Active Site Cysteine Research Articles

Development and Use of DJ-1 Affinity Microcolumns to Screen and Study Small Drug Candidates for Parkinson’s Disease

BackgroundDJ-1 is a protein whose mutation causes rare heritable forms of Parkinson’s disease (PD) and is of interest as a target for treating PD and other disorders. This work used high performance affinity microcolumns to screen and examine the binding of small molecules to DJ-1, as could be used to develop new therapeutics or to study the role of DJ-1 in PD. Non-covalent entrapment was used to place microgram quantities of DJ-1 in an unmodified form within microcolumns, which were then used in multiple studies to analyze binding by model compounds and possible drug candidates to DJ-1. ResultsSeveral factors were examined in optimizing the entrapment method, including the addition of a reducing agent to maintain a reduced active site cysteine residue in DJ-1, the concentration of DJ-1 employed, and the entrapment times. Isatin was used as a known binding agent (dissociation constant, ∼2.0 μM) and probe for DJ-1 activity. This compound gave good retention on 2.0 cm × 2.1 mm inner diameter DJ-1 microcolumns made under the final entrapment conditions, with a typical retention factor of 14 and elution in ∼8 min at 0.50 mL/min. These DJ-1 microcolumns were used to evaluate the binding of small molecules that were selected in silico to bind or not to bind DJ-1. A compound predicted to have good binding with DJ-1 gave a retention factor of 122, an elution time of ∼15 min at 0.50 mL/min, and an estimated dissociation constant for this protein of 0.5 μM. SignificanceThese chromatographic tools can be used in future work to screen additional possible binding agents for DJ-1 or adapted for examining drug candidates for other proteins. This work represents the first time protein entrapment has been deployed with DJ-1, and it is the first experimental confirmation of binding to DJ-1 by a small lead compound selected in silico.

Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine.

The kinase-like NiRAN domain of nsp12 in SARS-CoV-2 catalyzes the formation of the 5' RNA cap structure. This activity is required for viral replication, offering a new target for the development of antivirals. Here, we develop a high-throughput assay to screen for small molecule inhibitors targeting the SARS-CoV-2 NiRAN domain. We identified NCI-2, a compound with a reactive chloromethyl group that covalently binds to an active site cysteine (Cys53) in the NiRAN domain, inhibiting its activity. NCI-2 can enter cells, bind to, and inactivate ectopically expressed nsp12. A cryo-EM reconstruction of the SARS-CoV-2 replication-transcription complex (RTC) bound to NCI-2 offers a detailed structural blueprint for rational drug design. Although NCI-2 showed limited potency against SARS-CoV-2 replication in cells, our work lays the groundwork for developing more potent and selective inhibitors targeting the NiRAN domain. This approach presents a promising therapeutic strategy for effectively combating COVID-19 and potentially mitigating future coronavirus outbreaks.

Chemical Tools for Probing the Ub/Ubl Conjugation Cascades.

Conjugation of ubiquitin (Ub) and structurally related ubiquitin-like proteins (Ubls), essential for many cellular processes, employs multi-step reactions orchestrated by specific E1, E2 and E3 enzymes. The E1 enzyme activates the Ub/Ubl C-terminus in an ATP-dependent process that results in the formation of a thioester linkage with the E1 active site cysteine. The thioester-activated Ub/Ubl is transferred to the active site of an E2 enzyme which then interacts with an E3 enzyme to promote conjugation to the target substrate. The E1-E2-E3 enzymatic cascades utilize labile intermediates, extensive conformational changes, and vast combinatorial diversity of short-lived protein-protein complexes to conjugate Ub/Ubl to various substrates in a regulated manner. In this review, we discuss various chemical tools and methods used to study the consecutive steps of Ub/Ubl activation and conjugation, which are often too elusive for direct studies. We focus on methods developed to probe enzymatic activities and capture and characterize stable mimics of the transient intermediates and transition states, thereby providing insights into fundamental mechanisms in the Ub/Ubl conjugation pathways.

Identification of inhibitors of human ChaC1, a cytoplasmic glutathione degrading enzyme through high throughput screens in yeast.

The cytosolic glutathione-degrading enzyme, ChaC1, is highly up-regulated in several cancers, with the up-regulation correlating to poor prognosis. The ability to inhibit ChaC1 is therefore important in different pathophysiological situations, but is challenging owing to the high substrate Km of the enzyme. As no inhibitors of ChaC1 are known, in this study we have focussed on this goal. We have initially taken a computational approach where a systemic structure-based virtual screening was performed. However, none of the predicted hits proved to be effective inhibitors. Synthetic substrate analogs were also not inhibitory. As both these approaches targeted the active site, we shifted to developing two high-throughput, robust, yeast-based assays that were active site independent. A small molecule compound library was screened using an automated liquid handling system using these screens. The hits were further analyzed using in vitro assays. Among them, juglone, a naturally occurring naphthoquinone, completely inhibited ChaC1 activity with an IC50 of 8.7 µM. It was also effective against the ChaC2 enzyme. Kinetic studies indicated that the inhibition was not competitive with the substrate. Juglone is known to form adducts with glutathione and is also known to selectively inhibit enzymes by covalently binding to active site cysteine residues. However, juglone continued to inhibit a cysteine-free ChaC1 variant, indicating that it was acting through a novel mechanism. We evaluated different inhibitory mechanisms, and also analogues of juglone, and found plumbagin effective as an inhibitor. These compounds are the first inhibitor leads against the ChaC enzymes using a robust yeast screen.

TMX5/TXNDC15, a natural trapping mutant of the PDI family is a client of the proteostatic factor ERp44.

The ER is the organelle of nucleated cells that produces lipids, sugars, and proteins. More than 20 ER-resident members of the protein disulfide isomerase (PDI) family regulate formation, isomerization, and disassembly of covalent bonds in newly synthesized polypeptides. The PDI family includes few membrane-bound members. Among these, TMX1, TMX2, TMX3, TMX4, and TMX5 belong to the thioredoxin-related transmembrane (TMX) protein family. TMX5 is the least-known member of the family. Here, we establish that TMX5 covalently engages via its active site cysteine residue at position 220 a subset of secretory proteins, mainly single- and multipass Golgi-resident polypeptides. TMX5 also interacts non-covalently, and covalently, via non-catalytic cysteine residues, with the PDI family members PDI, ERp57, and ERp44. The association between TMX5 and ERp44 requires formation of a mixed disulfide between the catalytic cysteine residue 29 of ERp44 and the non-catalytic cysteine residues 114 and/or 124 of TMX5 and controls the ER localization of TMX5 in pre-Golgi compartments. Thus, TMX5 belongs to the family of proteins including Ero1α, Ero1β, Prx4, ERAP1, and SUMF1 that operate in pre-Golgi compartments but lack localization sequences required to position themselves and rely on ERp44 engagement for proper intercompartmental distribution.

Acylfulvenes covalently interact with thioredoxin as an additional cancer target

Maintaining cellular redox homeostasis is critical for cell viability and growth, with disruptions implicated in cellular responses to chemicals and drugs. This study investigates the interactions between acylfulvenes (AFs), a class of DNA alkylating drugs, and thioredoxin (Trx), a key redox regulating enzyme. AFs are semi-synthetic derivatives of the natural product illudin S. While their cytotoxic properties are widely attributed to DNA alkylation, they also react with cellular thiols, such as Trx, and the implications of these interactions remain poorly understood. Through biochemical assays with isolated E. Coli Trx, and cellular experiments in a human cell line (HeLa), we elucidate AFs’ impact on Trx activity and cellular levels. AFs, particularly hydroxymethylacylfulvene (HMAF), inhibited Trx activity by covalently modifying its active site cysteines. Drug exposure also altered cellular Trx levels and nuclear accumulation. In contrast, illudin S, which has a less selective toxicity profile for cancer cells, minimally inhibited isolated Trx. These data underscore Trx as a potential target contributing to the chemotherapeutic potential of AFs and provide insights into molecular interactions governing their impact on cancer cells.

Mechanistic Analysis of 5-Hydroxy γ-Pyrones as Michael Acceptor Prodrugs.

Substituted 5-hydroxy γ-pyrones have shown promise as covalent inhibitor leads against cysteine proteases and transcription factors, but their hydrolytic instability has hindered optimization efforts. Previous mechanistic proposals have suggested that these molecules function as Michael acceptor prodrugs, releasing a leaving group to generate an o-quinone methide-like structure. Addition to this electrophile of either water or an active site cysteine was purported to lead to inhibitor hydrolysis or enzyme inhibition, respectively. Through the use of kinetic nuclear magnetic resonance experiments, Hammett analysis, kinetic isotope effect studies, and density functional theory calculations, our findings suggest that enzyme inhibition and hydrolysis proceed by distinct pathways and are differentially influenced by substituent electronics. This mechanistic revision helps enable a more rational optimization for this class of promising compounds.

PTPN2 copper-sensing relays copper level fluctuations into EGFR/CREB activation and associated CTR1 transcriptional repression

Fluxes in human copper levels recently garnered attention for roles in cellular signaling, including affecting levels of the signaling molecule cyclic adenosine monophosphate. We herein apply an unbiased temporal evaluation of the signaling and whole genome transcriptional activities modulated by copper level fluctuations to identify potential copper sensor proteins responsible for driving these activities. We find that fluctuations in physiologically relevant copper levels modulate EGFR signal transduction and activation of the transcription factor CREB. Both intracellular and extracellular assays support Cu1+ inhibition of the EGFR phosphatase PTPN2 (and potentially PTPN1)–via ligation to the PTPN2 active site cysteine side chain–as the underlying mechanism. We additionally show i) copper supplementation drives weak transcriptional repression of the copper importer CTR1 and ii) CREB activity is inversely correlated with CTR1 expression. In summary, our study reveals PTPN2 as a physiological copper sensor and defines a regulatory mechanism linking feedback control of copper stimulated EGFR/CREB signaling and CTR1 expression.

The structure of the SufS–SufE complex reveals interactions driving protected persulfide transfer in iron-sulfur cluster biogenesis

Fe-S clusters are critical cofactors for redox chemistry in all organisms. The cysteine desulfurase, SufS, provides sulfur in the SUF Fe-S cluster bioassembly pathway. SufS is a dimeric, PLP-dependent enzyme that uses cysteine as a substrate to generate alanine and a covalent persulfide on an active site cysteine residue. SufS enzymes are activated by an accessory transpersulfurase protein, either SufE or SufU depending on the organism, which accepts the persulfide product and delivers it to downstream partners for Fe-S assembly. Here, using E. coli proteins, we present the first X-ray crystal structure of a SufS/SufE complex. There is a 1:1 stoichiometry with each monomeric unit of the EcSufS dimer bound to one EcSufE subunit, though one EcSufE is rotated ∼7° closer to the EcSufS active site. EcSufE makes clear interactions with the α16 helix of EcSufS and site-directed mutants of several α16 residues were deficient in EcSufE binding. Analysis of the EcSufE structure showed a loss of electron density at the EcSufS/EcSufE interface for a flexible loop containing the highly conserved residue R119. An R119A EcSufE variant binds EcSufS but is not active in cysteine desulfurase assays and fails to support Fe-S cluster bioassembly in vivo. 35S-transfer assays suggest that R119A EcSufE can receive a persulfide, suggesting the residue may function in a release mechanism. The structure of the EcSufS/EcSufE complex allows for comparison with other cysteine desulfurases to understand mechanisms of protected persulfide transfer across protein interfaces.

Structural and kinetic characterization of DUSP5 with a Di-phosphorylated tripeptide substrate from the ERK activation loop

Introduction: Dual specific phosphatases (DUSPs) are mitogen-activated protein kinase (MAPK) regulators, which also serve as drug targets for treating various vascular diseases. Previously, we have presented mechanistic characterizations of DUSP5 and its interaction with pERK, proposing a dual active site.Methods: Herein, we characterize the interactions between the DUSP5 phosphatase domain and the pT-E-pY activation loop of ERK2, with specific active site assignments. We also report the full NMR chemical shift assignments of DUSP5 that now enable chemical shift perturbation and dynamics studies.Results and Discussion: Both phosphates of the pT-E-pY tripeptide are dephosphorylated, based on 31P NMR; but, steady state kinetic studies of the tripeptide both as a substrate and as an inhibitor indicate a preference for binding and dephosphorylation of the phospho-tyrosine before the phospho-threonine. Catalytic efficiency (kcat/Km) is 3.7 M−1S−1 for T-E-pY vs 1.3 M−1S−1 for pT-E-Y, although the diphosphorylated peptide (pT-E-pY) is a better substrate than both, with kcat/Km = 18.2 M−1S−1 . Steady state inhibition studies with the pNPP substrate yields Kis values for the peptide inhibitors of: 15.82 mM (pT-E-Y), 4.932 mM (T-E-pY), 1.672 mM (pT-E-pY). Steady state inhibition studies with pNPP substrate and with vanadate or phosphate inhibitors indicated competitive inhibition with Kis values of 0.0006122 mM (sodium vanadate) and 17.32 mM (sodium phosphate), similar to other Protein Tyrosine Phosphatases with an active site cysteine nucleophile that go through a five-coordinate high energy transition state or intermediate. Molecular dynamics (MD) studies confirm preferential binding of the diphosphorylated peptide, but with preference for binding the pY over the pT reside in the catalytic site proximal to the Cys263 nucleophile. Based on MD, the monophosphorylated peptide binds tighter if phosphorylated on the Tyr vs the Thr. And, if the starting pose of the docked diphosphorylated peptide has pT in the catalytic site, it will adjust to have the pY in the catalytic site, suggesting a dynamic shifting of the peptide orientation. 2D 1H-15N HSQC chemical shift perturbation studies confirm that DUSP5 with tripeptide bound is in a dynamic state, with extensive exchange broadening observed—especially of catalytic site residues. The availability of NMR chemical shift assignments enables additional future studies of DUSP5 binding to the ERK2 diphosphorylated activation loop.Summary: These studies indicate a preference for pY before pT binding, but with ability to bind and dephosphorylate both residues, and with a dynamic active site pocket that accommodates multiple tripeptide orientations.

GSTO1 aggravates EGFR-TKIs resistance and tumor metastasis via deglutathionylation of NPM1 in lung adenocarcinoma.

Despite significantly improved clinical outcomes in EGFR-mutant lung adenocarcinoma, all patients develop acquired resistance and malignancy on the treatment of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Understanding the resistance mechanisms is crucial to uncover novel therapeutic targets to improve the efficacy of EGFR-TKI treatment. Here, integrated analysis using RNA-Seq and shRNAs metabolic screening reveals glutathione S-transferase omega 1 (GSTO1) as one of the key metabolic enzymes that is required for EGFR-TKIs resistance in lung adenocarcinoma cells. Aberrant upregulation of GSTO1 confers EGFR-TKIs resistance and tumor metastasis in vitro and in vivo dependent on its active-site cysteine 32 (C32). Pharmacological inhibition or knockdown of GSTO1 restores sensitivity to EGFR-TKIs and synergistically enhances tumoricidal effects. Importantly, nucleophosmin 1 (NPM1) cysteine 104 is deglutathionylated by GSTO1 through its active C32 site, which leads to activation of the AKT/NF-κB signaling pathway. In addition, clinical data illustrates that GSTO1 level is positively correlated with NPM1 level, NF-κB-mediated transcriptions and progression of human lung adenocarcinoma. Overall, our study highlights a novel mechanism of GSTO1 mediating EGFR-TKIs resistance and malignant progression via protein deglutathionylation, and GSTO1/NPM1/AKT/NF-κB axis as a potential therapeutic vulnerability in lung adenocarcinoma.

Unveiling a new strategy for PDIA1 inhibition: Integration of activity-based probes profiling and targeted degradation

The overexpression of PDIA1 in cancer has spurred the quest for effective inhibitors. However, existing inhibitors often bind to only one active site, limiting their efficacy. In our study, we developed a PROTAC-mimetic probe dPA by combining PACMA31 (PA) analogs with cereblon-directed pomalidomide. Through protein profiling and analysis, we confirmed dPA’s specific interaction with PDIA1’s active site cysteines. We further synthesized PROTAC variants with a thiophene ring and various linkers to enhance degradation efficiency. Notably, H4, featuring a PEG linker, induced significant PDIA1 degradation and inhibited cancer cell proliferation similarly to PA. The biosafety profile of H4 is comparable to that of PA, highlighting its potential for further development in cancer therapy. Our findings highlight a novel strategy for PDIA1 inhibition via targeted degradation, offering promising prospects in cancer therapeutics. This approach may overcome limitations of conventional inhibitors, presenting new avenues for advancing anti-cancer interventions.

Modeling the Initiation Phase of the Catalytic Cycle in the Glycyl-Radical Enzyme Benzylsuccinate Synthase.

The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site. Our data indicate that the hydrogen transfer reactions between the active site glycyl and cysteine are principally reversible, but their rates differ strongly depending on their stereochemical orientation, transfer of protium or deuterium, and the presence or absence of substrates or products in the active site. This is particularly evident for the isotope exchange of the remaining protium atom of the glycyl radical to deuterium, which appears dependent on substrate or product binding, explaining why the exchange is observed in some, but not all, glycyl-radical enzymes.

Phase 2 study to evaluate the novel mitochondrial PRX3 inhibitor, RSO-021, as an intrapleural monotherapy and in combination with IV paclitaxel in patients with malignant pleural effusion due to mesothelioma or another advanced solid tumor.

TPS8124 Background: Tumor cells generate elevated levels of reactive oxygen species (ROS), leading to increased expression and activity of critical ROS scavenging pathways. Peroxiredoxin 3 (PRX3) is the principal peroxide scavenging enzyme in mitochondria. RSO–021 is a novel small molecule PRX3 inhibitor that inactivates PRX3 through direct covalent adduction of active site cysteine residues, in turn inducing oxidative stress that is incompatible with tumor cell survival. Preclinical studies of RSO-021 show that it selectively inhibits mitochondrial PRX3 in vitro and is highly effective in a variety of tumor models, including mesothelioma and many solid tumors. RSO-021 has completed phase 1 evaluation in patients with advanced mesothelioma or solid tumors with predominant pleural disease and malignant pleural effusions. The phase 1 data supported weekly intrapleural dosing of RSO-021 at the 90 mg dose level, with acceptable safety and early signals of efficacy. Some patients were able to receive >30 weeks of treatment. Pre- and post-dose primary prophylaxis for local inflammatory reactions to intrapleural injection was instituted during dose escalation, using corticosteroids, NSAIDS and paracetamol. It also became clear that RSO-021 should not be administered to patients with a dry pleural tap, due to risk of extravasation. Methods: The objectives of this open-label, multi-center, 4-arm phase 2 study (MITOPE) are to confirm the efficacy, safety, tolerability, and pharmacology of weekly intrapleural RSO-021 administration. Exploratory endpoints include target engagement as well as biomarkers of response and inflammation. The study explores 2 different RSO-021 doses (90 mg/wk and 45 mg/wk) per FDA Project Optimus, both as monotherapy and in combination with IV paclitaxel 175 mg/m2 every 3 weeks at until progression or limiting toxicity. Each arm at each dose has a 2-stage Simon design (12-21 patients) requiring 1 response to trigger stage 2 (null/target response rate 3%/20% and type I/type II error 0.05/0.8) for a maximum of 168 patients in the trial. Three of the 4 study arms are investigating the antitumor activity of RSO-021 monotherapy (arms 1-3) and the fourth is assessing the paclitaxel combination. Eligibility (by arm) requires: newly diagnosed treatment-naïve mesothelioma with pleural effusion; mesothelioma with pleural effusion that progressed on ≥1 standard of care (SoC) regimen; solid tumor with predominant pleural/lung disease and effusion that progressed on ≥1 SoC regimen; and breast, epithelial ovarian or non-small cell lung cancer with predominant pleural/lung disease and effusion that progressed on ≥1 SoC regimen. The first patient was treated in 4Q2023 and the trial is actively recruiting to all 4 arms at 9 UK sites. Additional sites are planned. Clinical trial information: NCT05278975 .

The structure of the SufS-SufE complex reveals interactions driving protected persulfide transfer in iron-sulfur cluster biogenesis.

Fe-S clusters are critical cofactors for redox chemistry in all organisms. The cysteine desulfurase, SufS, provides sulfur in the SUF Fe-S cluster bioassembly pathway. SufS is a dimeric, PLP-dependent enzyme that uses cysteine as a substrate to generate alanine and a covalent persulfide on an active site cysteine residue. SufS enzymes are activated by an accessory transpersulfurase protein, either SufE or SufU depending on the organism, which accepts the persulfide product and delivers it to downstream partners for Fe-S assembly. Here, using E. coli proteins, we present the first X-ray crystal structure of a SufS/SufE complex. There is a 1:1 stoichiometry with each monomeric unit of the EcSufS dimer bound to one EcSufE subunit, though one EcSufE is rotated ~7° closer to the EcSufS active site. EcSufE makes clear interactions with the α16 helix of EcSufS and site-directed mutants of several α16 residues were deficient in EcSufE binding. Analysis of the EcSufE structure showed a loss of electron density at the EcSufS/EcSufE interface for a flexible loop containing the highly conserved residue R119. An R119A EcSufE variant binds EcSufS but is not active in cysteine desulfurase assays and fails to support Fe-S cluster bioassembly in vivo. 35S-transfer assays suggest that R119A EcSufE can receive a persulfide, suggesting the residue may function in a release mechanism. The structure of the EcSufS/EcSufE complex allows for comparison with other cysteine desulfurases to understand mechanisms of protected persulfide transfer across protein interfaces.

Anticancer Effects of the Trivalent Organoarsenical 2-Amino-4-(dihydroxyarsinoyl) Butanoate.

According to the National Cancer Institute, breast cancer is a leading cause of death in women. The lack of progesterone and estrogen receptors in triple-negative breast cancer (TNBC) cells results in a lack of response to hormonal, monoclonal, or tyrosine kinase inhibitor therapies. Despite intensive drug discovery, there is still no approved targeted treatment for TNBC. The metalloid arsenic has been used in herbal medicines, antibiotics, and chemotherapeutic drugs for centuries. This paper demonstrates that a trivalent arsenic-containing, nonproteogenic amino acid, R-AST-OH (2-amino-4-(dihydroxyarsinoyl) butanoate), inhibits kidney-type glutaminase (KGA), the enzyme that controls glutamine metabolism and is correlated with tumor malignancy. Cell-based assays using the TNBC MDA-MB-231 and HCC1569 cell lines showed that R-AST-OH kills TNBC cells and is not cytotoxic to a control cell line. The results of in silico molecular docking predictions indicate that R-AST-OH binds to the glutamine binding site and forms a covalent bond with an active site cysteine residue. We hypothesize that R-AST-OH is a single warhead for KGA that irreversibly binds to KGA through the formation of an As-S bond. We propose that R-AST-OH is a promising lead compound for the design of new drugs for the treatment of TNBC.

SARS-CoV-2 Mpro responds to oxidation by forming disulfide and NOS/SONOS bonds

The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein’s crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.

Discovery of a Tunable Heterocyclic Electrophile 4-Chloro-pyrazolopyridine That Defines a Unique Subset of Ligandable Cysteines.

Electrophilic small molecules with novel reactivity are powerful tools that enable activity-based protein profiling and covalent inhibitor discovery. Here, we report a reactive heterocyclic scaffold, 4-chloro-pyrazolopyridine (CPzP) for selective modification of proteins via a nucleophilic aromatic substitution (SNAr) mechanism. Chemoproteomic profiling reveals that CPzPs engage cysteines within functionally diverse protein sites including ribosomal protein S5 (RPS5), inosine monophosphate dehydrogenase 2 (IMPDH2), and heat shock protein 60 (HSP60). Through the optimization of appended recognition elements, we demonstrate the utility of CPzP for covalent inhibition of prolyl endopeptidase (PREP) by targeting a noncatalytic active-site cysteine. This study suggests that the proteome reactivity of CPzPs can be modulated by both electronic and steric features of the ring system, providing a new tunable electrophile for applications in chemoproteomics and covalent inhibitor design.

Diselenide-Containing Polymer Based on New Antitumor Mechanism as Efficient GSH Depletion Agent for Ferroptosis Therapy.

Glutathione (GSH) depletion-induced ferroptosis has emerged as a promising treatment for malignant cancer. It works by inactivating glutathione peroxidase 4 (GPX4) and facilitating lipid peroxidation. However, effectively delivering inducers and depleting intracellular GSH remains challenging due to the short half-lives and high hydrophobicity of small-molecule ferroptosis inducers. These inducers often require additional carriers. Herein, diselenide-containing polymers can consume GSH to induce ferroptosis for pancreatic cancer therapy. The diselenide bonds are controllably built into the backbone of the polycarbonate with a targeting peptide CRGD (Cys-Arg-Gly-Asp), which allows for self-assembly into stable nanoparticles (denoted CRNSe) for self-delivery. Significantly, at a concentration of 12 µg mL-1, CRNSe binds to the active site cysteine of GSH resulting in a thorough depletion of GSH. In contrast, the disulfide-containing analog only causes a slight decrease in GSH level. Moreover, the depletion of GSH inactivates GPX4, ultimately inducing ferroptosis due to the accumulation of lipid peroxide in BxPC-3 cells. Both in vitro and in vivo studies have demonstrated that CRNSe exhibits potent tumor suppressive ability with few side effects on normal tissue. This study validates the anti-tumor mechanism of diselenide-containing polymers in addition to apoptosis and also provides a new strategy for inherently inducing ferroptosis in cancer therapy.

RedOx regulation of LRRK2 kinase activity by active site cysteines

Mutations of the human leucine-rich repeat kinase 2 (LRRK2) have been associated with both, idiopathic and familial Parkinson’s disease (PD). Most of these pathogenic mutations are located in the kinase domain (KD) or GTPase domain of LRRK2. In this study we describe a mechanism in which protein kinase activity can be modulated by reversible oxidation or reduction, involving a unique pair of adjacent cysteines, the “CC” motif. Among all human protein kinases, only LRRK2 contains this “CC” motif (C2024 and C2025) in the Activation Segment (AS) of the kinase domain. In an approach combining site-directed mutagenesis, biochemical analyses, cell-based assays, and Gaussian accelerated Molecular Dynamics (GaMD) simulations we could attribute a role for each of those cysteines. We employed reducing and oxidizing agents with potential clinical relevance to investigate effects on kinase activity and microtubule docking. We find that each cysteine gives a distinct contribution: the first cysteine, C2024, is essential for LRRK2 protein kinase activity, while the adjacent cysteine, C2025, contributes significantly to redox sensitivity. Implementing thiolates (R-S-) in GaMD simulations allowed us to analyse how each of the cysteines in the “CC” motif interacts with its surrounding residues depending on its oxidation state. From our studies we conclude that oxidizing agents can downregulate kinase activity of hyperactive LRRK2 PD mutations and may provide promising tools for therapeutic strategies.