Cysteine Conjugate Research Articles

Metabolic activation of zolmitriptan mediated by CYP2D6

Zolmitriptan (ZOL), a member of triptans, has been used for the treatment of migraine with definite therapeutic effects. However, several cases of liver injury associated with ZOL have been reported and the underlying mechanisms remain unclear. The present study aimed to investigate the metabolic activation of ZOL in vitro and in vivo. ZOL-derived glutathione (GSH) and N-acetyl cysteine (NAC) conjugates were detected in rat liver microsomal incubations. In addition, the GSH and NAC conjugates were also found in bile and urine of rats given ZOL, respectively. ZOL-derived GSH conjugate M1 was also observed in ZOL-treated rat primary hepatocytes, and the formation of M1 was inhibited by pre-cultured with quinidine (a selective inhibitor of CYP2D6). Combining with recombinant P450 enzymes incubations, we found that CYP2D6 was the predominant enzyme responsible for the metabolic activation of ZOL. ZOL can be metabolized to an α,β-unsaturated imine intermediate by CYP2D6. Pre-treatment of primary hepatocytes with quinidine was able to reverse ZOL-induced cytotoxicity. The finding facilitates the understanding of the mechanisms involved in ZOL-associated liver adverse reactions.

Characterization of Cytosolic Glutathione S-Transferases Involved in the Metabolism of the Aromatase Inhibitor, Exemestane.

Exemestane (EXE) is a hormonal therapy used to treat estrogen receptor-positive breast cancer by inhibiting the final step of estrogen biosynthesis catalyzed by the enzyme aromatase. Cysteine conjugates of EXE and its active metabolite 17β-dihydro-EXE (DHE) are the major metabolites found in both the urine and plasma of patients taking EXE. The initial step in cysteine conjugate formation is glutathione conjugation catalyzed by the glutathione S-transferase (GST) family of enzymes. The goal of the present study was to identify cytosolic hepatic GSTs active in the GST-mediated metabolism of EXE and 17β-DHE. Twelve recombinant cytosolic hepatic GSTs were screened for their activity against EXE and 17β-DHE, and glutathionylated EXE and 17β-DHE conjugates were detected by ultra-performance liquid chromatography tandem mass spectrometry. GST α (GSTA) isoform 1, GST μ (GSTM) isoform 3 and isoform 1 were active against EXE, whereas only GSTA1 exhibited activity against 17β-DHE. GSTM1 exhibited the highest affinity against EXE with a Michaelis-Menten constant (KM) value that was 3.8- and 7.1-fold lower than that observed for GSTA1 and GSTM3, respectively. Of the three GSTs, GSTM3 exhibited the highest intrinsic clearance against EXE (intrinsic clearance = 0.14 nl·min-1·mg-1). The KM values observed for human liver cytosol against EXE (46 μM) and 17β-DHE (77 μM) were similar to those observed for recombinant GSTA1 (53 and 30 μM, respectively). Western blot analysis revealed that GSTA1 and GSTM1 composed 4.3% and 0.57%, respectively, of total protein in human liver cytosol; GSTM3 was not detected. These data suggest that GSTA1 is the major hepatic cytosolic enzyme involved in the clearance of EXE and its major active metabolite, 17β-DHE. SIGNIFICANCE STATEMENT: Most previous studies related to the metabolism of the aromatase inhibitor exemestane (EXE) have focused mainly on phase I metabolic pathways and the glucuronidation phase II metabolic pathway. However, recent studies have indicated that glutathionylation is the major metabolic pathway for EXE. The present study is the first to characterize hepatic glutathione S-transferase (GST) activity against EXE and 17β-dihydro-EXE and to identify GST α 1 and GST μ 1 as the major cytosolic GSTs involved in the hepatic metabolism of EXE.

Citotoxicity, mutagenicity, and genotoxicity of two metabolites of propyl-propane-thiosulfonate (PTSO) and thiosulfinate (PTS): Glutathione conjugate (GSSP) and cysteine conjugate (CSSP)

Citotoxicity, mutagenicity, and genotoxicity of two metabolites of propyl-propane-thiosulfonate (PTSO) and thiosulfinate (PTS): Glutathione conjugate (GSSP) and cysteine conjugate (CSSP)

Cysteine conjugate of paracetamol serves as substrate for kidney cysteine S-conjugates ß-lyase: implications for drug-induced acute kidney injury

Cysteine conjugate of paracetamol serves as substrate for kidney cysteine S-conjugates ß-lyase: implications for drug-induced acute kidney injury

Cyanobacteria Microcystis aeruginosa Contributes to the Severity of Fish Diseases: A Study on Spring Viraemia of Carp.

Fish are exposed to numerous stressors in the environment including pollution, bacterial and viral agents, and toxic substances. Our study with common carps leveraged an integrated approach (i.e., histology, biochemical and hematological measurements, and analytical chemistry) to understand how cyanobacteria interfere with the impact of a model viral agent, Carp sprivivirus (SVCV), on fish. In addition to the specific effects of a single stressor (SVCV or cyanobacteria), the combination of both stressors worsens markers related to the immune system and liver health. Solely combined exposure resulted in the rise in the production of immunoglobulins, changes in glucose and cholesterol levels, and an elevated marker of impaired liver, alanine aminotransferase (ALT). Analytical determination of the cyanobacterial toxin microcystin-LR (MC-LR) and its structurally similar congener MC-RR and their conjugates showed that SVCV affects neither the levels of MC in the liver nor the detoxification capacity of the liver. MC-LR and MC-RR were depurated from liver mostly in the form of cysteine conjugates (MC-LR-Cys, MC-RR-Cys) in comparison to glutathione conjugates (LR-GSH, RR-GSH). Our study brought new evidence that cyanobacteria worsen the effect of viral agents. Such inclusion of multiple stressor concept helps us to understand how and to what extent the relevant environmental stressors co-influence the health of the fish population.

Impact of Oral Microbiota on Flavor Perception: From Food Processing to In-Mouth Metabolization.

Flavor perception during food intake is one of the main drivers of food acceptability and consumption. Recent studies have pointed to the oral microbiota as an important factor modulating flavor perception. This review introduces general characteristics of the oral microbiota, factors potentially influencing its composition, as well as known relationships between oral microbiota and chemosensory perception. We also review diverse evidenced mechanisms enabling the modulation of chemosensory perception by the microbiota. They include modulation of the chemosensory receptors activation by microbial metabolites but also modification of receptors expression. Specific enzymatic reactions catalyzed by oral microorganisms generate fragrant molecules from aroma precursors in the mouth. Interestingly, these reactions also occur during the processing of fermented beverages, such as wine and beer. In this context, two groups of aroma precursors are presented and discussed, namely, glycoside conjugates and cysteine conjugates, which can generate aroma compounds both in fermented beverages and in the mouth. The two entailed families of enzymes, i.e., glycosidases and carbon–sulfur lyases, appear to be promising targets to understand the complexity of flavor perception in the mouth as well as potential biotechnological tools for flavor enhancement or production of specific flavor compounds.

Front Cover: Site‐Specific Small Molecule Labeling of an Internal Loop in JC Polyomavirus Pentamers Using the π‐Clamp‐Mediated Cysteine Conjugation (ChemBioChem 21/2021)

Few methods exist to site-specifically label loops of globular proteins without interfering with the function of the protein. A modular π-clamp reactive perfluoro-aromatic probe was synthesized in only two-steps and used to selectively modify an internal, surface exposed loop of the major capsid protein of JC polyomavirus (VP1). The perfluoro-biphenyl probe reacts selectively with the cysteine in the π-clamp (Phe-Cys-Pro-Phe) over 6 native cysteines present in VP1. Using microscopy, fluorescently labeled π-clamp VP1 can be observed binding and entering human glial cells. More information can be found in the Communication by F. M. Rossi, C. D. S. Nelson et al.

Glutathione conjugation and protein modification resulting from metabolic activation of venlafaxine in vitro and in vivo

Venlafaxine (VLF), an antidepressant agent, is widely used to combat major depressive disorders, particularly for the treatment of selective serotonin reuptake inhibitor-resistant depression. VLF has been shown to cause liver injury. The present study aimed to investigate the metabolic activation of VLF and explore the mechanisms of hepatotoxicity induced by VLF. One glutathione (GSH) conjugate and one cysteine conjugate were both detected in mouse and human liver microsomal incubations containing VLF and GSH or cysteine. The two conjugates were also detected in cultured mouse primary hepatocytes and bile of rats after exposure to VLF. The in vitro and in vivo studies demonstrated that VLF was metabolized to a quinone methide intermediate reactive to GSH and cysteine residues of hepatic protein. The observed protein covalent binding revealed dose-dependency. The metabolic activation of VLF was P450-dependent, and CYP3A4 was found as the predominant enzyme involved in the bioactivation process. These findings facilitate better understanding of the metabolic activation-hepatotoxicity relationship of VLF and provide chemists with information about new potential structural alerts during drug design process.

Cysteine-Based Coupling: Challenges and Solutions.

Antibody-drug conjugates (ADCs) have attracted great attention in recent years in the wake of an accelerated FDA approval rate and several large-scale acquisitions. To date, there are ten ADC drugs on the market and more than 70 in various stages of clinical trials. Yet, due to the complicated nature of ADC molecules, considerations need to cover many aspects for the success of ADCs, including target specificity, linker-payload stability, tumor permeability, and clearance rate. This topical review summarizes and discusses current methods used to increase stability and homogeneity of ADCs of cysteine conjugation. We believe that they will lead to improvement of efficacy and pharmacokinetics (PK) of ADC drugs.

Site-Specific Small Molecule Labeling of an Internal Loop in JC Polyomavirus Pentamers Using the π-Clamp-Mediated Cysteine Conjugation.

The major capsid protein VP1 of JC Polyomavirus assembles into pentamers that serve as a model for studying viral entry of this potentially severe human pathogen. Previously, labeling of viral proteins utilized large fusion proteins or non-specific amine- or cysteine-functionalization with fluorescent dyes. Imaging of these sterically hindered fusion proteins or heterogeneously labeled virions limits reproducibility and could prevent the detection of subtle trafficking phenomena. Here we advance the π-clamp-mediated cysteine conjugation for site-selective fluorescent labeling of VP1-pentamers. We demonstrate a one-step synthesis of a probe consisting of a bio-orthogonal click chemistry handle bridged to a perfluoro-biphenyl π-clamp reactive electrophile by a polyethylene glycol linker. We expand the scope of the π-clamp conjugation by demonstrating selective labeling of an internal, surface exposed loop in VP1. Thus, the π-clamp conjugation offers a general method to selectively bioconjugate tags-of-interest to viral proteins without impeding their ability to bind and enter cells.

Metabolic disposition of the EGFR covalent inhibitor furmonertinib in humans.

Furmonertinib was designed for the treatment of non-small cell lung cancer (NSCLC) patients with EGFR T790M mutation. In this study, we investigated the metabolic disposition and mass balance in humans and tissue distribution in rats. After a single oral administration of 97.9 μCi/81.5 mg [14C]-furmonertinib mesylate to six healthy male volunteers, the absorption process of furmonertinib was fast with a tmax of total plasma radioactivity at 0.75 h. Afterward, furmonertinib was extensively metabolized, with the parent drug and active metabolite AST5902 accounting for 1.68% and 0.97% of total radioactivity in plasma. The terminal t1/2 of total radioactivity in plasma was as long as 333 h, suggesting that the covalent binding of drug-related substances to plasma proteins was irreversible to a great extent. The most abundant metabolites identified in feces were desmethyl metabolite (AST5902), cysteine conjugate (M19), and parent drug (M0), which accounted for 6.28%, 5.52%, and 1.38% of the dose, respectively. After intragastric administration of 124 μCi/9.93 mg/kg [14C]-furmonertinib to rats, drug-related substances were widely and rapidly distributed in tissues within 4 h. The concentration of total radioactivity in the lung was 100-fold higher than that in rat plasma, which could be beneficial to the treatment of lung cancer. Mass balance in humans was achieved with 77.8% of the administered dose recovered in excretions within 35 days after administration, including 6.63% and 71.2% in urine and feces, respectively. In conclusion, [14C]-furmonertinib is completely absorbed and rapidly distributed into lung tissue, extensively metabolized in humans, presented mostly as covalent conjugates in plasma, and slowly eliminated mostly via fecal route.

Expression of N-Terminal Cysteine Aβ42 and Conjugation to Generate Fluorescent and Biotinylated Aβ42.

Fluorescent derivatives of the β-amyloid peptides (Aβ) are valuable tools for studying the interactions of Aβ with cells. Facile access to labeled expressed Aβ offers the promise of Aβ with greater sequence and stereochemical integrity, without impurities from amino acid deletion and epimerization. Here, we report methods for the expression of Aβ42 with an N-terminal cysteine residue, Aβ(C1-42), and its conjugation to generate Aβ42 bearing fluorophores or biotin. The methods rely on the hitherto unrecognized observation that expression of the Aβ(MC1-42) gene yields the Aβ(C1-42) peptide, because the N-terminal methionine is endogenously excised by Escherichia coli. Conjugation of Aβ(C1-42) with maleimide-functionalized fluorophores or biotin affords the N-terminally labeled Aβ42. The expression affords ∼14 mg of N-terminal cysteine Aβ from 1 L of bacterial culture. Subsequent conjugation affords ∼3 mg of labeled Aβ from 1 L of bacterial culture with minimal cost for labeling reagents. High-performance liquid chromatography analysis indicates the N-terminal cysteine Aβ to be >97% pure and labeled Aβ peptides to be 94-97% pure. Biophysical studies show that the labeled Aβ peptides behave like unlabeled Aβ and suggest that labeling of the N-terminus does not substantially alter the properties of the Aβ. We further demonstrate applications of the fluorophore-labeled Aβ peptides by using fluorescence microscopy to visualize their interactions with mammalian cells and bacteria. We anticipate that these methods will provide researchers convenient access to useful N-terminally labeled Aβ, as well as Aβ with an N-terminal cysteine that enables further functionalization.

Acetaminophen and the Developing Lung: Could There Be Lifelong Consequences?

Acetaminophen and the Developing Lung: Could There Be Lifelong Consequences?

Bioactivation of trichloroethylene to three regioisomeric glutathione conjugates by liver fractions and recombinant human glutathione transferases: Species differences and implications for human risk assessment

Enzymatic conjugation of glutathione (GSH) to trichloroethylene (TCE) followed by catabolism to the corresponding cysteine-conjugate, S-(dichlorovinyl)-L-cysteine (DCVC), and subsequent bioactivation by renal cysteine conjugate beta-lyases is considered to play an important role in the nephrotoxic effects observed in TCE-exposed rat and human.In this study, it is shown for the first time that three regioisomers of GSH-conjugates of TCE are formed by rat and human liver fractions, namely S-(1,2-trans-dichlorovinyl)-glutathione (1,2-trans-DCVG), S-(1,2-cis-dichlorovinyl)-glutathione (1,2-cis-DCVG) and S-(2,2-dichlorovinyl)-glutathione (2,2-DCVG). In incubations of TCE with rat liver fractions their amounts decreased in order of 1,2-cis-DCVG > 1,2-trans-DCVG > 2,2-DCVG. Human liver cytosol showed a more than 10-fold lower activity of GSH-conjugation, with amounts of regioisomers decreasing in order 2,2-DCVG > 1,2-trans-DCVG > 1,2-cis-DCVG. Incubations with recombinant human GSTs suggest that GSTA1-1 and GSTA2-2 play the most important role in human liver cytosol. GSTP1-1, which produces regioisomers in order 1,2-trans-DCVG > 2,2-cis-DCVG > 1,2-cis-DCVG, is likely to contribute to extrahepatic GSH-conjugation of TCE.Analysis of the products formed by a beta-lyase mimetic model showed that both 1,2-trans-DCVC and 1,2-cis-DCVC are converted to reactive products that form cross-links between the model nucleophile 4-(4-nitrobenzyl)-pyridine (NBP) and thiol-species. No NBP-alkylation was observed with 2,2-DCVC corresponding to its low cytotoxicity and mutagenicity.The lower activity of GSH-conjugation of TCE by human liver fractions, in combination with the lower fraction of potential nephrotoxic and mutagenic 1,2-DCVG-isomers, suggest that humans are at much lower risk for TCE-associated nephrotoxic effects than rats.

Impact of Drug Conjugation and Loading on Target Antigen Binding and Cytotoxicity in Cysteine Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) consist of a target-specific antibody that is covalently conjugated to a drug via a linker. ADCs are designed to deliver cytotoxic drugs (payloads), specifically to cancer cells, while minimizing systemic toxicity. Conventional cysteine conjugation typically results in the formation of ADC molecules containing a heterogeneous mixture of 2, 4, 6, and 8 drug-loaded species. The drug-to-antibody ratio (DAR) of the mixture represents the weighted average of these species. In this report, we have investigated the impact of the hydrophobicity of payloads and the overall drug loading on the in vitro binding and cytotoxicity of ADC species. Several ADCs were prepared by conventional cysteine conjugation using different payloads. ADC species with different DAR values were purified from the ADC mixture and characterized by standard analytical techniques. These ADC species were evaluated for target antigen binding using an immunoassay, enzyme-linked immunosorbent assay (ELISA). The potency was assessed using a cell-based cytotoxicity assay. These structure-function studies lead to a better understanding of factors that impact the in vitro target binding and cytotoxicity of ADC species. ADC species containing hydrophobic payloads with high DAR were found to have lower target binding by ELISA compared to that of the unconjugated antibody or the heterogeneous reference ADC with DAR ∼4. Under similar assay conditions, the ADCs conjugated to hydrophilic payloads did not show a significant impact on the target binding. The cytotoxic potency of ADC species increased with increasing level of drug loading in the cell-based cytotoxicity assay.

Glutathione conjugates of the mercapturic acid pathway and guanine adduct as biomarkers of exposure to CEES, a sulfur mustard analog.

Sulfur mustard (SM), a chemical warfare agent, is a strong alkylating compound that readily reacts with numerous biomolecules. The goal of the present work was to define and validate new biomarkers of exposure to SM that could be easily accessible in urine or plasma. Because investigations using SM are prohibited by the Organisation for the Prohibition of Chemical Weapons, we worked with 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM. We developed an ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) approach to the conjugate of CEES to glutathione and two of its metabolites: the cysteine and the N-acetylcysteine conjugates. The N7-guanine adduct of CEES (N7Gua-CEES) was also targeted. After synthesizing the specific biomarkers, a solid-phase extraction protocol and a UHPLC-MS/MS method with isotopic dilution were optimized. We were able to quantify N7Gua-CEES in the DNA of HaCaT keratinocytes and of explants of human skin exposed to CEES. N7Gua-CEES was also detected in the culture medium of these two models, together with the glutathione and the cysteine conjugates. In contrast, the N-acetylcysteine conjugate was not detected. The method was then applied to plasma from mice cutaneously exposed to CEES. All four markers could be detected. Our present results thus validate both the analytical technique and the biological relevance of new, easily quantifiable biomarkers of exposure to CEES. Because CEES behaves very similar to SM, the results are promising for application to this toxic of interest.

Eleven absorbed constituents and 91 metabolites of chuanxiong rhizoma decoction in rats

Objective: Chuanxiong Rhizoma (CR, the dried rhizomes of Ligusticum chuanxiong) is a well-known traditional Chinese medicine that promotes qi to activate blood, dispels wind, and relieves pain. To date, more than 118 constituents of CR have been isolated and identified. However, the in vivo mechanism of CR decoction is unclear and needs further investigation. In addition, to clarify the effective forms of CR, it is essential to reveal the absorbed constituents and metabolites of CR. Materials and Methods: The absorbed constituents and metabolites in urine and plasma samples of rats orally administered with CR decoction were screened and characterized using a high-performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry technique. Results: In total, 102 compounds, including 11 absorbed constituents (eight phthalides and three phthalic acids) and 91 metabolites (71 phthalide-related and 20 phenolic acid-related), were detected in drug-containing rat urine and plasma samples, among which 33 were new metabolites of either CR or its constituents. Based on the structures of these metabolites, six phthalides (ligustilide, senkyunolide I/H, senkyunolide J/N, and butylidenephthalide) and three phenolic acids (ferulic acid, isoferulic acid, and caffeic acid) were proposed as their precursors. They were also deduced to be the main absorbed constituents of CR decoction, which should have closer relationships with its pharmacological effects than other constituents. Phthalide-related metabolites were formed through the metabolic reactions of hydration, hydroxylation, cysteine conjugation, acetylcysteine conjugation, methanethiol conjugation, mercaptomethanol conjugation, glucuronidation, and sulfation, whereas the phenolic acid-related metabolites were mainly formed by glucuronidation and sulfation. Conclusions: Six phthalides and three phenolic acids were shown to be the main precursors of the metabolites of CR, and 33 compounds were new metabolites of either CR or its constituents. These results are helpful for further understanding of the in vivo mechanism and effective forms of CR.

The Construction of a Genetically Encoded, Phage-Displayed Cyclic-Peptide Library.

Due to the great potentials of cyclic peptides as therapeutic agents, several phage-displayed peptide libraries in which cyclization is achieved by the covalent linkage of cysteines have been previously demonstrated to identify cyclic-peptide ligands for therapeutic targets. While problems remain in these cysteine conjugation strategies, we have invented a phage display technique in which its displayed peptides are cyclized through a proximity-driven Michael addition reaction between a cysteine and an amber-codon-encoded Nε-acryloyl-lysine (AcrK). Using a randomized 6-mer library in which peptides were cyclized at two ends through a cysteine-AcrK linker, we demonstrated the successful selection of a potent ligand, CycH8a, for histone deacetylase 8 (HDAC8). We believe this approach will find broad applications in drug discovery.

Detection and Identification of Catalpol Metabolites in the Rat Plasma, Urine and Faeces Using Ultra-high Performance Liquid Chromatography-Q Exactive Hybrid Quadrupole-orbitrap High-resolution Accurate Mass Spectrometry.

Catalpol, an iridoid glycoside, is one of the richest bioactive components present in Rehmannia glutinosa. More and more metabolites of drugs have exhibited various pharmacological effects, thus providing guidance for clinical application. However, few researches have paid attention to the metabolism of catalpol. This study aimed to establish a rapid and effective method to identify catalpol metabolites and evaluate the biotransformation pathways of catalpol in rats. In this study, catalpol metabolites in rat urine, plasma and faeces were analyzed by UHPLC-Q-Exactive MS for the characterization of the metabolism of catalpol. Based on high-resolution extracted ion chromatograms (HREICs) and parallel reaction monitoring mode (PRM), metabolites of catalpol were identified by comparing the diagnostic product ions (DPIs), chromatographic retention times, neutral loss fragments (NLFs) and accurate mass measurement with those of catalpol reference standard. A total of 29 catalpol metabolites were detected and identified in both negative and positive ion modes. Nine metabolic reactions, including deglycosylation, hydroxylation, dihydroxylation, hydrogenation, dehydrogenation, oxidation of methylene to ketone, glucuronidation, glycine conjugation and cysteine conjugation, were proposed. A rapid and effective method based on UHPLC-Q-Exactive MS was developed to mine the metabolism information of catalpol. Results of metabolites and biotransformation pathways of catalpol suggested that when orally administrated, catalpol was firstly metabolized into catalpol aglycone, after which phase I and phase II reactions occurred. However, hydrophilic chromatography-mass spectrometry is still needed to further find the polar metabolites of catalpol.

Design and Validation of Linkers for Site-Specific Preparation of Antibody-Drug Conjugates Carrying Multiple Drug Copies Per Cysteine Conjugation Site.

First-generation cysteine-based site-specific antibody–drug conjugates (ADCs) are limited to one drug per cysteine. However, certain applications require a high drug to antibody ratio (DAR), such as when low-potency payloads are used. Higher drug load can be achieved using classical cysteine conjugation methods, but these result in heterogeneity, suboptimal efficacy and pharmacokinetics. Here, we describe the design, synthesis and validation of heterobifunctional linkers that can be used for the preparation of ADCs with a DAR of two, three and four in a site-specific manner per single cysteine conjugation site, resulting in site-specific ADCs with a DAR of four, six and eight. The designed linkers carry a sulfhydryl-specific iodoacetyl reactive group, and multiple cyclic diene moieties which can efficiently react with maleimide-carrying payloads through the Diels–Alder reaction. As a proof of concept, we synthesized site-specific DAR four, six and eight ADCs carrying tubulysin (AZ13601508) using engineered antibodies with a cysteine inserted after position 239 in the antibody CH2 domain. We evaluated and compared the in vitro cytotoxicity of ADCs obtained via the site-specific platform described herein, with ADCs prepared using classical cysteine conjugation. Our data validated a novel cysteine-based conjugation platform for the preparation of site-specific ADCs with high drug load for therapeutic applications.