Люминесценция дитолуоилметаната дифторида бора. Образование J-агрегатов

The processes of the formation of J-aggregates during the dissolution 2,2-difluoro-4,6-di(4’-methylphenyl)-1,3,2-dioxaborine crystals (1) and their subsequent dissociation have been studied by absorption and luminescence spectroscopy and quantum-chemical modeling. It is shown that two luminescent centers are observed in the solution 1: monomeric luminescence and luminescence of J-aggregates (dual luminescence). Evolution of absorption, luminescence excitation and luminescence spectra is observed over time, indicating a slow dissociation of J-aggregates.

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has become an attractive therapeutic strategy for lowering low-density lipoprotein cholesterol (LDL-C). In this study, a novel high affinity humanized IgG1 mAb (named h5E12-L230G) targeting the catalytic domain of human PCSK9 (hPCSK9) was generated by using CDR-grafting, alanine-scanning mutagenesis, and saturated site-directed mutagenesis. The heavy-chain constant region of h5E12-L230G was modified to eliminate the cytotoxic effector functions and mitigate the heterogeneity. The biolayer interferometry (BLI) binding assay and molecular docking study revealed that h5E12-L230G binds to the catalytic domain of hPCSK9 with nanomolar affinity (KD = 1.72 nM) and an extremely slow dissociation rate (koff, 4.84 × 10−5 s−1), which interprets its quite low binding energy (−54.97 kcal/mol) with hPCSK9. Additionally, h5E12-L230G elevated the levels of LDLR and enhanced the LDL-C uptake in HepG2 cells, as well as reducing the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to the positive control alirocumab. Our data indicate that h5E12-L230G is a high-affinity anti-PCSK9 antibody candidate with an extremely slow dissociation rate for favorably treating hypercholesterolemia and relevant cardiovascular diseases.

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis

Inhibition of Proprotein convertase subtilisin/kexin type 9 (PCSK9) has become an attractive therapeutic strategy for lowering low-density lipoprotein cholesterol (LDL-C). In this study, a novel high affinity humanized IgG1 mAb (named h5E12-L230G) targeting the catalytic domain of human PCSK9 (hPCSK9) was generated by using CDR-grafting, alanine-scanning mutagenesis, and saturated site-directed mutagenesis. To eliminate the cytotoxic effector functions and mitigate the heterogeneity, the heavy-chain constant region of h5E12-L230G was modified with L234A/L235A/N297G mutations and C-terminal lysine deletion. The biolayer interferometry (BLI) binding assay and molecular docking study revealed that h5E12-L230G binds to the catalytic domain of hPCSK9 with nanomolar affinity (KD =1.72 nM) and an extremely slow dissociation rate (koff, 4.84 × 10−5 s−1), which interprets its quite low binding energy (-54.97 kcal/mol) with hPCSK9. Additionally, h5E12-L230G elevated the levels of LDLR and enhanced the LDL-C uptake in HepG2 cells, as well as reduced the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to Alirocumab. Our data suggest that h5E12-L230G is a highly potent antibody binding to PCSK9 catalytic domain with slow dissociation rate which may be utilized as a therapeutic candidate for treating hypercholesterolemia and relevant cardiovascular diseases.

Psychomotor stimulant effects of α-pyrrolidinovalerophenone (αPVP) enantiomers correlate with drug binding kinetics at the dopamine transporter

α-Pyrrolidinovalerophenone (αPVP) is a psychostimulant and drug of abuse associated with severe intoxications in humans. αPVP exerts long-lasting psychostimulant effects, when compared to the classical dopamine transporter (DAT) inhibitor cocaine. Here, we compared the two enantiomeric forms of αPVP, the R- and the S-αPVP, with cocaine using a combination of in silico, in vitro and in vivo approaches. We found that αPVP enantiomers substantially differ from cocaine in their binding kinetics. The two enantiomers differ from each other in their association rates. However, they show similar slow dissociation rates leading to pseudo-irreversible binding kinetics at DAT. The pseudo-irreversible binding kinetics of αPVP is responsible for the observed non-competitive pharmacology and it correlates with persistent psychostimulant effects in mice. Thus, the slow binding kinetics of αPVP enantiomers profoundly differ from the fast kinetics of cocaine both in vitro and in vivo, suggesting drug-binding kinetics as a potential driver of psychostimulant effects in vivo.

High-throughput macromolecular polymorph screening via an NMR and X-ray powder diffraction synergistic approach: the case of human insulin co-crystallized with resorcinol derivatives

Regular injections of insulin provide life-saving benefits to millions of diabetics. Apart from native insulin and insulin analogue formulations, microcrystalline insulin suspensions are also commercially available. The onset of action of the currently available basal insulins relies on the slow dissociation of insulin hexamers in the subcutaneous space due to the strong binding of small organic ligands. With the aim of identifying insulin–ligand complexes with enhanced pharmacokinetic and pharmacodynamic profiles, the binding affinity of two resorcinol-based molecules (4-chlororesorcinol and 4-bromoresorcinol) and the structural characteristics of insulin upon co-crystallization with them were investigated in the present study. `In solution' measurements were performed via saturation transfer difference (STD) NMR. Co-crystallization upon pH variation resulted in the production of polycrystalline precipitates, whose structural characteristics (i.e. unit-cell symmetry and dimension) were assessed. In both cases, different polymorphs (four and three, respectively) of monoclinic symmetry (P21 and C2 space groups) were identified via X-ray powder diffraction. The results demonstrate the efficiency of a new approach that combines spectroscopy and diffraction techniques and provides an innovative alternative for high-throughput examination of insulin and other therapeutic proteins.

Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions

Recent advances in quantitative proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their physical associations with other proteins are ambiguous. A quantitative understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of transient associations between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow association kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissociation kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnology.

Dissecting the Interaction of FGF8 with Receptor FGFRL1

In mammals, the novel protein fibroblast growth factor receptor-like 1 (FGFRL1) is involved in the development of metanephric kidneys. It appears that this receptor controls a crucial transition of the induced metanephric mesenchyme to epithelial renal vesicles, which further develop into functional nephrons. FGFRL1 knockout mice lack metanephric kidneys and do not express any fibroblast growth factor (FGF) 8 in the metanephric mesenchyme, suggesting that FGFRL1 and FGF8 play a decisive role during kidney formation. FGFRL1 consists of three extracellular immunoglobulin (Ig) domains (Ig1-Ig2-Ig3), a transmembrane domain and a short intracellular domain. We have prepared the extracellular domain (Ig123), the three individual Ig domains (Ig1, Ig2, Ig3) as well as all combinations containing two Ig domains (Ig12, Ig23, Ig13) in recombinant form in human cells. All polypeptides that contain the Ig2 domain (Ig123, Ig12, Ig23, Ig2) were found to interact with FGF8 with very high affinity, whereas all constructs that lack the Ig2 domain (Ig1, Ig3, Ig13) poorly interacted with FGF8 as shown by ELISA and surface plasmon resonance. It is therefore likely that FGFRL1 represents a physiological receptor for FGF8 in the kidney and that the ligand primarily binds to the Ig2 domain of the receptor. With Biacore experiments, we also measured the affinity of FGF8 for the different constructs. All constructs containing the Ig2 domain showed a rapid association and a slow dissociation phase, from which a KD of 2–3 × 10−9 M was calculated. Our data support the hypothesis that binding of FGF8 to FGFRL1 could play an important role in driving the formation of nephrons in the developing kidney.

Clustering of human prion protein and α-synuclein oligomers requires the prion protein N-terminus

The interaction of prion protein (PrP) and α-synuclein (αSyn) oligomers causes synaptic impairment that might trigger Parkinson’s disease and other synucleinopathies. Here, we report that αSyn oligomers (αSynO) cluster with human PrP (huPrP) into micron-sized condensates. Multivalency of αSyn within oligomers is required for condensation, since clustering with huPrP is not observed for monomeric αSyn. The stoichiometry of the heteroassemblies is well defined with an αSyn:huPrP molar ratio of about 1:1. The αSynO−huPrP interaction is of high affinity, signified by slow dissociation. The huPrP region responsible for condensation of αSynO, residues 95−111 in the intrinsically disordered N-terminus, corresponds to the region required for αSynO-mediated cognitive impairment. HuPrP, moreover, achieves co-clustering of αSynO and Alzheimer’s disease-associated amyloid-β oligomers, providing a case of a cross-interaction of two amyloidogenic proteins through an interlinking intrinsically disordered protein region. The results suggest that αSynO-mediated condensation of huPrP is involved in the pathogenesis of synucleinopathies.