechanism for antidepressants, which may be closely related to the elevation of BDNF level at the same time.

Effect of pinacidil on nicotine-induced current in rat superior cervical ganglia / 中国药理学通报

AIM To study the effect of ATP sensitive potassium channel(K ATP ) opener pinacidil on the current induced by nicotine in cultured rat superior cervical ganglia. METHOD Whole cell patch clamp technique. RESULTS Pinacidil inhibited nicotine induced current when in higher dose(100 ?mol?L -1 ), but had no effect on the desensitization of nicotinic receptors. K ATP antagonist glibenclamide didn't influence the inhibitory effect of pinacidil on nicotinic current. CONCLUSION Pinacidil had an inhibitory effect on nicotine induced current in rat superior cervical ganglia and this effect wasn't mediated by K ATP .