Photoaffinity Labeling Experiments Research Articles

Design, Synthesis, and Evaluation of An Anti-trypanosomal [1,2,4]Triazolo[1,5-a]pyrimidine Probe for Photoaffinity Labeling Studies.

Studies have shown that depending on the substitution pattern, microtubule (MT)-targeting 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) can produce different cellular responses in mammalian cells that may be due to these compounds interacting with distinct binding sites within the MT structure. Selected TPDs are also potently bioactive against the causative agent of human African trypanosomiasis, Trypanosoma brucei, both in vitro and in vivo. So far, however, there has been no direct evidence of tubulin engagement by these TPDs in T. brucei. Therefore, to enable further investigation of anti-trypanosomal TPDs, a TPD derivative amenable to photoaffinity labeling (PAL) was designed, synthesized, and evaluated in PAL experiments using HEK293 cells and T. brucei. The data arising confirmed specific labeling of T. brucei tubulin. In addition, proteomic data revealed differences in the labeling profiles of tubulin between HEK293 and T. brucei, suggesting structural differences between the TPD binding site(s) in mammalian and trypanosomal tubulin.

Discovery of Bis-sulfonamides as Novel Inhibitors of Mitochondrial NADH-Quinone Oxidoreductase (Complex I).

Mitochondrial oxidative phosphorylation (OXPHOS) is an essential cellular metabolic process that generates ATP. The enzymes involved in OXPHOS are considered to be promising druggable targets. Through screening of an in-house synthetic library with bovine heart submitochondrial particles, we identified a unique symmetric bis-sulfonamide, KPYC01112 (1) as an inhibitor targeting NADH-quinone oxidoreductase (complex I). Structural modifications of KPYC01112 (1) led to the discovery of the more potent inhibitors 32 and 35 possessing long alkyl chains (IC50 = 0.017 and 0.014 μM, respectively). A photoaffinity labeling experiment using a newly synthesized photoreactive bis-sulfonamide ([125I]-43) revealed that it binds to the 49-kDa, PSST, and ND1 subunits which make up the quinone-accessing cavity of complex I.

Design and Evaluation of a Cyclobutane Diazirine Alkyne Tag for Photoaffinity Labeling in Cells.

Alkyl diazirines are frequently used in photoaffinity labeling to map small molecule-protein interactions in target identification studies. However, the alkyl diazirines can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a reactive alkyl diazo intermediate. Here, we explore the use of ring strain to alter these reactivity preferences and report the development of a cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity, termed PALBOX. We show that PALBOX possesses differential reactivity profiles as compared to other diazirine tags in vitro and is readily incorporated into small molecules to profile their binding interactions in cells. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label the known protein targets in cells with reduced labeling of known alkyl diazirine off-targets. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule-protein binding sites. Thus, PALBOX is a versatile diazirine-based photoaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule-protein interactions.

Designing a bis-azide photoaffinity probe in labeling influenza nucleoprotein trimer to give an insight into the binding mode

Targeting the highly conserved E330 … R416 salt bridge involved in nucleoprotein (NP) trimerization is considered a favorable approach to address the problem of drug resistance in influenza therapy. A photoaffinity probe (1) containing aryl and alkyl azide moieties was synthesized and applied to investigate its binding mode with influenza NP at the trimeric state. Compound 1 possesses appreciable anti-influenza activity with EC50 ​= ​9.1 ​μM. The photoaffinity labeling experiments were conducted with either the lysate of cells infected by A/WSN/1933(H1N1) virus or the recombinant NP proteins. The aryl azide moiety in bis-azide 1 was selectively activated at 300 ​nm or longer wavelength to enable covalent linkage with the binding site and adjacent amino acid residues. The remaining alkyl azide group was subjected to Cu(I)-catalyzed alkyne–azide cycloaddition with a biotin-tethered alkyne (20), followed by enrichment using streptavidin magnetic beads. Following trypsin digestion, the labeled peptides were identified by LC-MS/MS analysis and Mascot search. Of the five identified NP peptides, four are associated with the NP-trimer binding pocket. Molecular docking experiments show that the 2-anilino-1,3-thiazole-4-carboxamide backbone of compound 1 occupies almost the same position of E339 … R416 salt bridge to form hydrogen bonds with E339, providing evidence for interference with NP trimerization.

Bile acids regulate the epithelial Na+ channel in native tissues through direct binding at multiple sites.

Bile acids, originally known to emulsify dietary lipids, are now established signalling molecules that regulate physiological processes. Signalling targets several proteins that include the ion channels involved in regulating intestinal motility and bile viscosity. Studies show that bile acids regulate the epithelial sodium channel (ENaC) in cultured cell models and heterologous expression systems. ENaC plays both local and systemic roles in regulating extracellular fluids. Here we investigated whether bile acids regulate ENaC expressed in native tissues. We found that taurocholic acid and taurohyodeoxycholic acid regulated ENaC in both the distal nephron and distal colon. We also tested the hypothesis that regulation occurs through direct binding. Using photoaffinity labelling, we found evidence for specific binding to both the β and γ subunits of the channel. In functional experiments, we found that the α subunit was sufficient for regulation. We also found that regulation by at least one bile acid was voltage-sensitive, suggesting that one binding site may be closely associated with the pore-forming helices of the channel. Our data provide evidence that bile acids regulate ENaC by binding to multiple sites to influence the open probability of the channel. KEY POINTS: Recent studies have shown that bile acids regulate the epithelial sodium channel (ENaC) in vitro. Here we investigated whether bile acids regulate ENaC in native tissues and whether bile acids directly bind the channel. We found that bile acids regulate ENaC expressed in the mouse cortical collecting duct and mouse colon by modulating open probability. Photoaffinity labelling experiments showed specific binding to the β and γ subunits of the channel, while channels comprising only α subunits were sensitive to taurocholic acid in functional experiments using Xenopus oocytes. Taurocholic acid regulation of ENaC was voltage-dependent, providing evidence for binding to pore-forming helices. Our data indicate that bile acids are ENaC regulatory effectors that may have a role in the physiology and pathophysiology of several systems.

Diverse reaction behaviors of artificial ubiquinones in mitochondrial respiratory complex I

The ubiquinone (UQ) reduction step catalyzed by NADH-UQ oxidoreductase (mitochondrial respiratory complex I) is key to triggering proton translocation across the inner mitochondrial membrane. Structural studies have identified a long, narrow, UQ-accessing tunnel within the enzyme. We previously demonstrated that synthetic oversized UQs, which are unlikely to transit this narrow tunnel, are catalytically reduced by native complex I embedded in submitochondrial particles but not by the isolated enzyme. To explain this contradiction, we hypothesized that access of oversized UQs to the reaction site is obstructed in the isolated enzyme because their access route is altered following detergent solubilization from the inner mitochondrial membrane. In the present study, we investigated this using two pairs of photoreactive UQs (pUQm-1/pUQp-1 and pUQm-2/pUQp-2), with each pair having the same chemical properties except for a ∼1.0 Å difference in side-chain widths. Despite this subtle difference, reduction of the wider pUQs by the isolated complex was significantly slower than of the narrower pUQs, but both were similarly reduced by the native enzyme. In addition, photoaffinity-labeling experiments using the four [125I]pUQs demonstrated that their side chains predominantly label the ND1 subunit with both enzymes but at different regions around the tunnel. Finally, we show that the suppressive effects of different types of inhibitors on the labeling significantly changed depending on [125I]pUQs used, indicating that [125I]pUQs and these inhibitors do not necessarily share a common binding cavity. Altogether, we conclude that the reaction behaviors of pUQs cannot be simply explained by the canonical UQ tunnel model.

Mechanistic Insights of Sulfonamide‐Based NLRP3 Inhibitors for the Treatment of Neurodegenerative Diseases

Neuroinflammation has proven to play an important role in the pathogenesis of neurodegenerative diseases by encouraging the expression and maturation of pro‐inflammatory regulators. Studies have shown the upregulation of pro‐inflammatory cytokines interleukin (IL)‐1β and IL‐18 in neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), traumatic brain injury (TBI), and many more. The NLRP3 (NOD‐, LRR‐ and pyrin domain‐containing protein 3) inflammasome is the key regulator in the maturation and release of these inflammatory cytokines and is therefore an attractive target for suppressing inflammation. Upon assembly and activation of the multimeric NLRP3 inflammasome complex comprised of NLRP3, ASC protein, and pro‐caspase‐1, autoproteolytic cleavage of pro‐caspase‐1 occurs. Activated caspase‐1 is then able to cleave pro‐IL‐1β and pro‐IL‐18 and release their biologically active forms, IL‐1β and IL‐18, consequently leading to an inflammatory cell death known as pyroptosis (Figure 1). Our recent medicinal chemistry campaign has led to a series of novel small molecule inhibitors that are suggested to selectively target NLRP3 and show both in vitro and in vivo activity to inhibit the release of IL‐1β and IL‐18 with sub‐micromolar potencies. Through various assays and photoaffinity labeling experiments using a chemical probe, we have revealed and validated the mechanisms and binding of our lead compound, YQ128, to NLRP3. YQ128 is able to inhibit caspase‐1 and GSDMD cleavage as well as ASC speck formation in mouse macrophages; however, it has no effect on the NLRP3 expression, suggesting its target is upstream of ASC speck and caspase‐1 but downstream of priming. Through target identification studies using a photoaffinity labeling (PAL) technique, we found that YQ128 specifically interacts with NLRP3, but not other components of the NLRP3 inflammasome complex. Competition studies with the parent compound and other known NLRP3 inhibitors suggested the specific and unique binding of YQ128 to NLRP3. The results from other biological assays proposed its binding to the NACHT domain of NLRP3. Using our chemical probe in a pull‐down assay, we also discovered other potential targets within the inflammasome pathway, including a protein that was recently termed “undruggable”. These results suggest a novel mechanism of inhibition by our compounds and further studies are currently underway to improve these inhibitors as potential therapeutics for neuroinflammation in neurodegenerative diseases.

Bile acids directly bind and allosterically regulate the epithelial sodium channel

Bile acids are critical for the digestion and absorption of dietary fats and have important roles as signaling molecules. Bile acids are normally restricted to the enterohepatic circulation, but can be elevated in the blood and urine of patients with advanced liver diseases. The epithelial sodium channel (ENaC) mediates Na+ transport in the biliary epithelium, where it regulates bile viscosity, and in the aldosterone‐sensitive distal nephron, where it regulates extracellular fluid volume. We previously showed that bile acid regulation of ENaC depends on the position and stereochemistry of specific functional groups, but not on their chemico‐physical properties. To determine whether bile acids regulate ENaC in ex vivo native tissues, we performed patch clamp experiments on isolated split open renal tubules from mice, using recording pipettes backfilled with taurocholic acid (t‐CA) or taurohyodeoxycholic acid (t‐HDCA). Consistent with in vitro experiments, t‐CA increased ENaC open probability (PO) by 2‐fold while t‐HDCA decreased ENaC PO by nearly two‐thirds. To determine whether bile acids regulate ENaC through direct binding, we performed photoaffinity labeling experiments. We employed a bile acid derivative (p‐DCA) with diazirine and terminal alkyne groups that facilitate uv‐crosslinking and affinity purification, respectively. Using cultured Fisher Rat Thyroid cells transfected with ENaC, we found that the deoxycholic acid derivative (p‐DCA) crosslinked to the α, β, and γ subunits. To determine whether crosslinking was specific, we performed experiments in the presence of competing bile acids. Crosslinking to the β subunit was significantly reduced in the presence of deoxycholic acid (DCA). Crosslinking to the γ subunit was significantly reduced by taurocholic acid (t‐CA). These data suggest direct binding and multiple sites. To determine the effect of subunit composition on regulation, we measured the effect of t‐CA on Li+ currents in Xenopusoocytes expressing different functional ENaC subunit combinations. Channels composed of α subunits alone were strongly activated by t‐CA, followed by αβγ‐ and αβ‐ channels, suggesting that the α subunit is sufficient for ENaC sensitivity to t‐CA. Our data are consistent with bile acids directly binding all three ENaC subunits at multiple sites. Bile acid moieties provide for selective preferences for each of the sites, resulting in distinct effects for each bile acid.

Targeting the Mitotic Kinesin KIF18A in Chromosomally Unstable Cancers: Hit Optimization Toward an In Vivo Chemical Probe.

Chromosomal instability (CIN) is a hallmark of cancer that results from errors in chromosome segregation during mitosis. Targeting of CIN-associated vulnerabilities is an emerging therapeutic strategy in drug development. KIF18A, a mitotic kinesin, has been shown to play a role in maintaining bipolar spindle integrity and promotes viability of CIN cancer cells. To explore the potential of KIF18A, a series of inhibitors was identified. Optimization of an initial hit led to the discovery of analogues that could be used as chemical probes to interrogate the role of KIF18A inhibition. Compounds 23 and 24 caused significant mitotic arrest in vivo, which was sustained for 24 h. This would be followed by cell death either in mitosis or in the subsequent interphase. Furthermore, photoaffinity labeling experiments reveal that this series of inhibitors binds at the interface of KIF18A and tubulin. This study represents the first disclosure of KIF18A inhibitors with in vivo activity.

Comprehensive understanding of multiple actions of anticancer drug tamoxifen in isolated mitochondria

Tamoxifen has been widely used in the treatment of estrogen receptor (ER)-positive breast cancer, whereas it also exhibits ER-independent anticancer effects in various cancer cell types. As one of the convincing mechanisms underlying the ER-independent effects, induction of apoptosis through mitochondrial dysfunction has been advocated. However, the mechanism of action of tamoxifen even at the isolated mitochondrial level is not fully understood and remains controversial. Here, we attempted to comprehensively understand tamoxifen's multiple actions in isolated rat liver mitochondria through not only revisiting the actions hitherto reported but also conducting originally designed experiments. Using submitochondrial particles, we found that tamoxifen has potential as an inhibitor of both respiratory complex I and ATP synthase. However, these inhibitory effects were not elicited in intact mitochondria, likely because penetration of tamoxifen across the inner mitochondrial membrane is highly restricted owing to its localized positive charge (-N+H(CH3)2). This restricted penetration may also explain why tamoxifen is unable to function as a protonophore-type uncoupler in mitochondria. Moreover, tamoxifen suppressed opening of the mitochondrial permeability transition pore induced by Ca2+ overload through enhancing phosphate uptake into the matrix. The photoaffinity labeling experiments using a photolabile tamoxifen derivative (pTAM1) indicated that pTAM1 specifically binds to voltage-dependent anion channels (VDACs) 1 and 3, which regulate transport of various substances into mitochondria. The binding of tamoxifen to VDAC1 and/or VDAC3 could be responsible for the enhancement of phosphate uptake. Taking all the results together, we consider the principal impairment of mitochondrial functions caused by tamoxifen.

Anti-HIV Effects of Baculiferins Are Regulated by the Potential Target Protein DARS.

Baculiferins are a group of marine sponge-derived polycyclic alkaloids with anti-HIV (human immunodeficiency virus) activities. To identify additional baculiferin-based congeners for SAR analysis and to investigate the mode of action, a total of 18 new baculiferin-type derivatives were synthesized. The inhibitory activities of the congeners against the HIV-1 virus were evaluated in vitro, and the relevant SAR was discussed. Compound 18 exerted the most potent activity toward VSV-G-pseudotyped HIV-1 (IC50 of 3.44 μM) and HIV-1 strain SF33 (IC50 of 2.80 μM) in vitro. To identify the cellular targets, three photoaffinity baculiferin probes were simultaneously synthesized. Photoaffinity labeling experiments together with LC-MS/MS data identified aspartate-tRNA ligase (DARS) as a putative target protein of 18. The overexpression and knockdown of DARS in HEK293T cells provided additional data to demonstrate that DARS is a potential target protein in the regulation of HIV virus infection. The modes of antiviral baculiferins 13 and 18 binding to DARS were determined by a molecular docking simulation. Thus, baculiferin 18 is considered a promising lead as a new molecular target for the development of anti-HIV agents.

Labeling Preferences of Diazirines with Protein Biomolecules.

Diazirines are widely used in photoaffinity labeling (PAL) to trap noncovalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Herein, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while the aryl-fluorodiazirine labeling pattern reflects reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why alkyl diazirine probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome and will facilitate design and interpretation of biomolecular labeling experiments with diazirines.

Structure, Bonding, and Photoaffinity Labeling Applications of Dialkyldiazirines

AbstractDialkyldiazirine photoaffinity probes are unparalleled tools for the study of small molecule–protein interactions. Here we summarize the basic principles of structure, bonding, and photoreactivity of dialkyldiazirines, current methods for their synthesis, and their practical application in photoaffinity labeling experiments. We demonstrate the unique utility of dialkyldiazirine probes in the context of our recent photoaffinity crosslinking-mass spectrometry analysis to reveal a hidden cholesterol binding site in the Hedgehog morphogen proteins.1 Introduction2 Structure, Bonding, and Spectral Properties3 Photoreactivity4 Synthesis5 Application in Photoaffinity Labeling6 Discovery of a Cholesterol–Hedgehog Protein Interface7 Conclusions and Outlook

Inhibitors of a Na+-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function

The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na+-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Vibrio cholerae Na+-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp49 and Asp52 (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na+-NQR.

IACS-010759, a potent inhibitor of glycolysis-deficient hypoxic tumor cells, inhibits mitochondrial respiratory complex I through a unique mechanism

The small molecule IACS-010759 has been reported to potently inhibit the proliferation of glycolysis-deficient hypoxic tumor cells by interfering with the functions of mitochondrial NADH-ubiquinone oxidoreductase (complex I) without exhibiting cytotoxicity at tolerated doses in normal cells. Considering the significant cytotoxicity of conventional quinone-site inhibitors of complex I, such as piericidin and acetogenin families, we hypothesized that the mechanism of action of IACS-010759 on complex I differs from that of other known quinone-site inhibitors. To test this possibility, here we investigated IACS-010759's mechanism in bovine heart submitochondrial particles. We found that IACS-010759, like known quinone-site inhibitors, suppresses chemical modification by the tosyl reagent AL1 of Asp160 in the 49-kDa subunit, located deep in the interior of a previously proposed quinone-access channel. However, contrary to the other inhibitors, IACS-010759 direction-dependently inhibited forward and reverse electron transfer and did not suppress binding of the quinazoline-type inhibitor [125I]AzQ to the N terminus of the 49-kDa subunit. Photoaffinity labeling experiments revealed that the photoreactive derivative [125I]IACS-010759-PD1 binds to the middle of the membrane subunit ND1 and that inhibitors that bind to the 49-kDa or PSST subunit cannot suppress the binding. We conclude that IACS-010759's binding location in complex I differs from that of any other known inhibitor of the enzyme. Our findings, along with those from previous study, reveal that the mechanisms of action of complex I inhibitors with widely different chemical properties are more diverse than can be accounted for by the quinone-access channel model proposed by structural biology studies.

Design, Synthesis, and Biological Application of Novel Photoaffinity Probes of Dihydropyridine Derivatives, BAY R3401.

To explore the molecular mechanisms of BAY R3401, four types of novel photoaffinity probes bearing different secondary tags were synthesized. Their potency for glycogenolysis was evaluated in primary human liver HL-7702 cells and HepG2 cells. Probe 2d showed the best activity in primary human liver HL-7702 cells and HepG2 cells, with IC50 values of 4.45 μM and 28.49 μM, respectively. Likewise, probe 5d showed IC50 values of 6.46 μM in primary human liver HL-7702 cells and 15.29 μM in HepG2 cells, respectively. Photoaffinity labeling experiments were also performed and protein bands larger than 170 kDa were specifically tagged by probe 2d. The results suggest that the synthesized probe 2d might be a very promising tool for the isolation of the target proteins of BAY R3401.

Design and Synthesis of the Diazirine-based Clickable Photo-affinity Probe Targeting Sphingomyelin Synthase 2

Background:SMS family plays a very important role in sphingolipids metabolism and is involved in the membrane mobility and signaling transduction.Methods:SMS2 subtype was related to a variety of diseases and could be regarded as a promising potential drug target. However, the uncertainty of the binding sites and the molecular mechanism of action limited the development of SMS2 inhibitors. Herein, we discovered a photo-affinity probe PAL-1 targeting SMS2.Results:The enzyme inhibitory activity and the photo-affinity labeling experiments showed that PAL-1 could be mono-labeled on SMS2.Conclusion:In summary, starting from the N-arylbenzamides core structure and the minimalist terminal alkyne-containing diazirine photo-crosslinker, we designed and synthesized a photoaffinity probe PAL-1 targeting SMS2. The enzymatic inhibitory activity study showed that PAL-1 exhibited superior selectivities for SMS2 with an IC50 of 0.37 µM over SMS1.

Defining the mechanism of action of S1QELs, specific suppressors of superoxide production in the quinone-reaction site in mitochondrial complex I

Site-specific suppressors of superoxide production (named S1QELs) in the quinone-reaction site in mitochondrial respiratory complex I during reverse electron transfer have been previously reported; however, their mechanism of action remains elusive. Using bovine heart submitochondrial particles, we herein investigated the effects of S1QELs on complex I functions. We found that the inhibitory effects of S1QELs on complex I are distinctly different from those of other known quinone-site inhibitors. For example, the inhibitory potencies of S1QELs significantly varied depending on the direction of electron transfer (forward or reverse). S1QELs marginally suppressed the specific chemical modification of Asp160 in the 49-kDa subunit, located deep in the quinone-binding pocket, by the tosyl chemistry reagent AL1. S1QELs also failed to suppress the binding of a photoreactive quinazoline-type inhibitor ([125I]AzQ) to the 49-kDa subunit. Moreover, a photoaffinity labeling experiment with photoreactive S1QEL derivatives indicated that they bind to a segment in the ND1 subunit that is not considered to make up the binding pocket for quinone or inhibitors. These results indicate that unlike known quinone-site inhibitors, S1QELs do not occupy the quinone- or inhibitor-binding pocket; rather, they may indirectly modulate the quinone-redox reactions by inducing structural changes of the pocket through binding to ND1. We conclude that this indirect effect may be a prerequisite for S1QELs' direction-dependent modulation of electron transfer. This, in turn, may be responsible for the suppression of superoxide production during reverse electron transfer without significantly interfering with forward electron transfer.

Design, Synthesis, and Use of Novel Photoaffinity Probes in Measuring the Serum Concentration of Glycogen Phosphorylase.

A procedure to measure the serum concentration of glycogen phosphorylase during acute myocardial infarction is presented. This method was based on the synthesis of photoaffinity probes, and used the semiquantitative protein electrophoretic mobility shift technique. Three novel photoaffinity probes bearing different secondary tags were synthesized. Their potency was evaluated in an enzyme inhibition assay against rabbit muscle glycogen phosphorylase a (RMGPa). The inhibitory activity of probe 1 was only 100-fold less potent than the mother compound CP-320626. The photoaffinity labeling experiments were also performed, and a protein with molecular weight (MW) of about 90–100 kDa, which was consistent with the MW of GP, was clearly labeled by probe 1. A semiquantitative evaluation of the GP level in serum with probe 1 was also performed. The results showed that the protein band with a MW of about 90–100 kDa was tagged, and the concentration of the protein in serum was found to be between 25 and 50 ng/mL. Mass spectrometric analysis revealed that alpha-1,4 glucan phosphorylase (GPMM) was well-preserved in the bands.

Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches

NADH-quinone oxidoreductase (respiratory complex I) couples NADH-to-quinone electron transfer to the translocation of protons across the membrane. Even though the architecture of the quinone-access channel in the enzyme has been modeled by X-ray crystallography and cryo-EM, conflicting findings raise the question whether the models fully reflect physiologically relevant states present throughout the catalytic cycle. To gain further insights into the structural features of the binding pocket for quinone/inhibitor, we performed chemical biology experiments using bovine heart sub-mitochondrial particles. We synthesized ubiquinones that are oversized (SF-UQs) or lipid-like (PC-UQs) and are highly unlikely to enter and transit the predicted narrow channel. We found that SF-UQs and PC-UQs can be catalytically reduced by complex I, albeit only at moderate or low rates. Moreover, quinone-site inhibitors completely blocked the catalytic reduction and the membrane potential formation coupled to this reduction. Photoaffinity-labeling experiments revealed that amiloride-type inhibitors bind to the interfacial domain of multiple core subunits (49 kDa, ND1, and PSST) and the 39-kDa supernumerary subunit, although the latter does not make up the channel cavity in the current models. The binding of amilorides to the multiple target subunits was remarkably suppressed by other quinone-site inhibitors and SF-UQs. Taken together, the present results are difficult to reconcile with the current channel models. On the basis of comprehensive interpretations of the present results and of previous findings, we discuss the physiological relevance of these models.