Benzodioxane Carboxamide Derivatives As Novel Monoamine Oxidase B Inhibitors with Antineuroinflammatory Activity.

In this study, a series of N-phenyl-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide derivatives were designed, synthesized, and evaluated for their inhibitory activities against human MAO-B (hMAO-B). The structure-activity relationship (SAR) was investigated and summarized. Compound 1l (N-(3,4-dichlorophenyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide) showed the most potent inhibitory activity with an IC50 value of 0.0083 µM and the selectivity index (IC50 (hMAO-A)/IC50 (hMAO-B)) was >4819. Kinetics and reversibility studies confirmed that compound 1l acted as a competitive and reversible inhibitor of hMAO-B. Molecular docking studies revealed the enzyme-inhibitor interactions, and the rationale was provided. Additionally, compound 1l could effectively inhibit the release of NO, TNF-α, and IL-1ß in both LPS- and Aß1-42-stimulated BV2 cells and attenuate the cytotoxicity induced by Aß1-42. Since compound 1l exhibited low neurotoxicity, we believe that the hit compound with dual activities of inhibiting MAO-B and antineuroinflammation could be further investigated as a novel potential lead for future studies in vivo.

De Novo Screening and Mirror Image Isomerization of Linear Peptides Targeting α7 Nicotinic Acetylcholine Receptor.

As ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs) are widely distributed in the central and peripheral nervous systems and are associated with the pathogenesis of various degenerative neurological diseases. Here, we report the results of phage display-based de novo screening of an 11-residue linear peptide (named LKP1794) that targets the α7 nAChR, which is among the most abundant nAChR subtypes in the brain. Moreover, two d-peptides were generated through mirror image and/or primary sequence inverso isomerization (termed DRKP1794 and DKP1794) and displayed improved inhibitory effects (IC50 = 0.86 and 0.35 µM, respectively) on α7 nAChR compared with the parent l-peptide LKP1794 (IC50 = 2.48 µM), which markedly enhanced serum stability. A peptide-based fluorescence probe was developed using proteolytically resistant DKP1794 to specifically image the α7 nAChR in living cells. This work provides a new peptide tool to achieve inhibitory modulation and specifically image the α7 nAChR.

Modification of Low-Energy Surfaces Using Bicyclic Peptides Discovered by Phage Display.

Solid-binding peptides are a simple and versatile tool for the non-covalent modification of solid material surfaces, and a variety of peptides have been developed by reference to natural proteins or de novo design. Here, for the first time, we report the discovery of a bicyclic peptide targeting the heterogeneous material polypropylene by combining phage display technology and next-generation sequencing. We find that the enrichment properties of bicyclic peptides capable of binding to polypropylene are distinct from linear peptides, as reflected in amino acid abundance and a trend toward negative net charges and high hydrophobicity. The selected bicyclic peptide has a higher binding affinity for polypropylene compared with a previously reported linear peptide, enabling the hydrophilic and adhesive properties of the polypropylene to be more effectively enhanced. Our work paves the way for the exploration and utilization of conformational-restricted cyclic peptides as a new family of functionally evolvable agents for material surface modification.

Total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates using side-chain Boc/tBu protecting groups.

A practical strategy for the total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates was developed. In this strategy, the Boc/tBu protecting groups are utilized for the side chains of Trp, His, Arg, Asp, and Glu, and is deprotected in borate buffer at 90 °C to avoid depurination of the oligonucleotide caused by strong acid treatment. The advantage of this strategy is that the abovementioned amino acids are readily available in the market and the side reaction of deguanidination of the Arg residue can be avoided. This side-chain Boc/tBu protection strategy will expand the applicability of total stepwise synthesis in the preparation of peptide-oligonucleotide conjugates.

Parthenolide reveals an allosteric mode to inhibit the deISGylation activity of SARS-CoV­2 papain-like protease.

The coronavirus papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for viral polypeptide cleavage and the deISGylation of interferon-stimulated gene 15 (ISG15), which enable it to participate in virus replication and host innate immune pathways. Therefore, PLpro is considered an attractive antiviral drug target. Here, we show that parthenolide, a germacrane sesquiterpene lactone, has SARS-CoV-2 PLpro inhibitory activity. Parthenolide covalently binds to Cys-191 or Cys-194 of the PLpro protein, but not the Cys-111 at the PLpro catalytic site. Mutation of Cys-191 or Cys-194 reduces the activity of PLpro. Molecular docking studies show that parthenolide may also form hydrogen bonds with Lys-192, Thr-193, and Gln-231. Furthermore, parthenolide inhibits the deISGylation but not the deubiquitinating activity of PLpro in vitro. These results reveal that parthenolide inhibits PLpro activity by allosteric regulation.

Thioester-Assisted Sortase-A-Mediated Ligation.

Sortase A (SrtA)-mediated ligation, a popular method for protein labeling and semi-synthesis, is limited by its reversibility and dependence on the LPxTG motif, where "x" is any amino acid. Here, we report that SrtA can mediate the efficient and irreversible ligation of a protein/peptide containing a C-terminal thioester with another protein/peptide bearing an N-terminal Gly, with broad tolerance for a wide variety of LPxT-derived sequences. This strategy, the thioester-assisted SrtA-mediated ligation, enabled the expedient preparation of proteins bearing various N- or C-terminal labels, including post-translationally modified proteins such as the Ser139-phosphorylated histone H2AX and Lys9-methylated histone H3, with less dependence on the LPxTG motif. Our study validates the chemical modification of substrates as an effective means of augmenting the synthetic capability of existing enzymatic methods.

Structural insights into the activation of somatostatin receptor 2 by cyclic SST analogues.

The endogenous cyclic tetradecapeptide SST14 was reported to stimulate all five somatostatin receptors (SSTR1-5) for hormone release, neurotransmission, cell growth arrest and cancer suppression. Two SST14-derived short cyclic SST analogues (lanreotide or octreotide) with improved stability and longer lifetime were developed as drugs to preferentially activate SSTR2 and treat acromegalia and neuroendocrine tumors. Here, cryo-EM structures of the human SSTR2-Gi complex bound with SST14, octreotide or lanreotide were determined at resolutions of 2.85 Å, 2.97 Å, and 2.87 Å, respectively. Structural and functional analysis revealed that interactions between ß-turn residues in SST analogues and transmembrane SSTR2 residues in the ligand-binding pocket are crucial for receptor binding and functional stimulation of the two SST14-derived cyclic octapeptides. Additionally, Q1022.63, N2766.55, and F2947.35 could be responsible for the selectivity of lanreotide or octreotide for SSTR2 over SSTR1 or SSTR4. These results provide valuable insights into further rational development of SST analogue drugs targeting SSTR2.

Chemical Synthesis of a Full-Length G-Protein-Coupled Receptor ß2-Adrenergic Receptor with Defined Modification Patterns at the C-Terminus.

The ß2-adrenergic receptor (ß2AR) is a G-protein-coupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. ß2AR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length ß2AR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length ß2AR based on a combined native chemical ligation (NCL) and sortase ligation strategy. An array of homogeneous samples of full-length ß2ARs with distinct modification patterns, including a full-length ß2AR bearing both monoubiquitination and octaphosphorylation modifications, were successfully prepared for the first time. Using these homogeneously modified full-length ß2AR receptors, we found that different phosphorylation patterns mediate different interactions with ß-arrestin1 as reflected in different agonist binding affinities. Our experiments also indicated that ubiquitination can further modulate interactions between ß2AR and ß-arrestin1. Access to full-length ß2AR with well-defined and homogeneous modification patterns at the C-terminus opens a door to further in-depth mechanistic studies into the structure and dynamics of ß2AR complexes with downstream transducer proteins, including G proteins, arrestins, and GPCR kinases.

Structural mechanism of cooperative activation of the human calcium-sensing receptor by Ca2+ ions and L-tryptophan.

The human calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) responsible for maintaining Ca2+ homeostasis in the blood. The general consensus is that extracellular Ca2+ is the principal agonist of CaSR. Aliphatic and aromatic L-amino acids, such as L-Phe and L-Trp, increase the sensitivity of CaSR towards Ca2+ and are considered allosteric activators. Crystal structures of the extracellular domain (ECD) of CaSR dimer have demonstrated Ca2+ and L-Trp binding sites and conformational changes of the ECD upon Ca2+/L-Trp binding. However, it remains to be understood at the structural level how Ca2+/L-Trp binding to the ECD leads to conformational changes in transmembrane domains (TMDs) and consequent CaSR activation. Here, we determined the structures of full-length human CaSR in the inactive state, Ca2+- or L-Trp-bound states, and Ca2+/L-Trp-bound active state using single-particle cryo-electron microscopy. Structural studies demonstrate that L-Trp binding induces the closure of the Venus flytrap (VFT) domain of CaSR, bringing the receptor into an intermediate active state. Ca2+ binding relays the conformational changes from the VFT domains to the TMDs, consequently inducing close contact between the two TMDs of dimeric CaSR, activating the receptor. Importantly, our structural and functional studies reveal that Ca2+ ions and L-Trp activate CaSR cooperatively. Amino acids are not able to activate CaSR alone, but can promote the receptor activation in the presence of Ca2+. Our data provide complementary insights into the activation of class C GPCRs and may aid in the development of novel drugs targeting CaSR.

Structural insights into human acid-sensing ion channel 1a inhibition by snake toxin mambalgin1.

Acid-sensing ion channels (ASICs) are proton-gated cation channels that are involved in diverse neuronal processes including pain sensing. The peptide toxin Mambalgin1 (Mamba1) from black mamba snake venom can reversibly inhibit the conductance of ASICs, causing an analgesic effect. However, the detailed mechanism by which Mamba1 inhibits ASIC1s, especially how Mamba1 binding to the extracellular domain affects the conformational changes of the transmembrane domain of ASICs remains elusive. Here, we present single-particle cryo-EM structures of human ASIC1a (hASIC1a) and the hASIC1a-Mamba1 complex at resolutions of 3.56 and 3.90 Å, respectively. The structures revealed the inhibited conformation of hASIC1a upon Mamba1 binding. The combination of the structural and physiological data indicates that Mamba1 preferentially binds hASIC1a in a closed state and reduces the proton sensitivity of the channel, representing a closed-state trapping mechanism.

Design, synthesis, and evaluation of 1, 4-benzodioxan-substituted chalcones as selective and reversible inhibitors of human monoamine oxidase B.

The inhibition of monoamine oxidase B (MAO-B) could be an effective approach for the treatment of various neurological disorders. In this study, a series of 1, 4-benzodioxan-substituted chalcone derivatives were designed, synthesised and evaluated for their inhibitory activity against human MAO-B (hMAO-B). The majority of these compounds showed inhibitory activity and high selectivity. The most potent compound, (E)-1-(3-bromo-4-fluorophenyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)prop-2-en-1-one (22), exhibited an IC50 of 0.026 µM with a selectivity index greater than 1538. Kinetics and reversibility studies confirmed that the representative active compounds acted as competitive and reversible inhibitors of hMAO-B. The enzyme-inhibitor interactions were investigated by molecular docking studies and the rationale was provided. As these potent hMAO-B inhibitors exhibited low neurotoxicity and possessed promising drug-like properties, we believe that these active compounds could be further investigated as potential drug candidates for future in vivo studies.

A genetically encoded small-size fluorescent pair reveals allosteric conformational changes of G proteins upon its interaction with GPCRs by fluorescence lifetime based FRET.

The dynamics of GPCRs (G protein-coupled receptors) coupling for cognate G proteins play a critical role in signal transduction. Herein, we reported a site-specifically labelled small-sized fluorescent pair 7-HC/FlAsH ((7-hydroxycoumarin-4-yl)-ethylglycine/fluorescein arsenical hairpin) for fluorescence lifetime based FRET (fluorescence resonance energy transfer) to reveal conformational differences of Gαi1 (inhibitory G proteins) and Gαs (stimulatory G proteins) upon ß2AR (ß2-adrenergic receptor) coupling. It offers a new generally applicable method to probe protein dynamic interactions or conformational changes.

[Synthesis and monoamine oxidase B inhibitory activities of isoquiritigenin derivatives].

Isoquiritigenin,one of the active constituents in the Chinese herb liquorice,is found to have moderate inhibitory activity against rat monoamine oxidase B(MAO-B,IC5047. 2 µmol·L-1). However,the structure-activity relationship(SAR) remains unclear until now. In an attempt to reveal the SAR of inhibition by isoquiritigenin,and to identify more potent and selective inhibitors of MAOB,a series of 13 derivatives based on the scaffold of isoquiritigenin were prepared,and their purities and structures were confirmed by UPLC,1 H-NMR,13 C-NMR and HRMS. These compounds were then evaluated for their ability to inhibit the enzymatic activity of human MAO-B. The SAR of inhibition was summarized and a potent compound C8 with high inhibitory activity(IC501. 4 µmol·L-1) and selectivity(>57 folds over MAO-A) was identified. Enzyme kinetics studies suggested that C8 acted as a competitive inhibitor. In addition,C8 showed little cytotoxicity to glial cells in vitro,which could be a promising lead compound for further study.

A Reversible Colorimetric and Fluorescence "Turn-Off" Chemosensor for Detection of Cu2+ and Its Application in Living Cell Imaging.

Dual-function chemosensors that combine the capability of colorimetric and fluorimetric detection of Cu2+ are still relatively rare. Herein, we report that a 3-hydroxyflavone derivative (E)-2-(4-(dimethylamino)styryl)-3-hydroxy-4H-chromen-4-one (4), which is a red-emitting fluorophore, could serve as a reversible colorimetric and fluorescence "turn-off" chemosensor for the detection of Cu2+. Upon addition of Cu2+ to 4 in neutral aqueous solution, a dramatic color change from yellow to purple-red was clearly observed, and its fluorescence was markedly quenched, which was attributed to the complexation between the chemosensor and Cu2+. Conditions of the sensing process had been optimized, and the sensing studies were performed in a solution of ethanol/phosphate buffer saline (v/v = 3:7, pH = 7.0). The sensing system exhibited high selectivity towards Cu2+. The limit of naked eye detection of Cu2+ was determined at 8 × 10-6 mol/L, whereas the fluorescence titration experiment showed a detection limit at 5.7 × 10-7 mol/L. The complexation between 4 and Cu2+ was reversible, and the binding constant was found to be 3.2 × 104 M-1. Moreover, bioimaging experiments showed that 4 could penetrate the cell membrane and respond to the intracellular changes of Cu2+ within living cells, which indicated its potential for biological applications.

Examination of the Deubiquitylation Site Selectivity of USP51 by Using Chemically Synthesized Ubiquitylated Histones.

Histone ubiquitylation and deubiquitylation processes and the mechanisms of their regulation are closely relevant to the field of epigenetics. Recently, the deubiquitylating enzyme USP51 was reported to selectively cleave ubiquitylation on histone H2A at K13 or K15 (i.e., H2AK13Ub and H2AK15Ub), but not at K119 (i.e., H2AK119Ub), in nucleosomes in vivo. To elucidate the mechanism for the selectivity of USP51, we constructed structurally well-defined in vitro protein systems with a ubiquitin modification at precise sites. A total chemical protein synthesis procedure was developed, wherein hydrazide-based native chemical ligation was used to efficiently generate five ubiquitylated histones (H2AK13Ub, H2AK15Ub, H2AK119Ub, H2BK34Ub, and H2BK120Ub). These synthetic ubiquitylated histones were assembled into nucleosomes and subjected to in vitro USP51 deubiquitylation assays. Surprisingly, USP51 did not show preference between H2AK13/15Ub and H2AK119Ub, in contrast to previous in vivo observations. Accordingly, an understanding of the selectivity of USP51 may require consideration of other factors, such as alternative pre-existing histone modifications, competitive reader proteins, or different nucleosome quality among the in vivo extraction nucleosome and the in vitro reconstitution one. Further experiments established that USP51 in vitro could deubiquitylate a nucleosome carrying H2BK120Ub, but not H2BK34Ub. Molecular dynamics simulations suggested that USP51-catalyzed hydrolysis of ubiquitylated nucleosomes was affected by steric hindrance of the isopeptide bond.