Research on antitumor effects of small molecule inhibitors of deubiquitinases: new progress and new ideas / 药学学报

The abnormality of ubiquitin proteasome pathway is an important factor leading to the imbalance of protein homeostasis. In this process, the deubiquitinase responsible for removing the ubiquitin chain of protein substrate is very important. Its abnormal activity or expression can cause the functional changes of key oncogenic/tumor suppressor proteins, which directly or indirectly lead to the occurrence, development and malignant evolution of tumors. Based on this, the discovery and research of small molecule inhibitors targeting deubiquitinases have become a hot field of anti-tumor candidate drugs. This review will focus on the regulatory effect and mechanism of ubiquitin proteasome pathway, especially deubiquitinase on tumor, introduce the application of deubiquitinase small molecule inhibitors in tumor treatment, and discuss the research status and latest progress of small molecule inhibitors, so as to provide ideas for the research of new anti-tumor strategies based on deubiquitinase.

Targeting a cryptic allosteric site of SIRT6 with small-molecule inhibitors that inhibit the migration of pancreatic cancer cells

SIRT6 belongs to the conserved NAD+-dependent deacetylase superfamily and mediates multiple biological and pathological processes. Targeting SIRT6 by allosteric modulators represents a novel direction for therapeutics, which can overcome the selectivity problem caused by the structural similarity of orthosteric sites among deacetylases. Here, developing a reversed allosteric strategy AlloReverse, we identified a cryptic allosteric site, Pocket Z, which was only induced by the bi-directional allosteric signal triggered upon orthosteric binding of NAD+. Based on Pocket Z, we discovered an SIRT6 allosteric inhibitor named JYQ-42. JYQ-42 selectively targets SIRT6 among other histone deacetylases and effectively inhibits SIRT6 deacetylation, with an IC50 of 2.33 μmol/L. JYQ-42 significantly suppresses SIRT6-mediated cancer cell migration and pro-inflammatory cytokine production. JYQ-42, to our knowledge, is the most potent and selective allosteric SIRT6 inhibitor. This study provides a novel strategy for allosteric drug design and will help in the challenging development of therapeutic agents that can selectively bind SIRT6.

Modulating effects of RAMPs on signaling profiles of the glucagon receptor family

Receptor activity-modulating proteins (RAMPs) are accessory molecules that form complexes with specific G protein-coupled receptors (GPCRs) and modulate their functions. It is established that RAMP interacts with the glucagon receptor family of GPCRs but the underlying mechanism is poorly understood. In this study, we used a bioluminescence resonance energy transfer (BRET) approach to comprehensively investigate such interactions. In conjunction with cAMP accumulation, Gα q activation and β-arrestin1/2 recruitment assays, we not only verified the GPCR-RAMP pairs previously reported, but also identified new patterns of GPCR-RAMP interaction. While RAMP1 was able to modify the three signaling events elicited by both glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), and RAMP2 mainly affected β-arrestin1/2 recruitment by GCGR, GLP-1R and glucagon-like peptide-2 receptor, RAMP3 showed a widespread negative impact on all the family members except for growth hormone-releasing hormone receptor covering the three pathways. Our results suggest that RAMP modulates both G protein dependent and independent signal transduction among the glucagon receptor family members in a receptor-specific manner. Mapping such interactions provides new insights into the role of RAMP in ligand recognition and receptor activation.

Mechanism of action, clinical research and development, and application prospect of hepatitis B virus core protein allosteric modulators / 临床肝胆病杂志

Hepatitis B virus (HBV) infection is the main pathogenic factor for chronic hepatitis, and if it is not treated timely and effectively, it may have the risk of developing into end-stage liver diseases such as liver cirrhosis and hepatocellular carcinoma. Neither of the two types of antiviral drugs currently used in clinical practice can completely inhibit viral replication or eliminate viral transcriptional template, which means that covalently closed circular DNA (cccDNA) exists in infected liver cells for a long time, and thus patients with chronic hepatitis B require long-term or even lifelong medication. Therefore, it is of great importance to develop novel anti-HBV drugs. Core protein allosteric modulators (CpAM) are a type of novel anti-HBV drugs and can interfere with HBV nucleocapsid assembly and inhibit the depolymerization of mature nucleocapsid, the formation of cccDNA, and the biogenesis and secretion of HBeAg. CpAM have a great potential in clinical application since they act on various links of viral replication. This article reviews the function of CpAM target protein-core protein, the classification, action targets, and anti-HBV mechanism of CpAM, and the current research status, further development, and application prospect of CpAM.

Mechanism of allosteric activation of SIRT6 revealed by the action of rationally designed activators

The recent discovery of activator compounds binding to an allosteric site on the NAD

A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors

The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2 (SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2 (SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A. After initial screening of our in-house compound library (∼2300 compounds), we identified 4 new SHP2-PTP inhibitors (0.17% hit rate) and 28 novel allosteric SHP2 inhibitors (1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP (SYK-85: IC

Tyrosine phosphatase SHP2 inhibitors in tumor-targeted therapies

Src homology containing protein tyrosine phosphatase 2 (SHP2) represents a noteworthy target for various diseases, serving as a well-known oncogenic phosphatase in cancers. As a result of the low cell permeability and poor bioavailability, the traditional inhibitors targeting the protein tyrosine phosphate catalytic sites are generally suffered from unsatisfactory applied efficacy. Recently, a particularly large number of allosteric inhibitors with striking inhibitory potency on SHP2 have been identified. In particular, few clinical trials conducted have made significant progress on solid tumors by using SHP2 allosteric inhibitors. This review summarizes the development and structure-activity relationship studies of the small-molecule SHP2 inhibitors for tumor therapies, with the purpose of assisting the future development of SHP2 inhibitors with improved selectivity, higher oral bioavailability and better physicochemical properties.

Met-Controlled Allosteric Module of Neural Generation as A New Therapeutic Target in Rodent Brain Ischemia / 中国结合医学杂志

OBJECTIVE@#To investigate a Met-controlled allosteric module (AM) of neural generation as a potential therapeutic target for brain ischemia.@*METHODS@#We selected Markov clustering algorithm (MCL) to mine functional modules in the related target networks. According to the topological similarity, one functional module was predicted in the modules of baicalin (BA), jasminoidin (JA), cholic acid (CA), compared with I/R model modules. This functional module included three genes: Inppl1, Met and Dapk3 (IMD). By gene ontology enrichment analysis, biological process related to this functional module was obtained. This functional module participated in generation of neurons. Western blotting was applied to present the compound-dependent regulation of IMD. Co-immunoprecipitation was used to reveal the relationship among the three members. We used IF to determine the number of newborn neurons between compound treatment group and ischemia/reperfusion group. The expressions of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9) were supposed to show the changing circumstances for neural generation under cerebral ischemia.@*RESULTS@#Significant reduction in infarction volume and pathological changes were shown in the compound treatment groups compared with the I/R model group (P<0.05). Three nodes in one novel module of IMD were found to exert diverse compound-dependent ischemic-specific excitatory regulatory activities. An anti-ischemic excitatory allosteric module (AM@*CONCLUSIONS@#AM

Examples of biology-driven drug design / 药学学报

Pharmacological activity and drug likeness depend in principle upon the microscopic structure and macroscopic properties of drugs, which reside in their molecular structures. By means of medicinal chemistry the evolution of an active compound to a novel drug (NME) essentially makes the two pillars coexistence in one chemical structure, which either could merge as an intrinsic structure or connect from external fragments to each other with covalent bonds. Since the new millennium the advance in biology provides several knowledge and technologies, for example humanized monoclonal antibody, proteasome-ubiquitin system, allosteric modulation, natural macromolecules, structural biology, etc., for innovation of novel medicines. Taking several examples on marketed drugs or drug candidates in clinical trials, this article tries to concisely illustrate R & D conception of biology-driven drug design.

Mining Polo-Box domain of Polo-like kinase 1 as a new therapeutic target for cancer / 生物工程学报

Polo-like kinase 1 (Plk1) is widely regarded as one of the most promising targets for cancer therapy due to its essential role in cell division and tumor cell survival. At present, most Plk1 inhibitors have been developed based on kinase domain, some of which are in clinical trial. However, inhibitors targeting kinase domain face off-target effect and drug resistance owing to the conserved nature and the frequent mutations in the ATP-binding pocket. In addition to a highly conserved kinase domain, Plk1 also contains a unique Polo-Box domain (PBD), which is essential for Plk1's subcellular localization and mitotic functions. Inhibitors targeting Plk1 PBD show stronger selectivity and less drug resistance for cancer therapy. Therefore, Plk1 PBD is an attractive target for the development of anti-cancer agents. In this review, we will summarize the up-to date drug discovery for targeting Plk1 PBD, including the molecular structure and cellular functions of Plk1 PBD. Small-molecule inhibitors targeting Plk1 PBD not only provide an opportunity to specifically inhibit Plk1 activity for cancer treatment, but also unveil novel biological basis regarding the molecular recognition of Plk1 and its substrates.

Advances in core protein allosteric modulators / 中国药科大学学报

@#The core protein allosteric modulator targets the core protein and inhibits hepatitis B virus(HBV)replication by regulating the formation of covalently closed circular DNA(cccDNA), which is expected to completely cure hepatitis B and overcome the drug resistance of nucleoside drugs. This paper reviews the replication process of HBV, the function of core proteins, the mechanism, classification and research progress of core protein allosteric modulators, lists 12 drugs, and summarizes their mechanisms, categories, chemical structures, safety, anti-HBV effects, combined drug use, etc. In addition, the advantages and problems of core protein allosteric modulators are discussed to provide references for the development of new anti-HBV drugs.

Glycyrrhetinic acid binds to the conserved P-loop region and interferes with the interaction of RAS-effector proteins

Members of the proto-oncogene superfamily are indispensable molecular switches that play critical roles in cell proliferation, differentiation, and cell survival. Recent studies have attempted to prevent the interaction of RAS/GTP with RAS guanine nucleotide exchange factors (GEFs), impair RAS-effector interactions, and suppress RAS localization to prevent oncogenic signalling. The present study aimed to investigate the effect of the natural triterpenoic acid inhibitor glycyrrhetinic acid, which is isolated from the roots of plant species, on RAS stability. We found that glycyrrhetinic acid may bind to the P-loop of RAS and alter its stability. Based on our biochemical tests and structural analysis results, glycyrrhetinic acid induced a conformational change in RAS. Meanwhile, glycyrrhetinic acid abolishes the function of RAS by interfering with the effector protein RAF kinase activation and RAS/MAPK signalling.

Research progress on structural modification of α1-adrenergic receptor influencing its biological activities and its physiological function / 中国药理学通报

The α1-adrenergic receptor (α1-AR) belongs to G-protein coupled receptors (GPCRs) and contains three subtypes, namely α1A, α1B and α1D. Studies have shown that structural changes in the α1-ARs receptor can lead to changes in their physiological functions. In the present study, we summarize the structural changes in four aspects of α1-ARs receptors, including carboxy-terminal truncation, dimerization, allosteric effects, and point mutations that lead to endocytosis, phosphorylation, desensitization, and affinity for agonists.

Functionally diverse ligands modulate different activation states of the formyl peptide receptor 2, a G protein-coupled receptor / 中国药理学与毒理学杂志

OBJECTIVE To identify the mechanisms by which the formyl peptide receptor 2 (FPR2) mediates both inflammatory and anti-inflammatory signaling in an agonist-dependent manner. METHODS Cells expressing FPR2 were incubated with weak agonists, Aβ42 and Ac2-26, before stimulation with a strong agonist, WKYMVm. Calcium mobilization, cAMP inhibition and MAP kinase activation were measured. Intramolecular FRET were determined using FPR2 constructs with an ECFP attached to the C- terminus and a FlAsH binding motif embedded in the first or third intracellular loop (IL1 or IL3, respectively). RESULTS Aβ42 did not induce significant Ca2 + mobilization, but positively modulated WKYMVm-induced Ca2 + mobilization and cAMP reduction in a dose-variable manner within a narrow range of ligand concentrations. Treating FPR2-expressing cells with Ac2-26, a peptide with anti-inflam?matory activity, negatively modulated WKYMVm-induced Ca2 + mobilization and cAMP reduction. Intra?molecular FRET assay showed that stimulation of the receptor constructs with Aβ42 brought the C-terminal domain closer to IL1 but away from IL3. An opposite conformational change was induced by Ac2-26. The FPR2 conformation induced by Aβ42 corresponded to enhanced ERK phosphorylation and attenuated p38 MAPK phosphorylation, whereas Ac2-26 induced FPR2 conformational change corresponding to elevated p38 MAPK phosphorylation and reduced ERK phosphorylation. CONCLUSION Aβ42 and Ac2-26 induce different conformational changes in FPR2. These findings provide a structural basis for FPR2 mediation of inflammatory vs anti-inflammatory functions and identify a type of receptor modulation that differs from the classic positive and negative allosteric modulation.

Biphasic modulation of chemerin peptide-induced calcium flux and ERK phosphorylation by amyloid beta peptide / 中国药理学与毒理学杂志

OBJECTIVE The chemokine-like receptor 1 (CMKLR1, ChemR23) is a functional receptor for chemerin, the chemerin-derived nonapeptide (C9), and the amyloid β peptide 1-42 (Aβ42). Because these peptides share little sequence homology, studies were conducted to investigate their pharmaco?logical properties and regulation at CMKLR1. METHODS Cells expressing CMKLR1 were incubated with Aβ42 before stimulation with a strong agonist, the C9 peptide. Calcium mobilization, cAMP inhibition and MAP kinase activation were measured. Intramolecular FRET were determined using CMKLR1 constructs with an ECFP attached to the C- terminus and a FlAsH binding motif embedded in the first intracellular loop (IL1). RESULTS Binding of both Aβ42 and the C9 peptide induced CMKLR1 internal?ization, but only the Aβ42-induced receptor internalization involved clathrin-coated pits. Likewise, Aβ42 but not C9 stimulated β-arrestin 2 translocation to plasma membranes. A robust Ca2+ flux was observed following C9 stimulation, whereas Aβ42 was ineffective even at micromolar concentrations. Despite its low potency in calcium mobilization assay, Aβ42 was able to alter C9 -induced Ca2+ flux in dose-dependent manner: a potentiation effect at 100 pmol·L-1 of Aβ42 was followed by a suppression at 10 nmol·L-1 and further potentiation at 1 μmol·L-1. This unusual and biphasic modulatory effect was also seen in the C9-induced ERK phosphorylation but the dose curve was opposite to that of Ca2+ flux and cAMP inhibition, suggesting a reciprocal regulatory mechanism. Intramolecular FRET assay confirmed that Aβ42 modulates CMKLR1 rather than its downstream signaling pathways. CONCLUSION These findings suggest Aβ42 as an allosteric modulator that can both positively and negatively regulate the activation state of CMKLR1 in a manner that differs from existing allosteric modulatory mechanisms.

Operational models of allosteric modulation of G protein-coupled receptors / 药学学报

Since the Monod-Wyman-Changeux (MWC) model was initially proposed to explain the allosteric interactions between proteins and their ligands 50 years ago, there have been various models and hypotheses such as the induced-fit model on the interaction. These theoretical developments have been used broadly in the study of allosteric modulations of enzymes and receptors. In 1980, Lefkowitz and coworkers proposed a ternary complex model (TCM) for the regulatory mechanism of G protein-coupled receptors (GPCRs) that laid the theoretical foundation in the study of allosteric sites and ligands of GPCRs, the largest family of known receptors. The findings on how ligands interact with receptors to cause a functional response have significantly impacted the drug discovery field and accelerated the identification of allosteric modulators.

Steroid hormone receptors and prostate cancer: Role of structural dynamics in therapeutic targeting / 亚洲男科学杂志(英文版)

Steroid hormone receptors (SHRs) act in cell type- and gene-specific manner through interactions with coregulatory proteins to regulate numerous physiological and pathological processes at the level of gene regulation. Binding of steroid receptor modulator (SRM) ligand leads to allosteric changes in SHR to exert positive or negative effects on the expression of target genes. Due, in part, to the fact that current SRMs generally target ligand binding domain (LBD)/AF2 and neglect intrinsically disordered (ID) N-terminal domain (NTD)/AF1, clinically relevant SRMs lack selectivity and are also prone to the development of resistance over time. Therefore, to maximize the efficacy of SHR-based therapeutics, the possibility of developing unique modulators that act to control AF1 activity must be considered. Recent studies targeting androgen receptor′s (AR′s) ID AF1 domain for the castration-resistant prostate cancer has provided the possibility of therapeutically targeting ID NTD/AF1 surfaces by allosteric modulations to achieve desired effects. In this review article, we discuss how inter- and intra- molecular allosteric regulations controlled by AR′s structural flexibility and dynamics particularly the ID NTD/AF1 is an emerging area of investigation, which could be exploited for drug development and therapeutic targeting of prostate cancer.

Allosteric interactions between receptor site 3 and 4 of voltage-gated sodium channels: a novel perspective for the underlying mechanism of scorpion sting-induced pain

Background BmK I, a site-3-specific modulator of voltage-gated sodium channels (VGSCs), causes pain and hyperalgesia in rats, while BmK IT2, a site-4-specific modulator of VGSCs, suppresses pain-related responses. A stronger pain-related effect has been previously attributed to Buthus martensi Karsch (BmK) venom, which points out the joint pharmacological effect in the crude venom.Methods In order to detect the joint effect of BmK I and BmK IT2 on ND7-23 cells, the membrane current was measured by whole cell recording. BmK I and BmK IT2 were applied successively and jointly, and the synergistic modulations of VGSCs on ND7-23 cells were detected.Results Larger peak I Na and more negative half-activation voltage were elicited by joint application of BmK I and BmK IT2 than by application of BmK I or BmK IT2 alone. Compared to the control, co-applied BmK I and BmK IT2 also significantly prolonged the time constant of inactivation.Conclusions Our results indicated that site-4 toxin (BmK IT2) could enhance the pharmacological effect induced by site-3 toxin (BmK I), suggesting a stronger effect elicited by both toxins that alone usually exhibit opposite pharmacological effects, which is related to the allosteric interaction between receptor site 3 and site 4. Meanwhile, these results may bring a novel perspective for exploring the underlying mechanisms of scorpion sting-induced pain.

Allosteric interactions between receptor site 3 and 4 of voltage-gated sodium channels: a novel perspective for the underlying mechanism of scorpion sting-induced pain

Background BmK I, a site-3-specific modulator of voltage-gated sodium channels (VGSCs), causes pain and hyperalgesia in rats, while BmK IT2, a site-4-specific modulator of VGSCs, suppresses pain-related responses. A stronger pain-related effect has been previously attributed to Buthus martensi Karsch (BmK) venom, which points out the joint pharmacological effect in the crude venom.Methods In order to detect the joint effect of BmK I and BmK IT2 on ND7-23 cells, the membrane current was measured by whole cell recording. BmK I and BmK IT2 were applied successively and jointly, and the synergistic modulations of VGSCs on ND7-23 cells were detected.Results Larger peak I Na and more negative half-activation voltage were elicited by joint application of BmK I and BmK IT2 than by application of BmK I or BmK IT2 alone. Compared to the control, co-applied BmK I and BmK IT2 also significantly prolonged the time constant of inactivation.Conclusions Our results indicated that site-4 toxin (BmK IT2) could enhance the pharmacological effect induced by site-3 toxin (BmK I), suggesting a stronger effect elicited by both toxins that alone usually exhibit opposite pharmacological effects, which is related to the allosteric interaction between receptor site 3 and site 4. Meanwhile, these results may bring a novel perspective for exploring the underlying mechanisms of scorpion sting-induced pain.(AU)