Photoaffinity Labeling Studies Research Articles

Phenotype-based screening rediscovered benzopyran-embedded microtubule inhibitors as anti-neuroinflammatory agents by modulating the tubulin–p65 interaction

Neuroinflammation is one of the critical processes implicated in central nervous system (CNS) diseases. Therefore, alleviating neuroinflammation has been highlighted as a therapeutic strategy for treating CNS disorders. However, the complexity of neuroinflammatory processes and poor drug transport to the brain are considerable hurdles to the efficient control of neuroinflammation using small-molecule therapeutics. Thus, there is a significant demand for new chemical entities (NCEs) targeting neuroinflammation. Herein, we rediscovered benzopyran-embedded tubulin inhibitor 1 as an anti-neuroinflammatory agent via phenotype-based screening. A competitive photoaffinity labeling study revealed that compound 1 binds to tubulin at the colchicine-binding site. Structure–activity relationship analysis of 1’s analogs identified SB26019 as a lead compound with enhanced anti-neuroinflammatory efficacy. Mechanistic studies revealed that upregulation of the tubulin monomer was critical for the anti-neuroinflammatory activity of SB26019. We serendipitously found that the tubulin monomer recruits p65, inhibiting its translocation from the cytosol to the nucleus and blocking NF-κB-mediated inflammatory pathways. Further in vivo validation using a neuroinflammation mouse model demonstrated that SB26019 suppressed microglial activation by downregulating lba-1 and proinflammatory cytokines. Intraperitoneal administration of SB26019 showed its therapeutic potential as an NCE for successful anti-neuroinflammatory regulation. Along with the recent growing demands on tubulin modulators for treating various inflammatory diseases, our results suggest that colchicine-binding site-specific modulation of tubulins can be a potential strategy for preventing neuroinflammation and treating CNS diseases.

Discovery of the first tight‐binding reversible antagonists of Hedgehog protein autoprocessing

Hedgehog (Hh) signaling ligands (HhN) are expressed as precursors with an adjacent C‐terminal autoprocessing domain, HhC. Autoprocessing activity of HhC releases and cholesterylates the Hh ligand thereby activating the HhN for full biological activity. Inhibitors of HhC activity are of interest as probes of the Hh signaling pathway, and HhC inhibitors may also offer new therapeutic approaches to suppress oncogenic signaling activity of HhN. The intramolecular nature of the HhN/HhC system poses a challenge for reversible inhibitors, and to date most antagonists of HhC bind the active site covalently. Here we report we report the discovery and optimization of the first tight‐binding reversible inhibitor of HhC. Using a light‐to‐dark FRET activity assay for Drosophila melanogaster HhC, we screened a focused library of 1187 sterol‐like analogs in search of HhC inhibitors. Four structurally related hits were identified and further validated by dose‐response assays. The most effective inhibitor from the screen had an IC50 value of 2x10‐6M. A panel of analogs were prepared and tested for SAR analysis resulting in compound tBT‐HBT, a derivative with a sub‐micromolar IC50of 3x10‐7 M. Inhibition appears to be mediated by a previously unidentified allosteric binding site on HhC. Kinetic analysis, photoaffinity labeling, computational modeling, and point mutagenesis studies suggest that the binding site is nearby, but not overlapping with the cholesterol binding site of HhC.

Involvement of a Conserved Intramembrane Acidic Residue in Cholesterol Binding of Mitochondrial VDAC1 Revealed by Microsecond-Scale Simulations

Voltage-dependent anion channel 1 (VDAC1), a β-barrel membrane protein abundant in the mitochondrial outer membrane, mediates the exchange of ions and metabolites between the mitochondrial matrix and the cytosol. Both experimental and previous computational studies suggest that a conserved, membrane-facing residue in VDAC1, glutamate 73 (E73), plays an essential role in the function of the channel, including its oligomerization and interaction with other cytosolic partner proteins. In addition, a recent photoaffinity labeling study identified E73 as a specific binding site for cholesterol, an endogenous membrane lipid that also regulates VDAC functions. However, how cholesterol interacts with E73 and the effects of cholesterol binding at E73 remain poorly understood. Here, we performed an extensive set of microsecond-long atomistic molecular dynamics simulations on both wildtype and mutant forms and in different protonation states (totaling 14 μs) to characterize cholesterol interactions with VDAC1. Analysis of lipid occupancy showed that cholesterol exhibited distinct preferred binding sites from phospholipids on VDAC1. Spontaneous binding and unbinding of cholesterol to E73 in its deprotonated state was captured, a process accompanied by local membrane deformation and water solvation at the E73 side-chain in the middle of the lipid bilayer. Compared to VDAC1 in a pure phospholipid environment, the presence of cholesterol reduces the fluctuations of the β-strands in proximity to E73. Based on our results, we propose a stabilizing role of cholesterol in regulating the VDAC1 dynamics in the region close to E73, which might further modulate VDAC1 function as an ion channel.

Probing the Mechanism of Photoaffinity Labeling by Dialkyldiazirines through Bioorthogonal Capture of Diazoalkanes.

Dialkyldiazirines have emerged as reagents of choice for biological photoaffinity labeling studies. The mechanism of crosslinking has dramatic consequences for biological applications where instantaneous labeling is desirable, as carbene insertions display different chemoselectivity and are much faster than competing mechanisms involving diazo or ylide intermediates. Here, deuterium labeling and diazo compound trapping experiments are employed to demonstrate that both carbene and diazo mechanisms operate in the reactions of a dialkyldiazirine motif that is commonly utilized for biological applications. For the fraction of intermolecular labeling that does involve a carbene mechanism, direct insertion is not necessarily involved, as products derived from a carbonyl ylide are also observed. We demonstrate that a strained cycloalkyne can intercept diazo compound intermediates and serve as a bioorthogonal probe for studying the contribution of the diazonium mechanism of photoaffinity labeling on a model protein under aqueous conditions.

Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABAA) receptors

Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analysis, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABAAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ2 GABAARs by photoaffinity labeling with [3H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([3H]21-pTFDBzox-AP), a potent GABAAR PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the β subunit M4 (β3Pro-415, β3Leu-417, and β3Thr-418) and M3 (β3Arg-309) helices located at the base of a pocket in the β+-α- subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3β-OH analogs that are GABAAR antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABAAR PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABAAR β+-α- subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.

Design and synthesis of small molecule-conjugated photoaffinity nanoprobes for a streamlined analysis of binding proteins

We designed and synthesized a set of photoaffinity nanoprobes, which multivalently display a small molecule ligand and a photoreactive group on gold nanoparticles. Due to the typically hydrophobic nature of these two functional groups, a hydrophilic spacer was additionally introduced to co-functionalize the nanoprobes to maintain their dispersibility in aqueous buffer solutions. Photoaffinity labeling studies using the nanoprobes composed of different ratios of three functional groups showed that including high density of the spacer group attenuates crosslinking efficiency. Comparative analysis of the reactivity among three major photoreactive groups suggested that unlike in the context of conventional photoaffinity probes, arylazide group enables the most selective crosslinking of a model small molecule binding protein.

Alpha Synuclein Fibrils Contain Multiple Binding Sites for Small Molecules.

The fibrillary aggregation of the protein alpha synuclein (Asyn) is a hallmark of Parkinson's disease, and the identification of small molecule binding sites on fibrils is essential to the development of diagnostic imaging probes. A series of molecular modeling, photoaffinity labeling, mass spectrometry, and radioligand binding studies were conducted on Asyn fibrils. The results of these studies revealed the presence of three different binding sites within fibrillar Asyn capable of binding small molecules with moderate to high affinity. A knowledge of the amino acid residues in these binding sites will be important in the design of high affinity probes capable of imaging fibrillary species of Asyn.

Chemical Inhibition of Human Thymidylate Kinase and Structural Insights into the Phosphate Binding Loop and Ligand-Induced Degradation.

Targeting thymidylate kinase (TMPK) that catalyzes the phosphotransfer reaction for formation of dTDP from dTMP is a new strategy for anticancer treatment. This study is to understand the inhibitory mechanism of a previously identified human TMPK (hTMPK) inhibitor YMU1 (1a) by molecular docking, isothermal titration calorimetry, and photoaffinity labeling. The molecular dynamics simulation suggests that 1a prefers binding at the catalytic site of hTMPK, whereas the hTMPK inhibitors that bear pyridino[d]isothiazolone or benzo[d]isothiazolone core structure in lieu of the dimethylpyridine-fused isothiazolone moiety in 1a can have access to both the ATP-binding and catalytic sites. The binding sites of hTMPK inhibitors were validated by photoaffinity labeling and mass spectrometric studies. Taking together, 1a and its analogues stabilize the conformation of ligand-induced degradation (LID) region of hTMPK and block the catalytic site or ATP-binding site, thus attenuating the ATP binding-induced closed conformation that is required for phosphorylation of dTMP.

Functional and Molecular Interaction of Phenylalkylamines and Dihydropyridines with the Model Calcium Channel CavAb

Mammalian voltage-gated calcium channels (mCavs) are molecular targets of phenylalkylamines (PAAs) and dihydropyridines (DHPs), which are widely used antiarrhythmic and antihypertension drugs that block mCavs in a frequency- and voltage-dependent manner. The two distinct receptor sites for these drugs in mCavs have been elucidated through extensive photoaffinity labeling and mutagenesis studies, revealing key residues along the S5 and S6 transmembrane segments of domains III and IV of mCavs (Hockerman et al., 1997). We have recently reported the crystal structure of CavAb, which is a highly calcium-selective mutant of the prokaryotic sodium channel NavAb (Tang et al., 2014). Even though CavAb is separated evolutionarily from mCavs by more than one billion years, we found that PAAs and DHPs interact with CavAb in a similar state-dependent manner. The PAAs tested have Kd's in the range of 250 nM to 800 nM, and verapamil shows low-affinity block of the resting state and high-affinity use-dependent block of activated and/or inactivated states. Mutations of T206 in the center of segment S6 reduced PAA affinity by 20-fold. In the case of DHPs, nimodipine showed voltage-dependent block of activated CavAb with a Kd of 194 nM, but no resting-state block was observed in the tested concentration range. DHPs shift the voltage dependence of inactivation up to 100 mV negatively. This indicates that DHP interaction with CavAb is state-dependent and greatly favors the inactivated state, similar to mCavs. Mutation of I199 in the outer region of segment S6, which is critical for binding DHPs in mCavs, reduced binding affinity 40-fold. Our results reveal surprising similarity of drug action on CavAb and mCavs and thereby support use of CavAb as a molecular model to determine the three-dimensional structure of these important drug receptor sites.

Two‐Step Synthesis of a Clickable Photoaffinity Probe from an Anticancer Saponin OSW‐1 and its Photochemical Reactivity

AbstractOSW‐1 is a highly potent anticancer saponin with an unknown mechanism of action that is distinct from the currently used chemotherapeutic agents. Toward investigation of its binding proteins, we designed and synthesized a clickable photoaffinity probe from OSW‐1 and characterized its photochemical reactivity. A mild, two‐step procedure involving a Me2SnCl2‐mediated acylation reaction was developed to site‐selectively derivatize OSW‐1 with a linker that bears a photoreactive group and an alkyne tag. The OSW‐1‐based photoaffinity probe retained potent anticancer activity similar to that of the parent natural product, thereby providing a cell‐permeable analogue of OSW‐1. Photoaffinity labeling studies demonstrated that the probe enabled crosslinking of a model sterol‐binding protein in an affinity‐dependent fashion, which could be efficiently detected by conjugating a fluorophore or biotin by click chemistry.

Photoaffinity labeling studies of the carbohydrate-binding proteins with different affinities

Photoaffinity labeling has been used as a promising approach to detection and isolation of carbohydrate-binding proteins, which are typically characterized by low binding affinity and selectivity. When there are several specific binding proteins, it is desirable that a photoaffinity probe is capable of simultaneously crosslinking them and that the crosslinking yields depend on the relative binding affinities. In this study, we describe the design and synthesis of carbohydrate photoaffinity probes and their ability to capture lectins of different binding affinities.

Syntheses of photoreactive cardiolipins for a photoaffinity labeling study

The photoaffinity labeling technique using photoreactive cardiolipin (CL) is a powerful means for investigating the molecular mechanism of the formation of a specific cytochrome c–cardiolipin complex. Using phosphoramidite chemistry, we synthesized three photoreactive CLs, that possess an unstable diazirine ring at different positions; that is, the acyl chain in the sn-1 or sn-2 position and the central glycerol moiety.

Chemical modifications of respiratory complex I for structural and functional studies.

Studies on chemical modifications of bacterial and mitochondrial complex I by synthetic chemical probes as well as endogenous chemicals have provided useful information on the structural and functional aspects of this enzyme. We herein reviewed recent studies that investigated chemical modifications of complex I by endogenous chemicals (e.g. Cys-S-nitrosation, Cys-S-glutathionylation, and Ser-O-phosphorylation) and synthetic reagents (e.g. Cys-SH modification by SH-reagents and the cross-linking of nearby subunits by bifunctional cross-linkers). We also reviewed recent photoaffinity labeling studies using complex I inhibitors, which can be recognized as "site-specific modification" by synthetic chemicals. In addition, we discussed the possibility of site-specific modification by various functional probes via ligand-directed tosylate (LDT) chemistry as a promising approach for unique biophysical studies on complex I.

PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal Mechanism

The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg·ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.

Structural and functional insights into the juxtamembranous amino‐terminal tail and extracellular loop regions of class B GPCRs

Class B guanine nucleotide-binding protein GPCRs share heptahelical topology and signalling via coupling with heterotrimeric G proteins typical of the entire superfamily of GPCRs. However, they also exhibit substantial structural differences from the more extensively studied class A GPCRs. Even their helical bundle region, most conserved across the superfamily, is predicted to differ from that of class A GPCRs. Much is now known about the conserved structure of the amino-terminal domain of class B GPCRs, coming from isolated NMR and crystal structures, but the orientation of that domain relative to the helical bundle is unknown, and even less is understood about the conformations of the juxtamembranous amino-terminal tail or of the extracellular loops linking the transmembrane segments. We now review what is known about the structure and function of these regions of class B GPCRs. This comes from indirect analysis of structure-function relationships elucidated by mutagenesis and/or ligand modification and from the more direct analysis of spatial approximation coming from photoaffinity labelling and cysteine trapping studies. Also reviewed are the limited studies of structure of some of these regions. No dominant theme was recognized for the structures or functional roles of distinct regions of these juxtamembranous portions of the class B GPCRs. Therefore, it is likely that a variety of molecular strategies can be engaged for docking of agonist ligands and for initiation of conformational changes in these receptors that would be expected to converge to a common molecular mechanism for activation of intracellular signalling cascades.

ChemInform Abstract: Medicinal Chemistry and Chemical Biology of Diketopiperazine‐Type Antimicrotubule and Vascular‐Disrupting Agents

AbstractReview: 63 refs.

Modulation of multidrug resistance 1 expression and function in retinoblastoma cells by curcumin

Objective:To determine the possible interaction of curcumin with P-glycoprotein (P-gp) expression and function by in vitro and in silico studies.Materials and Methods:In this study, curcumin was compared for its potential to modulate the expression and function of P-gp in Y79 RB cells by western blot, RT-PCR (reverse transcription polymerase chain reaction) and functional assay. Further, in silico molecular modeling and docking simulations were performed to deduce the inhibitory binding mode of curcumin.Results:Western blot and RT-PCR analysis decreased the expression of P-gp in a dose-dependent manner. The effect of curcumin on P-gp function was demonstrated by Rhodamine 123 (Rh123) accumulation and efflux study. Curcumin increased the accumulation of Rh123 and decreased its efflux in retinoblastoma (RB) cells. In addition, curcumin inhibited verapamil stimulated ATPase activity and photoaffinity labeling study showed no effect on the binding of 8-azido-ATP-biotin, indicating its interaction at the substrate binding site. Moreover, molecular docking studies concurrently infer the binding of curcumin into the substrate binding site of P-gp with a binding energy of -7.66 kcal/mol.Conclusion:These findings indicate that curcumin suppresses the MDR1 expression and function, and therefore may be useful as modulators of multidrug resistance in RB tumor.

Molecular basis of peptide activation of the GLP-1 receptor

Molecular basis of peptide activation of the GLP-1 receptor

Design and synthesis of biotin- or alkyne-conjugated photoaffinity probes for studying the target molecules of PD 404182

To investigate the mechanism of action of the potent antiviral compound PD 404182, three novel photoaffinity probes equipped with a biotin or alkyne indicator were designed and synthesized based on previous structure–activity relationship studies. These probes retained the potent anti-HIV activity of the original pyrimidobenzothiazine derivatives. In photoaffinity labeling studies using HIV-1-infected H9 cells (H9IIIB), eight potential proteins were observed to bind PD 404182.

Medicinal Chemistry and Chemical Biology of Diketopiperazine-Type Antimicrotubule and Vascular-Disrupting Agents

Certain antimicrotubule agents displaying colchicine-like tubulin-depolymerizing activity can act as both cytotoxic and vascular-disrupting agents (VDAs). VDAs constitute a new class of anticancer drugs and are currently in clinical trials. We have developed a VDA clinical candidate (phase II) diketopiperazine (DKP)-type antimicrotubule agent called plinabulin (7) derived from the natural DKP phenylahistin (5), which displays colchicine-like tubulin-depolymerizing activity. To develop more potent antimicrotubule DKP derivatives, we performed an intensive structure-activity relationship study examining the phenyl group of compound 7. This study identified more potent DKP derivatives (2,5-difluoro derivatives [29] and benzophenone derivatives [36]) with vascular-disrupting activities. The benzophenone moiety of compound 36 was further modified to yield the most potent cytotoxic derivative yet discovered, the 4-fluorobenzophenone derivative 38 m, which inhibited the growth of HT-29 cells in vitro at subnanomolar levels. As both VDAs and cytotoxic agents, these potent DKP derivatives are valuable second-generation drug candidates. The chemical biology of plinabulin was examined by designing and synthesizing biotin-tagged photoaffinity probes 40-42 that could be used to indicate the binding mode of compound 7 with tubulin. A tubulin photoaffinity labeling study suggested that compound 7 binds at the interface between the α- and β-tubulins near the colchicine-binding site and not inside the colchicine-binding cavity. A water-soluble prodrug of the poorly water-soluble 7 was next designed in an effort to improve the pharmacokinetics and chemotherapeutic indices. The lead compound 56 revealed high water solubility and a half-life profile appropriate for an injected drug.