药学学报

	110203040*50
1	dahksevahr fkdlgeenfk alvliafaqy lqqcpfedhv klvnevtefa
51	ktcvadesae ncdkslhtlf gdklctvatl retygemadc cakqeperne
101	cflqhkddnp nlprlvrpev dvmctafhdn eetflkkyly eiarrhpyfy
151	apellffakr ykaafteccq aadkaacllp kldelrdegk assakqrlkc
201	aslqkfgera fkawavarls qrfpkaefae vsklvtdltk vhtecchgdl
251	lecaddradl akyicenqds issklkecce kpllekshci aevendemra
301	dlpslaadfv eskdvcknya eakdvflgmf lyeyarrhpd ysvvlllrla
351	ktyettlekc caaadphecy akvfdefkpl veepqnlikq ncelfeqlge
401	ykfqnallvr ytkkvpqvst ptlvevsrnl gkvgskcckh peakrmpcae
451	dylsvvlnql cvlhektpvs drvtkcctes lvnrrpcfsa levdetyvpk
501	efnaetftfh adictlseke rqikkqtalv elvkhkpkat keqlkavmdd
551	faafvekcck addketcfae egkklvaasq aalgl

	110203040*50
1	dahksevahr fkdlgeenfk alvliafaqy lqqcpfedhv klvnevtefa
51	ktcvadesae ncdkslhtlf gdklctvatl retygemadc cakqeperne
101	cflqhkddnp nlprlvrpev dvmctafhdn eetflkkyly eiarrhpyfy
151	apellffakr ykaafteccq aadkaacllp kldelrdegk assakqrlkc
201	aslqkfgera fkawavarls qrfpkaefae vsklvtdltk vhtecchgdl
251	lecaddradl akyicenqds issklkecce kpllekshci aevendemra
301	dlpslaadfv eskdvcknya eakdvflgmf lyeyarrhpd ysvvlllrla
351	ktyettlekc caaadphecy akvfdefkpl veepqnlikq ncelfeqlge
401	ykfqnallvr ytkkvpqvst ptlvevsrnl gkvgskcckh peakrmpcae
451	dylsvvlnql cvlhektpvs drvtkcctes lvnrrpcfsa levdetyvpk
501	efnaetftfh adictlseke rqikkqtalv elvkhkpkat keqlkavmdd
551	faafvekcck addketcfae egkklvaasq aalgl

Drug	Reactive motif	Drug-HSA adduct	Number of modified sites	Modified amino acid	Adducts formed
Amoxicillin^[25]	Beta-lactam	√	√	1	Lys-190 (in vitro and in vivo)	Amide
Aztreonam^[26]	Beta-lactam	√	NR	11	Lys-4, 12, 137, 190, 199, 212, 351, 432, 436, 525, 541 (in vitro)	Amide
Benzylpenicillin (BP), benzylpenicillin acid (BA)^{[27, 28]}	Beta-lactam	√	√	BP: 12/14 BA: 16	BP: Lys-20, 137, 159, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro) BP: Lys-20, 137, 159, 190, 195, 199, 212, 351, 372, 432, 436, 525, 541, 545 (in vivo) BA: Lys-20, 137, 159, 162, 190, 195, 199, 212, 351, 372, 432, 436, 475, 525, 541, 545 (in vitro)	Amide
Cefazolin, cephaloridine, cephalothin, cefotaxime^[29]	Beta-lactam	√	NR	NR	NR	Amide
Flucloxacillin (5-hydroxymethyl flucloxacillin)^[30]	Beta-lactam	√	√	10/10	Lys-162, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro) Lys-137, 162, 190, 195, 199, 212, 351, 432, 525, 541 (in vivo)	Amide
Meropenem^[26]	Beta-lactam	√	NR	7	Lys-12, 137, 190, 199, 212, 351, 545 (in vitro)	Amide
Piperacillin^{[26, 31]}	Beta-lactam	√	√	13/4	Lys-4, 12, 137, 162, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro) Lys-190, 195, 432, 541 (in vivo)	Amide
Benoxaprofen AG^[32]	Acyl glucuronide	√	Drug-plasma protein adducts	7	Lys-159, 199; Ser-312, 470, 480, 489; Arg-222 (in vitro)	Amide or ketoamine
Diclofenac AG^[33]	Acyl glucuronide	√	√	10/7	Lys-137, 162, 190, 195, 199, 351, 432, 436, 525, 541 (in vitro) Lys-190, 195, 199, 432, 436, 525, 541 (in vivo)	Amide or ketoamine
Tolmetin AG^{[34, 35]}	Acyl glucuronide	√	Drug-plasma protein adducts	9	Lys-195, 199, 525, 541; Ser-220, 232, 480; Arg-222, 521 (in vitro)	Amide or ketoamine
Zomepirac AG^{[36, 37]}	Acyl glucuronide	√	Drug-plasma protein adducts	3	Lys-195, 199, 351 (in vitro)	Amide or ketoamine
Beclobric acid, carprofen, diflunisal, fenoprofen, furosemide, ibuprofen, naproxen, salicyl acid, and suprofen AG^[38-43]	Acyl glucuronide	√	Drug-plasma protein adducts (beclobrate, carprofen, fenoprofen, furosemide, ibuprofen, salicyl acid AG)	NR	NR	Amide or ketoamine
Afatinib^[44]	Acrylamide	√	Drug-plasma protein adducts	1	Lys-190 (in vitro)	Michael adducts
Evobrutinib^[45]	Acrylamide	√	NR	4	Lys-137, 351, 402, 525 (in vitro)	Michael adducts
Furmonertinib^[12]	Acrylamide	√	Drug-plasma protein adducts	2	Lys-195, 199 (in vitro)	Michael adducts
Ibrutinib^{[19, 45, 46]}	Acrylamide	√	Drug-plasma protein adducts	6	Cys-34; Lys-351, 402, 414, 525; Arg-348 (in vitro)	Michael adducts
Neratinib^[11]	Acrylamide	√	Drug-plasma protein adducts	1	Lys-190 (in vitro)	Michael adducts
Olmutinib^[45]	Acrylamide	√	NR	6	Lys-106, 137, 351, 414, 525; Cys-34 (in vitro)	Michael adducts
Osimertinib^[12]	Acrylamide	√	Drug-plasma protein adducts	2	Lys-195, 199 (in vitro)	Michael adducts
Pyrotinib^[47]	Acrylamide	√	Drug-plasma protein adducts	1	Lys-190 (in vitro)	Michael adducts
Remibrutinib^[45]	Acrylamide	√	NR	2	Lys-351, 414 (in vitro)	Michael adducts
Tolebrutinib^[45]	Acrylamide	√	NR	6	Lys-137, 351, 402, 414, 525, 545 (in vitro)	Michael adducts
Zanubrutinib^[45]	Acrylamide	√	NR	3	Lys-262, 351, 414 (in vitro)	Michael adducts
Nitroso sulfamethoxazole^[48]	Nitroso	√	√	1/1	Cys-34 (in vitro and in vivo)	Sulfinamide
Acetaminophen metabolite (N-acetyl-p-benzoquinoneimine)^[49-51]	Quinone imine	√	√	1/1	Cys-34 (in vitro and in vivo)	Thioether
α-Hydroxy trimethoprim^[52]	Phenylmethanol	√	NR	2	Cys-34, Lys-313 or His-288 (in vitro)	Thioether
Frusemide (F) F glucuronide^[53]	Chlorobenzene	√	NR	NR	Warfarin-binding site of HSA	NR
Isoniazid^[54]	Hydrazide	√	√	6/3	Lys-137, 190, 199, 212, 351, 525 (in vitro) Lys-137, 190, 525 (in vivo)	Amide
Abacavir aldehyde^[55]	Enone	√	√	7/3	Lys-190, His-146, Cys-34, Lys-159/Arg-160, His-146/Arg-145, Cys-34/Gln-33 (in vitro) Cys-34, His-146, Cys-34/Gln-33 (in vivo)	Michael adducts cross-linking adducts
Ethacrynic acid^[56]	Olefinic link	√	NR	1	Cys-34 (in vitro)	Thioether
Houttuynin^[57]	β-keto aldehyde	√	NR	5	Lys-212, 414, 525 (Schiff base) (in vitro) Lys-414, 432 (pyridinium) (in vitro)	Schiff base adducts and pyridinium adducts
12-Sulfoxyl nevirapine^[58]	Sulfate	√	√	4/1	His-146, 242, 338; Cys-34 (in vitro) His-146 (in vivo)	Michael adducts
Carbamazepine 10, 11-epoxide^[59]	Arene oxide epoxide	√	NR	2	His-146, 338 (in vitro)	Epoxide ring- opening
Aspirin^[60]	Acetyl	√	√	1/1	Lys-199 (in vitro and in vivo)	Amide
Captopril^[61]	Thiol	√	NR	1	Cys-34 (in vitro)	Thioether
Butaselen (Phase Ⅱ)^[62]	Se	√	√	1/1	Cys-34 (in vitro and in vivo)	Se-S bond
Nornitrogen mustard, mechlorethamine, chlorambucil, melphalan, cyclophosphamide^[63]	Chlorethamin	√	Drug-plasma protein adducts (melphalan, cyclophosphamide)	1	Cys-34 (in vitro)	Thioether

Drug	Reactive motif	Drug-HSA adduct	Number of modified sites	Modified amino acid	Adducts formed
Amoxicillin^[25]	Beta-lactam	√	√	1	Lys-190 (in vitro and in vivo)	Amide
Aztreonam^[26]	Beta-lactam	√	NR	11	Lys-4, 12, 137, 190, 199, 212, 351, 432, 436, 525, 541 (in vitro)	Amide
Benzylpenicillin (BP), benzylpenicillin acid (BA)^{[27, 28]}	Beta-lactam	√	√	BP: 12/14 BA: 16	BP: Lys-20, 137, 159, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro) BP: Lys-20, 137, 159, 190, 195, 199, 212, 351, 372, 432, 436, 525, 541, 545 (in vivo) BA: Lys-20, 137, 159, 162, 190, 195, 199, 212, 351, 372, 432, 436, 475, 525, 541, 545 (in vitro)	Amide
Cefazolin, cephaloridine, cephalothin, cefotaxime^[29]	Beta-lactam	√	NR	NR	NR	Amide
Flucloxacillin (5-hydroxymethyl flucloxacillin)^[30]	Beta-lactam	√	√	10/10	Lys-162, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro) Lys-137, 162, 190, 195, 199, 212, 351, 432, 525, 541 (in vivo)	Amide
Meropenem^[26]	Beta-lactam	√	NR	7	Lys-12, 137, 190, 199, 212, 351, 545 (in vitro)	Amide
Piperacillin^{[26, 31]}	Beta-lactam	√	√	13/4	Lys-4, 12, 137, 162, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro) Lys-190, 195, 432, 541 (in vivo)	Amide
Benoxaprofen AG^[32]	Acyl glucuronide	√	Drug-plasma protein adducts	7	Lys-159, 199; Ser-312, 470, 480, 489; Arg-222 (in vitro)	Amide or ketoamine
Diclofenac AG^[33]	Acyl glucuronide	√	√	10/7	Lys-137, 162, 190, 195, 199, 351, 432, 436, 525, 541 (in vitro) Lys-190, 195, 199, 432, 436, 525, 541 (in vivo)	Amide or ketoamine
Tolmetin AG^{[34, 35]}	Acyl glucuronide	√	Drug-plasma protein adducts	9	Lys-195, 199, 525, 541; Ser-220, 232, 480; Arg-222, 521 (in vitro)	Amide or ketoamine
Zomepirac AG^{[36, 37]}	Acyl glucuronide	√	Drug-plasma protein adducts	3	Lys-195, 199, 351 (in vitro)	Amide or ketoamine
Beclobric acid, carprofen, diflunisal, fenoprofen, furosemide, ibuprofen, naproxen, salicyl acid, and suprofen AG^[38-43]	Acyl glucuronide	√	Drug-plasma protein adducts (beclobrate, carprofen, fenoprofen, furosemide, ibuprofen, salicyl acid AG)	NR	NR	Amide or ketoamine
Afatinib^[44]	Acrylamide	√	Drug-plasma protein adducts	1	Lys-190 (in vitro)	Michael adducts
Evobrutinib^[45]	Acrylamide	√	NR	4	Lys-137, 351, 402, 525 (in vitro)	Michael adducts
Furmonertinib^[12]	Acrylamide	√	Drug-plasma protein adducts	2	Lys-195, 199 (in vitro)	Michael adducts
Ibrutinib^{[19, 45, 46]}	Acrylamide	√	Drug-plasma protein adducts	6	Cys-34; Lys-351, 402, 414, 525; Arg-348 (in vitro)	Michael adducts
Neratinib^[11]	Acrylamide	√	Drug-plasma protein adducts	1	Lys-190 (in vitro)	Michael adducts
Olmutinib^[45]	Acrylamide	√	NR	6	Lys-106, 137, 351, 414, 525; Cys-34 (in vitro)	Michael adducts
Osimertinib^[12]	Acrylamide	√	Drug-plasma protein adducts	2	Lys-195, 199 (in vitro)	Michael adducts
Pyrotinib^[47]	Acrylamide	√	Drug-plasma protein adducts	1	Lys-190 (in vitro)	Michael adducts
Remibrutinib^[45]	Acrylamide	√	NR	2	Lys-351, 414 (in vitro)	Michael adducts
Tolebrutinib^[45]	Acrylamide	√	NR	6	Lys-137, 351, 402, 414, 525, 545 (in vitro)	Michael adducts
Zanubrutinib^[45]	Acrylamide	√	NR	3	Lys-262, 351, 414 (in vitro)	Michael adducts
Nitroso sulfamethoxazole^[48]	Nitroso	√	√	1/1	Cys-34 (in vitro and in vivo)	Sulfinamide
Acetaminophen metabolite (N-acetyl-p-benzoquinoneimine)^[49-51]	Quinone imine	√	√	1/1	Cys-34 (in vitro and in vivo)	Thioether
α-Hydroxy trimethoprim^[52]	Phenylmethanol	√	NR	2	Cys-34, Lys-313 or His-288 (in vitro)	Thioether
Frusemide (F) F glucuronide^[53]	Chlorobenzene	√	NR	NR	Warfarin-binding site of HSA	NR
Isoniazid^[54]	Hydrazide	√	√	6/3	Lys-137, 190, 199, 212, 351, 525 (in vitro) Lys-137, 190, 525 (in vivo)	Amide
Abacavir aldehyde^[55]	Enone	√	√	7/3	Lys-190, His-146, Cys-34, Lys-159/Arg-160, His-146/Arg-145, Cys-34/Gln-33 (in vitro) Cys-34, His-146, Cys-34/Gln-33 (in vivo)	Michael adducts cross-linking adducts
Ethacrynic acid^[56]	Olefinic link	√	NR	1	Cys-34 (in vitro)	Thioether
Houttuynin^[57]	β-keto aldehyde	√	NR	5	Lys-212, 414, 525 (Schiff base) (in vitro) Lys-414, 432 (pyridinium) (in vitro)	Schiff base adducts and pyridinium adducts
12-Sulfoxyl nevirapine^[58]	Sulfate	√	√	4/1	His-146, 242, 338; Cys-34 (in vitro) His-146 (in vivo)	Michael adducts
Carbamazepine 10, 11-epoxide^[59]	Arene oxide epoxide	√	NR	2	His-146, 338 (in vitro)	Epoxide ring- opening
Aspirin^[60]	Acetyl	√	√	1/1	Lys-199 (in vitro and in vivo)	Amide
Captopril^[61]	Thiol	√	NR	1	Cys-34 (in vitro)	Thioether
Butaselen (Phase Ⅱ)^[62]	Se	√	√	1/1	Cys-34 (in vitro and in vivo)	Se-S bond
Nornitrogen mustard, mechlorethamine, chlorambucil, melphalan, cyclophosphamide^[63]	Chlorethamin	√	Drug-plasma protein adducts (melphalan, cyclophosphamide)	1	Cys-34 (in vitro)	Thioether

药物与人血清白蛋白共价加合物的鉴定研究进展

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刘晓云 ¹^,² , 刁星星 ¹ , 钟大放 ¹^,^*

药学学报 | 综述 2024,59(4): 886-898

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药学学报 | 综述 2024, 59(4): 886-898

药物与人血清白蛋白共价加合物的鉴定研究进展

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刘晓云¹^,², 刁星星¹, 钟大放¹^,^*

作者信息

1.中国科学院上海药物研究所, 上海 201203

2.贝达药业股份有限公司, 浙江杭州 311100

通讯作者:

^*钟大放, Tel: 13482825842, E-mail: dfzhong@simm.ac.cn

Advancements in the identification of adducts of drug-human serum albumin

Xiao-yun LIU¹^,², Xing-xing DIAO¹, Da-fang ZHONG¹^,^*

Affiliations

1. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

2. Betta Pharmaceuticals Co., Ltd., Hangzhou 311100, China

出版时间: 2024-04-12 doi: 10.16438/j.0513-4870.2023-0922

文章导航

摘要

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药物及其代谢物与蛋白质的共价结合形成药物-蛋白质加合物, 可能导致机体的不良反应。加合物组学技术的进展, 有助于系统研究药物与人血浆蛋白的共价加合。对于许多药物而言, 如β-内酰胺抗生素、酰基葡萄糖苷酸、共价酪氨酸激酶抑制剂及反应性代谢物, 人血清白蛋白是形成药物-蛋白质加合物的潜在靶标和生物标志物。本综述将叙述相关的技术进展, 阐述药物与人血清白蛋白共价加合物的鉴定方法, 定义形成加合物的化学反应, 并初步探讨药物与人血清白蛋白共价加合在药物不良反应中的作用以及对药动学的潜在影响。

关键词

人血清白蛋白 / 共价加合物 / 加合物组学 / 药物不良反应 / 药动学

Abstract

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The covalent binding of drugs and their metabolites to proteins forms drug-protein adducts, which may cause adverse reactions in the body. The development of adductomics technology is helpful for the identification of covalent adducts between drugs and human plasma proteins. For many drugs, such as beta-lactam antibiotics, acyl glucuronides, covalent tyrosine kinases inhibitors, and reactive metabolites, human serum albumin (HSA) is a potential target and biomarker for the formation of drug-protein adducts. In this review, we will describe the relevant technical advances, describe the methods for the identification of covalent adducts of drugs and HSA, define the chemical reactions that form adducts, and preliminarily explore the role of drug-HSA adducts in adverse drug reactions and the potential effect on pharmacokinetics.

Key words

human serum albumin / covalent adduct / adductomics / adverse drug reaction / pharmacokinetics

引用本文

刘晓云, 刁星星, 钟大放. 药物与人血清白蛋白共价加合物的鉴定研究进展. 药学学报, 2024 , 59 (4) : 886 -898 . DOI: 10.16438/j.0513-4870.2023-0922

Xiao-yun LIU, Xing-xing DIAO, Da-fang ZHONG. Advancements in the identification of adducts of drug-human serum albumin[J]. Acta Pharmaceutica Sinica, 2024 , 59 (4) : 886 -898 . DOI: 10.16438/j.0513-4870.2023-0922

正文

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药物-蛋白质加合物由蛋白质与化学活性的药物或代谢物的反应产生。许多药物通过对蛋白质的共价修饰发挥药理活性, 然而也可能通过某些机制导致药物的毒性或不良反应, 包括诱导不希望的免疫响应和药物过敏, 或者导致药动学的显著改变。确定这些药物分子或反应性代谢物的靶标蛋白质以及这些加合物的功能, 是一项困难的任务。近年来, 以人血清白蛋白(human serum albumin, HSA) 为基础的药物递送系统因其具有生物降解性、非免疫原形、生物相容性和较长的半衰期等特点, 受到了广泛关注, 已有文章综述基于HSA药物递送系统的研究进展, 本文不再赘述^[1]。

在药物与HSA的共价结合研究中, 研究较多的包括β-内酰胺抗生素、非甾体抗炎药的酰基葡萄糖苷酸(acyl glucuronides, AG) 代谢物和共价酪氨酸激酶抑制剂(tyrosine kinases inhibitors, TKIs)。已有研究表明, 前2类药物产生的不良反应与形成药物-蛋白质加合物有关, 共价TKIs与HSA发生共价结合则会影响其临床药动学。质谱技术和加合物组学技术的进展有助于药物-蛋白质加合物的结构确证。

本综述将叙述相关的技术进展, 阐述药物与HSA加合物的鉴定方法, 定义形成加合物的化学反应, 并初步探讨药物-HSA共价加合在药物不良反应中的作用以及可能对药动学的影响。

1 药物-血浆蛋白加合物的鉴定

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在生物分析或体外试验中, 当药物与血浆孵育并发生共价结合时, 共价结合部分的药物无法被有机溶剂提取, 从而表现为药物在血浆中“消失”的情况。本文主要介绍放射性分析技术和质谱技术在药物-血浆蛋白加合物鉴定的应用。除此之外, 药物-蛋白质加合物的晶体结构也是鉴定和解析共价加合物的重要手段之一。结合放射性检测及自上而下的质谱分析, 可以确认药物是否与血浆蛋白发生共价结合, 以及与哪一种血浆蛋白发生共价结合。采用自下而上的质谱分析, 则可以研究确认药物与血浆蛋白的结合位点。

1.1 人血清白蛋白

血浆蛋白结合率(binding rate of plasma protein, BRPP) 广泛应用于药物发现与开发, 包括预测药物-药物相互作用(drug-drug interactions, DDI)、评估治疗窗和建立药代动力学/药效动力学(pharmacokinetics/pharmacodynamics, PK/PD) 关系等。HSA和α1-酸性糖蛋白(α1-acid glycoprotein, AAG) 是2种与药物结合的最重要的血浆蛋白, 它们的正常血浆浓度分别是500~700 μmol·L^-1和10~20 μmol·L^-1, 半衰期分别是18~21天和5天^[2]。白蛋白是血浆蛋白的主要成分, 约占血浆蛋白总量的60%。HSA是由585个氨基酸组成的单链, 分子质量为66.5 kDa, 包含3个结构域, 分别是Ⅰ (1~195), Ⅱ (196~383), 和Ⅲ (384~585), 每个结构域包含2个亚域(A和B)^[3]。HSA含有35个半胱氨酸残基, 形成了17对二硫键, 并包含1个游离半胱氨酸, 其蛋白氨基酸序列和三维结构分别见表 1和图 1^{[4, 5]}。HSA的主要生理功能是调节胶体渗透压和毛细血管膜通透性、配体结合和转运、清除自由基以及抗氧化和保护循环系统^[3]。影响加合物形成程度的因素包括亲核物质的亲核性和pK_a, 以及影响反应性亲电物质接近亲核位点的立体因素。在生理pH (7.4) 时, 去质子化的亲核位点有利于加合, 因为它们有自由电子对, 能参与共价键的形成^[6]。HSA中的亲核位点如半胱氨酸巯基和组氨酸、赖氨酸以及N-末端氨基酸的胺基官能团能与药物形成加合物^[7]。

1.2 放射性检测

放射性标记药物在药物研发过程中发挥着重要作用, 可用于未知代谢物定量、组织分布、排泄和代谢物鉴定研究^[8]。³H和¹⁴C标记的药物较常用于药物代谢研究中, 可用于药物与肝细胞或血浆蛋白的共价结合研究^[9]。³H和¹⁴C放射性核素能自发地放出β射线, 被放射性探测仪所检测, 从而实现对标记化合物的定量和定位。放射性同位素示踪技术具有高灵敏度、专属性强、适用性广、检测方法简便等优点^[10]。放射性分析技术包括放射性流动检测(radio flow detection, RFD)、液体闪烁计数(liquid scintillation counting, LSC)、微板闪烁技术(microplate scintillation counting, MSC)、停流高效液相色谱(high performance liquid chromatography, HPLC)-RFD、动态流HPLC-RFD、加速器质谱(accelerator mass spectrometry, AMS) 等^[8]。放射性分析与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 相结合, 是目前检测复杂体系中药物-蛋白质加合物最广泛应用的方法^[11-13]。

1.3 质谱分析

1.3.1 自上而下的质谱分析

在复杂基质样品中, 当无法确认药物或代谢物具体与某一蛋白质发生共价结合时, 可采用自上而下(top down) 的质谱分析来发现加合物。自上而下的质谱分析是将完整的蛋白质引入到质谱进行分析, 而不需要提前进行化学或酶学水解^[14]。典型的工作流程包括三个主要步骤: 样品预处理、蛋白质分离与MS/MS分析、数据分析。在过去的十年中, 受益于突破性的技术进步, 自上而下的蛋白质组学经历了快速发展, 自上而下的蛋白质组学在理解基本生物功能、解开疾病机制和发现新的生物标志物方面表现出巨大潜力^[15]。傅里叶变换离子回旋共振(Fourier transform ion cyclotron resonance, FTICR)、Orbitrap、四级杆-飞行时间(quadrupole time-of-flight, Q-TOF)、基质辅助激光解吸电离飞行时间(matrix-assisted laser desorption/ionization time-of-flight, MALDI-TOF) 等高分辨质谱仪可应用于自上而下的蛋白质组学分析^{[16, 17]}。此外, 多维分离技术的应用, 如正交分离平台的耦合, 包括反向液相色谱法(reverse phase liquid chromatography, RPLC)、毛细管电泳(capillary electrophoresis, CE)、或基于尺寸的分离, 提高了分离效率, 从而达到对完整的蛋白质进行深度的蛋白质形态鉴定^[18]。

已有较多研究采用自上而下的质谱分析研究共价TKIs与HSA的加合物。例如在体外将¹⁴C标记的来那替尼(neratinib)、奥希替尼(osimertinib) 和伏美替尼(furmonertinib) 分别与血浆进行孵育后, 经LC分离后, 可经Orbitrap检测到药物-HSA加合物^{[11, 12]}。此外, 文献报道可使用Q-TOF的MS^e采集模式, 通过对药物-蛋白质加合物进行碰撞诱导裂解(collision-induced dissociation, CID), 检测小分子特征性碎片离子, 从而在复杂的蛋白质基质中检测药物-蛋白质加合物离子^[19]。

1.3.2 自下而上的质谱分析

在自下而上(bottom up) 的质谱分析中, 蛋白质被水解成多肽后, 再经LC-MS/MS分析。与完整的蛋白质相比, 多肽具有更容易溶解、分离和离子化的优势。与自上而下的质谱分析相比, 自下而上需要增加蛋白还原、烷基化和酶解步骤。在样品预处理中, 溶液中消化和过滤辅助的样品制备是典型和广泛使用的方法, 也有报道膜上消化、磁珠酶解、固定化的酶解和悬浮液捕集等预处理方法^[20]。胰蛋白酶是酶解中应用最为广泛的丝氨酸蛋白酶, 它能将多肽链中赖氨酸或精氨酸残基中的羧基侧切断。已报道的替代蛋白酶包括胰凝乳蛋白酶、Glu-C、Lys-C、Asp-N、Arg-C和Proalanase等。研究表明使用一种替代蛋白酶或多种蛋白酶可以增加蛋白质序列的覆盖率、翻译后修饰的鉴定数量和覆盖的剪接连接的数量^[21]。在数据分析中, 利用X!Tandem、Mascot、Sequest、Comet、Maxquant、Byonic、MSFragger和Open-pFind等数据库对获得的原始MS/MS数据进行搜索, 对肽和蛋白质进行定性和定量研究^[20]。自下而上的质谱分析是研究药物与HSA的共价结合位点的重要技术手段, 此外, 合成药物-氨基酸加合物的标准品有助于鉴定结合位点。

1.4 基于质谱的蛋白质共价加合物鉴定研究

蛋白质共价加合物是蛋白暴露于内部或外部的反应性化学物质时形成的, 其鉴定研究通常分为2类: ①靶向性, 研究暴露于特定化学物质时形成的共价加合物; ②非靶向性, 旨在全面描述与蛋白质的特定亲核残基结合的共价加合物的整体特征。非靶向性研究通常被称为加合物组学, 用于描述反应性亲电体的暴露特征; 靶向性研究也被称为靶向加合物组学^[22]。已有综述详细介绍了靶向或非靶向蛋白质加合物组学的研究策略、方法, 以及定量研究^{[6, 22-24]}。3种主要基于质谱的策略被应用于靶向和非靶向加合物组学研究, 分别是: ① “自下而上”策略, 将蛋白质水解成肽段再进行MS分析; ②使用非特异性水解酶如链霉蛋白酶(将蛋白质水解成单一的氨基酸) 或酸将蛋白质水解成氨基酸, 随后进行MS分析; ③ N-烷基Edman降解, 在与异硫氰酸酯反应后, 末端加合氨基酸作为乙内酰脲选择性分离, 然后进行质谱分析^[22]。关于HSA和人血红蛋白(Hb) 的加合物组学已有较充分的研究, 修饰位点关注HSA的Cys-34残基和Hb的N端Val残基^[6]。

2 药物-HSA加合物类别

收起

以下将从β-内酰胺抗生素、酰基葡萄糖苷酸、共价酪氨酸激酶抑制剂和其他药物这四个类别来介绍药物-HSA加合物的共价结合机制和特点。本文汇总了药物与HSA共价结合位点、反应基团、生成的代谢物等信息, 见表 2^[25-63]。

2.1 β-内酰胺抗生素

β-内酰胺抗生素广泛用于抗细菌治疗, 过敏反应是其临床应用中较常见的不良反应(AE)。这类药物结构中的β-内酰胺具有内在反应性, 以阿莫西林为例, HSA的Lys-190残基的ε-NH₂能与阿莫西林的β-内酰胺发生亲核加成反应, 生成阿莫西林-HSA (AX-HSA) 加合物(图 2A)^[25]。2名健康受试者口服阿莫西林(1 g/次, 8 h/次, 连续给药6次) 后, 在第1次给药前、第3次给药后(24 h)、第6次给药后(48 h) 采集全血, 分离血清样品进行检测。通过靶向质谱方法鉴定阿莫西林仅与HSA的Lys-190残基发生共价结合。通过Orbitrap的单离子色谱图(single ion chromatogram, SIC) 模式对AX-HSA加合物进行半定量, AX-HSA加合物约占血浆HSA总量的1%~2% (6~12 μmol·L^-1)^[25]。不同结构的β-内酰胺抗生素具有不同的反应性, 其与HSA的共价结合位点信息详见表 2。

β-内酰胺抗生素对HSA的赖氨酸残基的共价修饰具有时间和浓度依赖性^{[26-28, 30]}。体外研究表明, 哌拉西林(piperacillin)、美罗培南(meropenem) 和氨曲南(aztreonam) 以时间和浓度依赖的方式修饰Lys-190和至少另外6个赖氨酸残基^{[26, 31]}。青霉素G (benzylpenicillin, BP) 和重排代谢产物青霉素酸(benzylpenicillin acid, BA) 在低浓度下与HSA短时间孵育后, 分别选择性修饰Lys-199和Lys-525残基。随着孵育时间或药物浓度的增加, 检测到修饰HSA的多个位点。分子模拟表明, 在共价修饰之前, 选择性是一个非共价相互作用的函数^{[27, 28]}。氟氯西林(flucloxacillin) 和代谢物5-羟甲基氟氯西林(5-hydroxymethyl flucloxacillin) 对HSA的共价修饰以浓度、时间和修饰位点依赖性的方式发生。在8位接受氟氯西林治疗的患者中, 对HSA赖氨酸残基的共价修饰多达9个位点, 每个患者都能检测到对HSA Lys-190和Lys-212的共价修饰, 与体外结果相吻合^[30]。

将头孢唑林(cefazolin)、头孢噻啶(cephaloridine)、头孢菌素(cephalothin)、头孢噻肟(cefotaxime) 与HSA进行体外孵育, 当孵育体系的pH从7增加到10时, 药物-HSA加合物的比例增加^[29]。

2.2 酰基葡萄糖苷酸

含羧酸结构的药物在体内可经代谢生成1-O-β-酰基葡萄糖苷酸(acyl glucuronide, AG), 该代谢物可共价修饰蛋白质的亲核氨基酸残基, 如赖氨酸、丝氨酸、精氨酸和天冬氨酸等。AG代谢物可通过2种作用机制共价修饰蛋白质, 以双氯芬酸(diclofenac) 为例, 一种途径是HSA的亲核基团如Lys残基上的ε-NH₂进攻羰基, 发生亲核取代, 生成酰基转移的加合物(acyl adduct); 另一种途径是葡萄糖醛酸的羟基对酯羰基进行亲核攻击, 导致酰基转移, 分别生成2-O-β-AG、3-O-β-AG和4-O-β-AG。AG异构体经去环化和异构化, 生成醛基, 并与赖氨酸残基上的ε-NH₂发生缩合反应, 形成羟亚胺糖基化加合物(glycation adduct)。随后发生Amadori重排, 生成化学更稳定的1-胺基-2-酮产物(图 2B)^[33]。也有研究鉴定了苯洛酚(benoxaprofen)、托美丁(tolmetin)、佐美酸(zomepirac) 的AG代谢物与HSA的共价结合位点^{[32, 34-37]}。

对6名服用双氯芬酸(100~150 mg/天) 至少1年的患者采集全血分离HSA进行共价结合位点鉴定, 共检测到7个Lys残基被修饰, 生成7种酰基转移的加合物和3种羟亚胺糖基化加合物; 分别在6位和4位患者的HSA检测到Lys-195和Lys-199的共价修饰^[33]。可能是因为Lys-195和Lys-199残基位于HSA药物位点1 (drug site 1) 的宽阔而灵活的入口处, 这是一个主要的极性配体结合口袋^[64]。托美丁和佐美酸AG代谢物也都能共价修饰HSA的Lys-195和Lys-199残基^{[34, 36]}。6名健康受试者单次口服400 mg托美丁后, 血浆中托美丁-蛋白质加合物的C_max为1.14~3.76 ng·mg^-1 protein; 连续10天口服托美丁(400 mg/次, BID) 后, 血浆中托美丁-蛋白质加合物的C_max为18.8~37.1 ng·mg^-1 protein, 加合物的半衰期为3.7~6.1天^[35]。佐美酸AG体外与HSA孵育研究表明, 其共价结合的程度为时间和pH依赖性。孵育时间达6 h后, 加合物的生成量最高, 随着孵育时间延长, 加合物的量轻微下降, 可能是由于加合物缓慢水解造成的。在pH 5~9范围内, 加合物的生成速率随着pH的增加而增加。在更高的pH下, 加合物发生水解, 并通过酰基转移发生重排。6例健康受试者分别单药口服100 mg佐美酸或联用丙磺舒(500 mg, BID) 后, 其共价加合物的血浆浓度与佐美酸AG的药时曲线下面积(area under the plasma concentration versus time curve, AUC) 成正比, 合用丙磺舒增加了血浆中佐美酸AG的AUC, 并使共价加合物的生成量增加^[37]。与此类似, 布洛芬(ibuprofen) AG与血浆蛋白共价结合的量与血浆中布洛芬AG的AUC成正比(r = 0.966)^[43]。

体外研究了水杨酸(salicylic acid)、萘普生(naproxen)、二氟尼柳(diflunisal)、舒洛芬(suprofen) 与HSA的共价结合机制^{[38-40, 42]}。对9个药物的AG代谢物进行了体外研究, 结果表明, 每个药物AG代谢物的降解速率(分子内重排和水解) 与共价结合到HSA的程度成正比(r² = 0.995)。对6个药物在人体内的研究表明, 血浆加合物的生成量经AG血浆暴露量归一化后, 其与AG体外降解速率常数具有良好的相关性(r² = 0.873), 可用于预测体内的共价结合^[41]。结构-反应性研究表明, α-碳原子取代度较高的羧酸表现出更高的稳定性^[65]。

2.3 共价酪氨酸激酶抑制剂

共价酪氨酸激酶抑制剂结构中的丙烯酰胺或丙炔酰胺基团具有弱亲电性, 可与靶标蛋白的Cys残基的游离SH发生Michael加成, 从而抑制下游信号通路的传导, 发挥良好的抗肿瘤疗效。除了与靶蛋白发生共价结合外, 共价TKI也可能与其他蛋白如HSA蛋白发生共价结合。以伏美替尼为例, 结构中的丙烯酰胺基团能与HSA的Lys-195和Lys-199发生Michael加成, 生成伏美替尼-HSA加合物(图 2C)^[12]。丙烯酰胺广泛应用于针对EGFR T790M突变的二代和三代表皮生长因子受体(EGFR) 抑制剂、人表皮生长因子受体-2 (HER-2) 和HER-4抑制剂, 以及布鲁顿酪氨酸激酶(BTK) 抑制剂等。研究表明, 阿法替尼(afatinib)、evobrutinib、伏美替尼(furmonertinib)、依鲁替尼(ibrutinib)、来那替尼(neratinib)、奥莫替尼(olmutinib)、奥希替尼(osimertinib)、吡咯替尼(pyrotinib)、remibrutinib、tolebrutinib、泽布替尼(zanubrutinib) 等能与HSA发生共价结合^{[11, 12, 19, 44-47]}。已有文献^[66]综述共价TKI的代谢和药动学, 值得关注的是共价TKI与血浆蛋白发生共价结合后, 表现为血浆总放射性半衰期长于原形或代谢物半衰期。

6名健康受试者单次口服97.9 μCi/81.5 mg ¹⁴C-甲磺酸伏美替尼后, 血浆中原药和活性代谢物AST5902分别占血浆总放射性的1.68%和0.97%, 原药、AST5902和血浆总放射性的半衰期分别是37.2、62.1和333 h (图 3)^[67]。血浆代谢物鉴定结果表明, 除原药和AST5902 (M6-3) 外, 仅检测到微量的半胱氨酸结合物M19、双氧化物M10和N-去甲基代谢物M6-2, 表明血浆中绝大多数的药物相关物质与血浆蛋白发生共价结合^[67]。

将¹⁴C-伏美替尼与血浆共孵育后, 经体积排阻色谱(SEC) 分离, 收集放射性流出物经链霉蛋白酶水解, 检测到¹⁴C-伏美替尼-赖氨酸加合物, 并合成标准品进行确认。将孵育后样品经SDS-PAGE胶分离后, 经磷屏显影和胶内胰蛋白酶水解后, 进行高分辨质谱仪肽图分析和放射性检测, 确认伏美替尼能共价结合到HSA的Lys-195和Lys-199。经质谱检测到¹⁴C-伏美替尼- ASSAKQR ([M+3H]³⁺ m/z 439.894 0) 和¹²C-伏美替尼- ASSAKQR ([M+3H]³⁺ m/z 439.226 2) 加合物。经y2⁺、y*4⁺、y*5⁺和y*6⁺碎片离子确认, 伏美替尼可结合到Lys-195。与此类似, 经质谱检测到¹⁴C-伏美替尼- LKCASLQK ([M+3H]³⁺ m/z 506.601 8) 和¹²C-伏美替尼-LKCASLQK ([M+3H]³⁺ m/z 505.934 1) 加合物。经y1⁺、y4⁺和y6⁺碎片离子确认, 伏美替尼可结合到Lys- 199^[12]。

HSA的半衰期为18~21天, 长于伏美替尼的血浆总放射性半衰期, 结合体外共价结合物的可逆性研究, 表明伏美替尼与HSA的共价结合在体内部分可逆^{[12, 68]}。奥希替尼的人体物质平衡研究中, 血浆总放射性平均半衰期为474 h, 与HSA的生理半衰期接近, 结合体外研究结果, 表明奥希替尼与HSA的共价结合为不可逆^{[9, 68]}。共价TKI与血浆蛋白的共价结合具有种属特异性^{[9, 11, 68]}。来那替尼与HSA的共价结合依赖于孵育pH值、时间和温度, 但不依赖于治疗范围内的底物浓度, 来那替尼与HSA的共价结合具有可逆性^[69]。

共价TKIs与靶蛋白的结合可分为2步, 第一步是药物与靶蛋白特定结合口袋发生非共价结合, 形成非共价复合物。第2步是在非共价复合物中, 共价TKIs的弹头与靶蛋白中的亲核性氨基酸残基自发形成共价键^[70]。尽管HSA中Cys-34残基的游离巯基的反应性是赖氨酸侧链ε-NH₂的10倍, 但Cys-34残基位于HSA的表面, 其硫原子朝向内部, 被Pro-35、His-39、Val-77和Tyr-84侧链包围^[11]。大多数共价TKIs没有检测到与HSA的Cys-34残基的加合物的可能解释是, 共价TKIs与Cys-34残基没有足够的非共价结合, 以至于Cys-34残基的巯基没有足够的时间与共价TKIs形成共价键。另一方面, 观察到HSA中至少4个赖氨酸残基的显著加合作用, 这只是HSA中总共59个赖氨酸残基的一小部分, 这4个赖氨酸残基可能被部分掩埋在分子三维结构的口袋里, 药物可以接近, 并且至少部分以中性状态存在^[47]。

2.4 其他药物

当药物结构含有苯胺基团时, 可经代谢活化后, 与HSA发生共价结合。磺胺甲恶唑(sulfamethoxazole) 苯环上的氨基被代谢生成亚硝基磺胺甲恶唑(nitroso sulfamethoxazole) 后, 可与HSA的Cys-34残基的巯基发生共价结合, 生成亚磺酰胺^[48]。对乙酰氨基酚(Acetaminophen) 经活化生成N-乙酰基-对苯醌亚胺(NAPQI), 体内外都能与HSA的Cys-34残基的巯基发生共价结合, 生成2个同分异构体, 巯基分别结合到羟基或氨基的邻位^[49-51]。甲氧苄啶(trimethoprim) 结构中的苄基可被代谢成羟基, 苄醇可与HSA的Cys-34、Lys-313或His-288残基发生共价结合^[52]。呋塞米(frusemide) 及其AG代谢物结构中的苯氯能与HSA的华法林结合位点处发生共价结合, 结合位点未确认^[53]。异烟肼结构中的肼可自动氧化成偶氮, 随后与HSA的Lys残基反应生成酰胺^[54]。

α, β-不饱和醛酮具有一定的亲电性, 能与亲核基团发生共价结合。阿巴卡韦(abacavir) 经代谢生成α, β-不饱和醛, 体外孵育可检测与HSA的Lys-190、His-146和Cys-34残基发生Michael加成, 也能修饰HSA的Lys-159/Arg-160、His-146/Arg-145、Cys-34/Gln-33残基, 生成交联加合物; 在患者血浆中可检测到与HSA的Cys-34、His-146残基发生Michael加成, 也能修饰Cys-34/Gln-33残基生成交联加合物^[55]。依他尿酸(ethacrynic acid) 结构中的α, β-不饱和酮也能与HSA的Cys-34残基发生共价结合^[56]。鱼腥草素(houttuynin) 结构中含有β-酮醛结构, 体外研究表明, 其能与HSA的Lys-212、Lys-414和Lys-525残基生成席夫碱加合物, 与Lys-414和Lys-432残基生成吡啶加合物^[57]。

奈韦拉平(nevirapine) 经代谢后生成12-硫酸酯奈韦拉平, 硫酸酯是良好的离去基团, 离去后生成亚甲基醌中间体, 在体外检测到与HSA的His-146、His-242、His-338和Cys-34残基发生共价结合, 在体内检测到与HSA的His-146残基发生共价结合^[58]。卡马西平(carbamazepine) 经代谢生成卡马西平10, 11-环氧化物, 环氧乙烷具有反应性, 可与HSA的His-146和His-338残基发生共价结合^[59]。

阿司匹林(aspirin) 能将乙酰基转移到HSA的Lys-199残基上, 生成酰胺^[60]。HSA的Cys-34残基作为多种内源性配体(如NO), 金属离子(如Au(I)、Pt(II)、Ag(I) 和Hg(II)) 和药物[包括乙酸、有机化合物和含巯基或二硫键的药物, 如卡托普利(captopril) 和双硫仑(disulfiram)] 的结合位点^[3]。患者服用卡托普利后, 可在血浆中检测到卡托普利-HSA二硫化物, 证明了卡托普利结构中的巯基可与HSA的Cys-34残基的游离巯基反应生成二硫键^[61]。丁烷硒啉(butaselen) 结构中的硒原子(Se) 能与HSA的Cys-34残基的游离巯基反应生成Se-S键^[62]。甲氮芥及氮芥类药物结构中的氯原子(Cl) 可被HSA的Cys-34残基的游离巯基取代, 从而共价修饰HSA^[63]。药物的结构及反应基团见图 4。

3 研究药物-血浆蛋白加合物的意义

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3.1 药物不良反应

β-内酰胺类抗生素的不良反应被认为是由蛋白质的共价修饰引起的。从过敏患者中分离出的CD4⁺、CD8⁺或CD4⁺CD8⁺ T细胞克隆在使用哌拉西林、美罗培南或氨曲南刺激后, 发生增殖并释放细胞因子。研究鉴定了哌拉西林, 美罗培南和氨曲南与不同HSA残基共价结合后的半抗原结构^[26]。合成的哌拉西林-HSA加合物在体外能激活哌拉西林特异性T细胞^[31]。此外, 已证明青霉素-HSA加合物和青霉素酸-HSA加合物都能激活来自过敏患者的青霉素特异性T细胞, 且后者的刺激能力更强^{[27, 71]}。对β-内酰胺类的研究表明, 白蛋白加合物本身不足以诱导药物超敏反应, 免疫反应的诱导是药物的化学结构和患者生物学共同作用的结果^{[27, 30, 31]}。上述研究表明多种β-内酰胺药物发生过敏可能是通过不同药物抗原对天然T细胞的刺激而引发的。

对于酰基葡萄糖苷酸的潜在毒性, 有三种推测的作用机制, 分别是: ①由于酰基葡萄糖苷酸的共价结合导致蛋白质分子发生化学变化, 从而直接破坏其功能; ②蛋白质的共价修饰形成新抗原, 随后激活免疫系统间接导致毒性, 引起超敏反应或自身免疫反应; ③酰基葡萄糖苷酸对较小的亲核物(如谷胱甘肽, glutathione, GSH) 的反应性可能是某些酸性药物产生毒性的原因之一, 并可能通过耗竭GSH增强其他外源性物质的毒性^[72]。佐美酸由于过敏性反应而被撤市。研究表明, 佐美酸AG与细胞骨架蛋白(尤其是微管蛋白) 形成共价加合物, 后者可能会导致细胞分裂过程中微管的组装受到干扰^[73]。长期服用丙戊酸的癫痫患者血浆中可检测到丙戊酸-蛋白加和物, 并检测到针对加合物的抗体, 但抗体滴度非常低, 与临床上较低的超敏反应发生率相吻合^[74]。

抗菌药物磺胺甲恶唑相关的免疫介导毒性(血液异常、皮肤不良反应和肝毒性) 发生率相对较高, 与苯胺基团生物活化为相应的羟胺和亚硝基中间体有关。亚硝基代谢物与微粒体蛋白、人体中性粒细胞、大鼠T细胞共价结合, 或与GSH反应生成相应的亚磺酰胺衍生物^{[48, 75-77]}。磺胺甲恶唑还可在角质细胞中被氧化, 这意味着生物活化和T细胞致敏作用可直接在皮肤上发生, 导致该药物引起严重的皮肤反应^[77]。大量证据表明, 药物-蛋白加合物存在于迁移到引流淋巴结的树突状细胞中, 对于外源药物的皮肤反应的发展至关重要; 树突状细胞可通过细胞内生物活化、摄取反应性代谢物或摄取邻近细胞(如角质细胞) 形成和释放的加合物获得与药物相关的半抗原化蛋白^[78]。

据报道, HSA的Cys-34、Met-87、Met-123、Met-298、Met-329、Met-446和Met-548残基具有抗氧化活性, Cys-34残基主要作为自由基清除剂, 而Met残基主要作为金属螯合剂。HSA的氧化修饰, 包括糖基化、二硫键形成和羰基化, 可能改变其结合性质, 降低或增加配体亲和力。糖尿病、肝病和肾病被认为与发生氧化应激, 并改变HSA功能有关^[3]。

一般认为, 药物的共价结合与药物毒性有关, 药物代谢产物与蛋白质的选择性共价结合可能会导致特定的器官毒性。蛋白质加合物诱导毒性的机制在很大程度上尚未明确, 但一般认为, 药物-蛋白质加合物是通过损害修饰蛋白质的功能或通过免疫介导机制引起毒性的。许多因素对确定蛋白质共价结合在药物诱导毒性中的作用非常重要, 这些因素包括药物相关因素、药物-蛋白质加合物形成和降解的动力学、受影响的蛋白质和器官以及患者的遗传和病理条件^[79]。

3.2 对药动学的影响

HSA经外源性药物共价修饰后, 其结合特性可能会发生改变。研究表征了HSA经依他尿酸共价修饰后的二级结构和结合性质: Cys-34残基的共价修饰未改变HSA的蛋白二级结构, 但对HSA的结合性质有所影响。无论是与I位点结合的药物, 如苯丁酮, 还是与Ⅱ位点结合的药物, 如地西泮, 对高亲和力结合位点的亲和力都没有改变, 而已知与Ⅲ位点结合的胆红素, 其亲和力显著增加。然而, 与位点Ⅰ或位点Ⅱ结合的药物在修饰蛋白的低亲和力结合位点的亲和力显著降低^[56]。与此类似, 研究了经青霉素共价修饰后的HSA的结合性质。与天然蛋白相比, 修饰蛋白的二级结构没有明显变化。分别采用苯丁酮、地西泮和胆红素作为标记物, 结果表明修饰后的HSA对高亲和力结合位点的亲和力降低^[28]。

在人体放射性物质平衡研究中, 血浆总放射性半衰期是血浆中所有药物相关物质半衰期的综合结果。当药物不与血浆蛋白发生共价结合时, 其血浆总放射性半衰期与药物或代谢物相当, 且血浆中大部分放射性可被有机溶剂提取鉴定, 如呋喹替尼(fruquintinib) 和氟唑帕利(fuzuloparib)^{[80, 81]}。呋喹替尼、代谢物M9、代谢物M11、血浆总放射性和全血总放射性平均半衰期分别是33.4、37.1、51.5、41.1和44.1 h, 血浆平均AUC_0-t (从零时至可定量浓度时间点的药时曲线下面积) 分别是4 719、371、1 511、9 922、4 964 ng·h·mL^-1, 原药和代谢物暴露量总和占血浆总放射性暴露量的66.5%^[80]。氟唑帕利和血浆总放射性的平均半衰期分别是18.0和20.7 h, 血浆平均AUC_0-t分别是113和156 ng·h·mL^-1, 原药暴露量占血浆总放射性暴露量的72.4%^[81]。共价TKI如奥希替尼、伏美替尼、吡咯替尼、来那替尼、阿法替尼和依鲁替尼在人体物质平衡研究中, 血浆中有大量的放射性物质不能被有机溶剂提取, 与血浆蛋白共价结合的药物相关物质是血浆中主要药物相关成分^{[9, 11, 46, 47, 67, 82]}。由于奥希替尼和伏美替尼与血浆蛋白的共价结合分别为不可逆或部分可逆, 血浆总放射性半衰期远高于原药或代谢物, 分别为474和333 h, 血浆中原药和主要代谢物的暴露量总和分别仅占血浆总放射性暴露量的0.95%和2.65%^{[9, 67]}。据推测, 该类药物的血浆蛋白加合物将随着HSA的自然分解, 释放出药物-赖氨酸结合物, 并进一步代谢或排泄。这尚待实验证明。在这种情况下, 与血浆蛋白的共价结合可被认为是药物的消除途径, 延长了药物在体内的消除时间。因此在奥希替尼和伏美替尼的人体物质平衡研究中, 样品的收集需要持续到给药后84天, 且平均总放射性回收率分别仅为81.9%和77.8%^{[9, 67]}。对于吡咯替尼、来那替尼、阿法替尼和依鲁替尼等药物而言, 其与血浆蛋白的共价结合是可逆的, 可作为药物储库持续释放原药。当与血浆蛋白共价结合的药物相关物质是血浆中主要药物相关成分时, 药物-蛋白质加合物及其共价结合位点亟待研究。加合物的确认有助于后续对HSA的功能影响、药物的安全性和药代动力学研究。

4 结论与展望

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药物-蛋白质加合物是导致药物不良反应可能原因之一, 但其诱导毒性的机制有待深入研究。药物生物活化和与蛋白质的共价结合的临床结果是不可预测的, 这取决于与所给药物和患者相关的许多因素。加合物的鉴定可作为物质基础, 用于研究药物相关不良反应的作用机制及对药动学的影响。当建立血浆共价加合物与不良反应的相关性后, 可考虑评估血浆中加合物的水平来预测药物引起的不良反应。近年来, 共价抑制剂方法作为药物发现的宝贵工具正迅速获得认可, 在药物开发过程中, 及早发现与重要细胞蛋白和/或药物代谢酶共价结合的问题药物, 可以节省时间和资金。幸运的是, 由于质谱技术的发展和蛋白质组学方法的高通量能力, 将其应用于药物-蛋白质加合物的研究有助于提高筛选效率。

作者贡献: 刘晓云负责文献检索、论文撰写和修改; 刁星星负责修改文章; 钟大放负责文章选题、指导写作、修改及校对文章。

利益冲突: 所有作者均声明不存在利益冲突。

基金

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国家自然科学基金资助项目(81521005)
国家自然科学基金资助项目(82373938)

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2024年第59卷第4期

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doi: 10.16438/j.0513-4870.2023-0922

接收时间：2023-07-30
首发时间：2025-11-28
出版时间：2024-04-12

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收稿日期：2023-07-30
修回日期：2023-09-06

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国家自然科学基金资助项目(81521005)

国家自然科学基金资助项目(82373938)

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1.中国科学院上海药物研究所, 上海 201203

2.贝达药业股份有限公司, 浙江杭州 311100

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^*钟大放, Tel: 13482825842, E-mail: dfzhong@simm.ac.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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51	ktcvadesae ncdkslhtlf gdklctvatl retygemadc cakqeperne
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301	dlpslaadfv eskdvcknya eakdvflgmf lyeyarrhpd ysvvlllrla
351	ktyettlekc caaadphecy akvfdefkpl veepqnlikq ncelfeqlge
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Drug	Reactive motif	Drug-HSA adduct	Number of modified sites	Modified amino acid	Adducts formed
Amoxicillin^[25]	Beta-lactam	√	√	1	Lys-190 (in vitro and in vivo)	Amide
Aztreonam^[26]	Beta-lactam	√	NR	11	Lys-4, 12, 137, 190, 199, 212, 351, 432, 436, 525, 541 (in vitro)	Amide
Benzylpenicillin (BP), benzylpenicillin acid (BA)^{[27, 28]}	Beta-lactam	√	√	BP: 12/14 BA: 16	BP: Lys-20, 137, 159, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro) BP: Lys-20, 137, 159, 190, 195, 199, 212, 351, 372, 432, 436, 525, 541, 545 (in vivo) BA: Lys-20, 137, 159, 162, 190, 195, 199, 212, 351, 372, 432, 436, 475, 525, 541, 545 (in vitro)	Amide
Cefazolin, cephaloridine, cephalothin, cefotaxime^[29]	Beta-lactam	√	NR	NR	NR	Amide
Flucloxacillin (5-hydroxymethyl flucloxacillin)^[30]	Beta-lactam	√	√	10/10	Lys-162, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro) Lys-137, 162, 190, 195, 199, 212, 351, 432, 525, 541 (in vivo)	Amide
Meropenem^[26]	Beta-lactam	√	NR	7	Lys-12, 137, 190, 199, 212, 351, 545 (in vitro)	Amide
Piperacillin^{[26, 31]}	Beta-lactam	√	√	13/4	Lys-4, 12, 137, 162, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro) Lys-190, 195, 432, 541 (in vivo)	Amide
Benoxaprofen AG^[32]	Acyl glucuronide	√	Drug-plasma protein adducts	7	Lys-159, 199; Ser-312, 470, 480, 489; Arg-222 (in vitro)	Amide or ketoamine
Diclofenac AG^[33]	Acyl glucuronide	√	√	10/7	Lys-137, 162, 190, 195, 199, 351, 432, 436, 525, 541 (in vitro) Lys-190, 195, 199, 432, 436, 525, 541 (in vivo)	Amide or ketoamine
Tolmetin AG^{[34, 35]}	Acyl glucuronide	√	Drug-plasma protein adducts	9	Lys-195, 199, 525, 541; Ser-220, 232, 480; Arg-222, 521 (in vitro)	Amide or ketoamine
Zomepirac AG^{[36, 37]}	Acyl glucuronide	√	Drug-plasma protein adducts	3	Lys-195, 199, 351 (in vitro)	Amide or ketoamine
Beclobric acid, carprofen, diflunisal, fenoprofen, furosemide, ibuprofen, naproxen, salicyl acid, and suprofen AG^[38-43]	Acyl glucuronide	√	Drug-plasma protein adducts (beclobrate, carprofen, fenoprofen, furosemide, ibuprofen, salicyl acid AG)	NR	NR	Amide or ketoamine
Afatinib^[44]	Acrylamide	√	Drug-plasma protein adducts	1	Lys-190 (in vitro)	Michael adducts
Evobrutinib^[45]	Acrylamide	√	NR	4	Lys-137, 351, 402, 525 (in vitro)	Michael adducts
Furmonertinib^[12]	Acrylamide	√	Drug-plasma protein adducts	2	Lys-195, 199 (in vitro)	Michael adducts
Ibrutinib^{[19, 45, 46]}	Acrylamide	√	Drug-plasma protein adducts	6	Cys-34; Lys-351, 402, 414, 525; Arg-348 (in vitro)	Michael adducts
Neratinib^[11]	Acrylamide	√	Drug-plasma protein adducts	1	Lys-190 (in vitro)	Michael adducts
Olmutinib^[45]	Acrylamide	√	NR	6	Lys-106, 137, 351, 414, 525; Cys-34 (in vitro)	Michael adducts
Osimertinib^[12]	Acrylamide	√	Drug-plasma protein adducts	2	Lys-195, 199 (in vitro)	Michael adducts
Pyrotinib^[47]	Acrylamide	√	Drug-plasma protein adducts	1	Lys-190 (in vitro)	Michael adducts
Remibrutinib^[45]	Acrylamide	√	NR	2	Lys-351, 414 (in vitro)	Michael adducts
Tolebrutinib^[45]	Acrylamide	√	NR	6	Lys-137, 351, 402, 414, 525, 545 (in vitro)	Michael adducts
Zanubrutinib^[45]	Acrylamide	√	NR	3	Lys-262, 351, 414 (in vitro)	Michael adducts
Nitroso sulfamethoxazole^[48]	Nitroso	√	√	1/1	Cys-34 (in vitro and in vivo)	Sulfinamide
Acetaminophen metabolite (N-acetyl-p-benzoquinoneimine)^[49-51]	Quinone imine	√	√	1/1	Cys-34 (in vitro and in vivo)	Thioether
α-Hydroxy trimethoprim^[52]	Phenylmethanol	√	NR	2	Cys-34, Lys-313 or His-288 (in vitro)	Thioether
Frusemide (F) F glucuronide^[53]	Chlorobenzene	√	NR	NR	Warfarin-binding site of HSA	NR
Isoniazid^[54]	Hydrazide	√	√	6/3	Lys-137, 190, 199, 212, 351, 525 (in vitro) Lys-137, 190, 525 (in vivo)	Amide
Abacavir aldehyde^[55]	Enone	√	√	7/3	Lys-190, His-146, Cys-34, Lys-159/Arg-160, His-146/Arg-145, Cys-34/Gln-33 (in vitro) Cys-34, His-146, Cys-34/Gln-33 (in vivo)	Michael adducts cross-linking adducts
Ethacrynic acid^[56]	Olefinic link	√	NR	1	Cys-34 (in vitro)	Thioether
Houttuynin^[57]	β-keto aldehyde	√	NR	5	Lys-212, 414, 525 (Schiff base) (in vitro) Lys-414, 432 (pyridinium) (in vitro)	Schiff base adducts and pyridinium adducts
12-Sulfoxyl nevirapine^[58]	Sulfate	√	√	4/1	His-146, 242, 338; Cys-34 (in vitro) His-146 (in vivo)	Michael adducts
Carbamazepine 10, 11-epoxide^[59]	Arene oxide epoxide	√	NR	2	His-146, 338 (in vitro)	Epoxide ring- opening
Aspirin^[60]	Acetyl	√	√	1/1	Lys-199 (in vitro and in vivo)	Amide
Captopril^[61]	Thiol	√	NR	1	Cys-34 (in vitro)	Thioether
Butaselen (Phase Ⅱ)^[62]	Se	√	√	1/1	Cys-34 (in vitro and in vivo)	Se-S bond
Nornitrogen mustard, mechlorethamine, chlorambucil, melphalan, cyclophosphamide^[63]	Chlorethamin	√	Drug-plasma protein adducts (melphalan, cyclophosphamide)	1	Cys-34 (in vitro)	Thioether

Drug

Reactive motif

Drug-HSA adduct

Number of modified sites

Modified amino acid

Adducts formed

In vitro

In vivo

Amoxicillin^[25]

Beta-lactam

√

Lys-190 (in vitro and in vivo)

Amide

Aztreonam^[26]

Beta-lactam

√

Lys-4, 12, 137, 190, 199, 212, 351, 432, 436, 525, 541 (in vitro)

Amide

Benzylpenicillin (BP),
benzylpenicillin acid (BA)^{[27, 28]}

Beta-lactam

√

BP: 12/14
BA: 16

BP: Lys-20, 137, 159, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro)
BP: Lys-20, 137, 159, 190, 195, 199, 212, 351, 372, 432, 436, 525, 541, 545 (in vivo)
BA: Lys-20, 137, 159, 162, 190, 195, 199, 212, 351, 372, 432, 436, 475, 525, 541, 545 (in vitro)

Amide

Cefazolin, cephaloridine, cephalothin, cefotaxime^[29]

Beta-lactam

√

Amide

Flucloxacillin
(5-hydroxymethyl flucloxacillin)^[30]

Beta-lactam

√

10/10

Lys-162, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro)
Lys-137, 162, 190, 195, 199, 212, 351, 432, 525, 541 (in vivo)

Amide

Meropenem^[26]

Beta-lactam

√

Lys-12, 137, 190, 199, 212, 351, 545 (in vitro)

Amide

Piperacillin^{[26, 31]}

Beta-lactam

√

13/4

Lys-4, 12, 137, 162, 190, 195, 199, 212, 351, 432, 525, 541, 545 (in vitro)
Lys-190, 195, 432, 541 (in vivo)

Amide

Benoxaprofen AG^[32]

Acyl glucuronide

√

Drug-plasma protein adducts

Lys-159, 199; Ser-312, 470, 480, 489; Arg-222 (in vitro)

Amide or ketoamine

Diclofenac AG^[33]

Acyl glucuronide

√

10/7

Lys-137, 162, 190, 195, 199, 351, 432, 436, 525, 541 (in vitro)
Lys-190, 195, 199, 432, 436, 525, 541 (in vivo)

Amide or ketoamine

Tolmetin AG^{[34, 35]}

Acyl glucuronide

√

Drug-plasma protein adducts

Lys-195, 199, 525, 541; Ser-220, 232, 480; Arg-222, 521 (in vitro)

Amide or ketoamine

Zomepirac AG^{[36, 37]}

Acyl glucuronide

√

Drug-plasma protein adducts

Lys-195, 199, 351 (in vitro)

Amide or ketoamine

Beclobric acid, carprofen,
diflunisal, fenoprofen, furosemide, ibuprofen, naproxen, salicyl acid, and suprofen AG^[38-43]

Acyl glucuronide

√

Drug-plasma protein adducts (beclobrate, carprofen, fenoprofen, furosemide, ibuprofen, salicyl acid AG)

Amide or ketoamine

Afatinib^[44]

Acrylamide

√

Drug-plasma protein adducts

Lys-190 (in vitro)

Michael adducts

Evobrutinib^[45]

Acrylamide

√

Lys-137, 351, 402, 525 (in vitro)

Michael adducts

Furmonertinib^[12]

Acrylamide

√

Drug-plasma protein adducts

Lys-195, 199 (in vitro)

Michael adducts

Ibrutinib^{[19, 45, 46]}

Acrylamide

√

Drug-plasma protein adducts

Cys-34; Lys-351, 402, 414, 525; Arg-348 (in vitro)

Michael adducts

Neratinib^[11]

Acrylamide

√

Drug-plasma protein adducts

Lys-190 (in vitro)

Michael adducts

Olmutinib^[45]

Acrylamide

√

Lys-106, 137, 351, 414, 525; Cys-34 (in vitro)

Michael adducts

Osimertinib^[12]

Acrylamide

√

Drug-plasma protein adducts

Lys-195, 199 (in vitro)

Michael adducts

Pyrotinib^[47]

Acrylamide

√

Drug-plasma protein adducts

Lys-190 (in vitro)

Michael adducts

Remibrutinib^[45]

Acrylamide

√

Lys-351, 414 (in vitro)

Michael adducts

Tolebrutinib^[45]

Acrylamide

√

Lys-137, 351, 402, 414, 525, 545 (in vitro)

Michael adducts

Zanubrutinib^[45]

Acrylamide

√

Lys-262, 351, 414 (in vitro)

Michael adducts

Nitroso sulfamethoxazole^[48]

Nitroso

√

1/1

Cys-34 (in vitro and in vivo)

Sulfinamide

Acetaminophen metabolite (N-acetyl-p-benzoquinoneimine)^[49-51]

Quinone imine

√

1/1

Cys-34 (in vitro and in vivo)

Thioether

α-Hydroxy trimethoprim^[52]

Phenylmethanol

√

Cys-34, Lys-313 or His-288 (in vitro)

Thioether

Frusemide (F)
F glucuronide^[53]

Chlorobenzene

√

Warfarin-binding site of HSA

Isoniazid^[54]

Hydrazide

√

6/3

Lys-137, 190, 199, 212, 351, 525 (in vitro)
Lys-137, 190, 525 (in vivo)

Amide

Abacavir aldehyde^[55]

Enone

√

7/3

Lys-190, His-146, Cys-34, Lys-159/Arg-160, His-146/Arg-145, Cys-34/Gln-33 (in vitro)
Cys-34, His-146, Cys-34/Gln-33 (in vivo)

Michael adducts
cross-linking adducts

Ethacrynic acid^[56]

Olefinic link

√

Cys-34 (in vitro)

Thioether

Houttuynin^[57]

β-keto aldehyde

√

Lys-212, 414, 525 (Schiff base) (in vitro)
Lys-414, 432 (pyridinium) (in vitro)

Schiff base adducts and pyridinium adducts

12-Sulfoxyl nevirapine^[58]

Sulfate

√

4/1

His-146, 242, 338; Cys-34 (in vitro)
His-146 (in vivo)

Michael
adducts

Carbamazepine 10, 11-epoxide^[59]

Arene oxide epoxide

√

His-146, 338 (in vitro)

Epoxide ring- opening

Aspirin^[60]

Acetyl

√

1/1

Lys-199 (in vitro and in vivo)

Amide

Captopril^[61]

Thiol

√

Cys-34 (in vitro)

Thioether

Butaselen (Phase Ⅱ)^[62]

√

1/1

Cys-34 (in vitro and in vivo)

Se-S bond

Nornitrogen mustard, mechlorethamine, chlorambucil, melphalan, cyclophosphamide^[63]

Chlorethamin

√

Drug-plasma protein adducts (melphalan, cyclophosphamide)

Cys-34 (in vitro)

Thioether