A Novel Method for Studying the interaction of Macromolecule with Small Molecule by Means of Affinity Capillary Electrophoresis

A novel capillary electrophoresis method coupled with on-line microdialysis using an attachable electrode has been developed to study the interaction of macromolecule with small molecule. The binding constants of bovine serum album (BSA) with D,L-tryptophan (Trp), sulfamethoxazole (SMZ) with trypsin and chymotrypsin were determined. These values are 2.3 x 10(4)L/mol for BSA-L-Trp; 1.77 x 10(3) L/mol for BSA-D-Trp in pH 7.4, 50 mmol/ L phosphate; 1.4 x 10(4) L/mol for SMZ- trypsin and 6.0 x 10(3) L/mol for SMZ-chymotrypsin in pH 6.5, 25 mmol/L Tris buffer. The proposed method has merits of speed, low sample consumption and readily available to be performed in desired physiological conditions.

Inetetamab combined with pyrotinib and chemotherapy in the treatment of breast cancer brain metastasis: A case report

BACKGROUND Breast cancer brain metastasis(BCBM)is an advanced breast disease that is difficult to treat and is associated with a high risk of death.Patient prognosis is usually poor,with reduced quality of life.In this context,we report the case of a patient with HER-2-positive BCBM treated with a macromolecular mAb(ine-tetamab)combined with a small molecule tyrosine kinase inhibitor(TKI).CASE SUMMARY The patient was a 58-year-old woman with a 12-year history of type 2 diabetes.She was compliant with regular insulin treatment and had good blood glucose control.The patient was diagnosed with invasive carcinoma of the right breast(T3N1M0 stage IIIa,HER2-positive type)through aspiration biopsy of the ipsilateral breast due to the discovery of a breast tumor in February 2019.Immunohistochemistry showed ER(-),PR(-),HER-2(3+),and Ki-67(55-60%+).Preoperative neoadjuvant chemotherapy,i.e.,the AC-TH regimen(epirubicin,cyclophosphamide,docetaxel-paclitaxel,and trastuzumab),was administered for 8 cycles.She underwent modified radical mastectomy of the right breast in November 2019 and received tocilizumab targeted therapy for 1 year.Brain metastasis was found 9 mo after surgery.She underwent brain metastasectomy in August 2020.Immunohistochemistry showed ER(-)and PR.(-),HER-2(3+),and Ki-67(10-20%+).In November 2020,the patient experienced headache symptoms.After an examination,tumor recurrence in the original surgical region of the brain was observed,and the patient was treated with inetetamab,pyrotinib,and capecitabine.Whole-brain radiotherapy was recommended.The patient and her family refused radiotherapy for personal reasons.In September 2021,a routine examination revealed that the brain tumor was considerably larger.The original systemic treatment was continued and combined with intensity-modulated radiation therapy for brain metastases,followed by regular hospitalization and routine examinations.The patient’s condition is generally stable,and she has a relatively high quality of life.This case report demonstrates that in patients with BCBM and resistance to trastuzumab,inetetamab combined with pyrotinib and chemotherapy can prolong survival.CONCLUSION Inetetamab combined with small molecule TKI drugs,chemotherapy and radiation may be an effective regimen for maintaining stable disease in patients with BCBM.

Discovery of a small-molecule bromodomain-containing protein 4 inhibitor that induces AMP-activated protein kinase-modulated autophagy-associated cell death in breast cancer

OBJECTIVE To discover a small-molecule bromodomain-containing protein 4(BRD4)inhibitor that induces AMP-activated protein kinase-modulated autophagy-associated cell death in breast cancer and exploreits potential mechanisms.METHODS BRD4 interactors were analyzed by PPI network prediction and The Cancer Genome Atlas(TCGA)analysis.The interaction between BRD4 and AMPK was confirmed by co-immunoprecipitation assay.Novel BRD4 inhibitors were designed and synthesized based upon pharmacophore analysis of BRD4(1),then screened by antiproliferative activity and Alpha Screen of BRD4(1).The selectivity of the best candidate compound 8f was validated by co-crystallization,FRET assay and co-immuno precipitation assay.The mechanisms of 8f were investigated by fluorescence microscopy,electron microscopy,Western blotting,immunocytochemistry,si RNA and GFP-m RFP-LC3 plasmid transfections,as well as immunohistochemistry and immunofluorescence.Potential mechanisms were discovered by i TRAQ-based proteomics analysis and the therapeutic effect of 8f was assessed by xenograft breast cancer mouse and zebrafish models.RESULTS We identified that BRD4 interacted with AMPK,which was remarkably downregulated in breast cancer.We next designed and synthesized 49 candidate compounds,and eventually discovered a selective small-molecule inhibitor of BRD4(8f).Subsequently,8f was discovered to induce autophagyassociated cell death(ACD)by BRD4-AMPK interaction,and thus activating AMPK-m TOR-ULK1-modulated autophagic pathway in breast cancer cells.Interestingly,the i TRAQ-based proteomics analyses revealed that 8f induced ACD pathways,involved in HMGB1,VDAC1/2 and e EF2.Moreover,8f displayed a therapeutic potential on both xenograft breast cancer mouse and zebrafish models.CONCLUSION We discovered a novel small-molecule inhibitor of BRD4 that induces BRD4-AMPK-modulated ACD in breast cancer,which may provide a candidate drug for future cancer therapy.

Two Small Molecules, ZCL278 and AZA197 Show Promise in Influencing Protein Interactions Involving the Ras-Related Protein Cell division cycle 42 [Cdc42] to Modulate Its Oncogenic Potential

Cdc42 is a member of the Rho subfamily of Ras-related proteins, which were among the first oncogenic proteins to be identified as playing a sig-nificant role in a variety of cellular events [Barbacaid, 1987, Ann. Rev. Biochem]. Equally important, Protein-Protein Interactions [PPIs] involving Cdc42 continue to highlight the role of Ras-related proteins’ relevance to cancer. As these proteins have been considered incapable of being “druggable”, due to a perceived lack of binding surface[s] that are amenable to small molecule targeting, there remains limited development of therapies to tackle diseased states caused by Cdc42-stimulated hyperactivity. Thusly, it has become important to characterize molecular details, including dynamics, of PPIs involving Cdc42 that may lend themselves as potential targets for therapeutic approaches. Recently, two small molecules, ZCL278 and AZA197, have shown promise in directly targeting Cdc42 to influence PPIs that are capable of causing Cdc42-stimulated abnormal signaling. In this editorial, we highlight recent studies that show case how these two small molecules may influence Cdc42-protein interactions.

Small molecules targeting protein-protein interactions for cancer therapy

Protein-protein interactions(PPIs)are fundamental to many biological processes that play an important role in the occurrence and development of a variety of diseases.Targeting the interaction between tumour-related proteins with emerging small molecule drugs has become an attractive approach for treatment of human diseases,especially tumours.Encouragingly,selective PPI-based therapeutic agents have been rapidly advancing over the past decade,providing promising perspectives for novel therapies for patients with cancer.In this review we comprehensively clarify the discovery and development of small molecule modulators of PPIs from multiple aspects,focusing on PPIs in disease,drug design and discovery strategies,structure-activity relationships,inherent dilemmas,and future directions.

The disruption of protein-protein interactions with co-chaperones and client substrates as a strategy towards Hsp90 inhibition

The 90-kilo Dalton(kD) heat shock protein(Hsp90) is a ubiquitous,ATP-dependent molecular chaperone whose primary function is to ensure the proper folding of several hundred client protein substrates.Because many of these clients are overexpressed or become mutated during cancer progression,Hsp90 inhibition has been pursued as a potential strategy for cancer as one can target multiple oncoproteins and signaling pathways simultaneously.The first discovered Hsp90 inhibitors,geldanamycin and radicicol,function by competitively binding to Hsp90’s N-terminal binding site and inhibiting its ATPase activity.However,most of these N-terminal inhibitors exhibited detrimental activities during clinical evaluation due to induction of the pro-survival heat shock response as well as poor selectivity amongst the four isoforms.Consequently,alternative approaches to Hsp90 inhibition have been pursued and include C-terminal inhibition,isoform-selective inhibition,and the disruption of Hsp90 protein-protein interactions.Since the Hsp90 protein folding cycle requires the assembly of Hsp90 into a large heteroprotein complex,along with various co-chaperones and immunophilins,the development of small molecules that prevent assembly of the complex offers an alternative method of Hsp90 inhibition.

Molecular glues modulate protein functions by inducing protein aggregation:A promising therapeutic strategy of small molecules for disease treatment

Molecular glues can specifically induce aggregation between two or more proteins to modulate biological functions.In recent years,molecular glues have been widely used as protein degraders.In addition,however,molecular glues play a variety of vital roles,such as complex stabilization,interactome modulation and transporter inhibition,enabling challenging therapeutic targets to be druggable and offering an exciting novel approach for drug discovery.Since most molecular glues are identified serendipitously,exploration of their systematic discovery and rational design are important.In this review,representative examples of molecular glues with various physiological functions are divided into those mediating homo-dimerization,homo-polymerization and hetero-dimerization according to their aggregation modes,and we attempt to elucidate their mechanisms of action.In particular,we aim to highlight some biochemical techniques typically exploited within these representative studies and classify them in terms of three stages of molecular glue development:starting point,optimization and identification.

Modulation of protein fate decision by small molecules:targeting molecular chaperone machinery

Modulation of protein fate decision and protein homeostasis plays a significant role in altering the protein level,which acts as an orientation to develop drugs with new mechanisms.The molecular chaperones exert significant biological functions on modulation of protein fate decision and protein homeostasis under constantly changing environmental conditions through extensive protein-protein interactions(PPIs)with their client proteins.With the help of molecular chaperone machinery the processes of protein folding,trafficking,quality control and degradation of client proteins could be arranged properly.The core members of molecular chaperones,including heat shock proteins(HSPs)family and their co-chaperones,are emerging as potential drug targets since they are involved in numerous disease conditions.Development of small molecule modulators targeting not only chaperones themselves but also the PPIs among chaperones,co-chaperones and clients is attracting more and more attention.These modulators are widely used as chemical tools to study chaperone networks as well as potential drug candidates for a broader set of diseases.Here,we reviewed the key checkpoints of molecular chaperone machinery HSPs as well as their co-chaperones to discuss the small molecules targeting on them for modulation of protein fate decision.

Understanding the interactions between tris(pentafluoroethyl)-trifluorophosphate-based ionic liquid and small molecules from molecular dynamics simulation

Tris(pentafluoroethyl)trifluorophosphate ([FEP])-based ionic liquids have been widely applied in many fields. For better understanding the properties of [FEP]-based ionic liquids, the interactions between 1-hexyl-3-methylimidazolium ([hmim])[FEP] and small molecules were investigated by molecular dynamics simulations in this work. The small molecules are water, methanol and dimethyl ether. The united-atom (UA) force fields were proposed for methanol and dimethyl ether based on AMBER force field. The densities, enthalpies of vaporization, excess molar properties, and diffusion coefficients of the mixtures were calculated, as well as the microscopic structures characterized by radial distribution functions. Both of the results of the excess energies and microscopic properties show that the strongest interaction is between [hmim][FEP] and dimethyl ether, whereas the interaction between [hmim][FEP] and water is the weakest. Moreover, [hmim][FEP] is more hydrophobic than [hmim] hexafluorophosphate ([PF6]), and the three solutes are mainly distributed around [FEP] anion.

小分子与生物大分子间非共价相互作用分析方法研究进展

对小分子与生物大分子间非共价相互作用分析方法的研究进展作了较详细的评述。重点介绍了光谱、电化学、核磁共振、质谱等方法在小分子与生物大分子间相互作用研究中的应用及进展,总结了这些分析方法的优缺点,引用文献56篇。

药物小分子与生物大分子相互作用的研究方法进展

评述了药物小分子与血清白蛋白、DNA相互作用的模式和近几年来国内外相关研究方法进展。引用文献57篇。

应用平衡透析法分析药物小分子与蛋白质相互作用

蛋白质的相关信息一直是生命科学研究的重点,其中药物小分子与蛋白质的相互作用成为近年来的研究热点。平衡透析法是研究药物小分子与蛋白质相互作用的经典方法,通过该方法可以定量的讨论药物小分子与蛋白质结合的结合数量、结合常数。就平衡透析法用于分析药物小分子与蛋白质的作用方式、作用模型及国内外研究进展进行综述。

平衡透析法用于有机小分子和蛋白质相互作用研究进展

生物大分子是近年来生命科学研究的热点和难点之一,有关蛋白质的各类研究也是人们比较感兴趣的课题.平衡透析法是定量研究蛋白质与有机小分子相互作用的经典方法.通过平衡透析法的研究我们可以讨论蛋白质与有机小分子的结合数目、结合平衡常数及作用力情况等.近年来,国内外学者在此方面做了大量的工作,提出了各种各样的结合模型.本文就此方面的研究进行综述.

质谱技术在中药小分子与生物大分子相互作用研究中的应用

中药发挥药理作用具有多组分,多靶点的重要特点。研究中药化学成分与生物大分子之间的相互作用不仅能够为阐明中药发挥药理作用的机理和物质基础提供科学依据,而且能够为新药设计提供理论指导。软电离质谱技术,尤其是电喷雾质谱(ESI-MS)和基质辅助激光解吸电离质谱(MALDI-MS)在一定条件下能够使药物与生物大分子形成的复合物完整地转移到气相中并被检测到,在中药小分子与生物大分子相互作用的研究中具有很大的优势。同时,色谱-质谱联用技术在中药复杂体系与生物大分子相互作用的研究中也显示出很大的应用潜力。本文介绍了质谱技术在药物与生物大分子相互作用研究中的应用原理,并总结了近年来软电离质谱技术在中药小分子与生物大分子相互作用研究中的应用。

亲脂性乳化剂的性质和用量对W/O/W型复合乳状液稳定性的影响

研究了亲脂性表面活性剂的性质和用量对W/O/W型复合乳状液稳定性的影响,采用两种类型的亲脂性表面活性剂:新型的聚合型高分子表面活性剂聚氧乙烯(30)二聚羟基硬脂酸脂(ArlacelP135)和传统的小分子表面活性剂Span80和Tween80的混合物(质量比9∶1,HLB=5.4)制备复乳。采用离心保留率、光学显微镜和黏度等方法评价初乳和复乳的稳定性。结果表明,与小分子表面活性剂Span80和Tween80的混合物相比,大分子聚合表面活性剂ArlacelP135可以显著地提高复合乳状液的稳定性,在优化的条件下复乳的稳定性可以达到2000r/min离心15min不分层。

DNA与小分子化合物相互作用的研究进展与展望

脱氧核糖核酸(DNA)是生物体中重要的遗传物质,它对遗传信息的储存、复制及转录具有重要的作用.为了进一步探索和认识DNA的性质、结构、行为、形态,揭示生命的奥秘,人们研究了不同类型的小分子化合物与DNA之间的相互作用.通常小分子与DNA作用有三种结合方式:嵌插作用、沟结合和静电作用.现有许多技术和方法来研究小分子化合物与DNA的相互作用,例如紫外光谱、荧光光谱、凝胶电泳和粘度测量等多种研究手段.

靶向MLL1-WDR5蛋白-蛋白相互作用抑制剂的研究进展

Mixed lineage leukemia 1(MLL1)是组蛋白甲基转移酶SET家族的成员之一。MLL1与WDR5、RbBP5、Ash2L和DPY-30组成MLL1甲基转移酶复合物调控组蛋白H3的第4位赖氨酸的甲基化水平,对造血系统的发育和血细胞的更新至关重要。部分白血病患者体内存在因MLL1基因易位而产生的致癌蛋白——MLL1融合蛋白,MLL1融合蛋白在发挥其致癌作用时需要功能完整的MLL1酶复合物,故靶向MLL1-WDR5的蛋白-蛋白相互作用成为治疗MLL1融合型白血病的潜在策略。本文对MLL1-WDR5蛋白-蛋白相互作用的生物学机制、结构信息以及抑制剂进行了系统的总结,并结合已报道数据对该领域进行了展望,以期为后续研究提供参考。

光谱法研究小分子与蛋白质间相互作用的进展

小分子与蛋白质之间相互作用会影响蛋白质构象变化,研究小分子与蛋白质相互作用的机理,对阐明小分子在生物体内的吸收、分布、代谢等过程具有重要意义。综述了近年来紫外光谱法、荧光光谱法、傅里叶变换红外光谱法和圆二色光谱法等技术在该领域的研究进展,分析了这些方法的特点和应用,展望了外源性小分子与蛋白质之间相互作用的发展前景。

小分子物质与蛋白质相互作用研究方法的现状与进展

小分子与蛋白质相互作用的研究是化学生物学的重要研究内容。蛋白质能与许多内源性和外源性小分子物质结合形成超分子复合物。通过实验方法可以获得蛋白质与小分子配体作用的结合常数、结合位点数、结合位置、作用力类型以及在客体小分子作用下蛋白质结构与功能的变化等信息。简述紫外可见光谱、荧光光谱法、傅立叶变换红外光谱和分子模拟法及其他分析方法在小分子与蛋白质相互作用方面的应用和研究进展。