Photoaffinity Analog Research Articles

Synthesis of portimines reveals the basis of their anti-cancer activity.

Marine-derived cyclic imine toxins, portimine A and portimine B, have attracted attention because of their chemical structure and notable anti-cancer therapeutic potential1-4. However, access to large quantities of these toxins is currently not feasible, and the molecular mechanism underlying their potent activity remains unknown until now. To address this, a scalable and concise synthesis of portimines is presented, which benefits from the logic used in the two-phase terpenoid synthesis5,6 along with other tactics such as exploiting ring-chain tautomerization and skeletal reorganization to minimize protecting group chemistry through self-protection. Notably, this total synthesis enabled a structural reassignment of portimine B and an in-depth functional evaluation of portimine A, revealing that it induces apoptosis selectively in human cancer cell lines with high potency and is efficacious in vivo in tumour-clearance models. Finally, practical access to the portimines and their analogues simplified the development of photoaffinity analogues, which were used in chemical proteomic experiments to identify a primary target of portimine A as the 60S ribosomal export protein NMD3.

Identification of antimalarial targets of chloroquine by a combined deconvolution strategy of ABPP and MS-CETSA

BackgroundMalaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine (CQ) has played an indispensable role, however, its mechanism of action (MoA) is not fully understood.MethodsWe used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling (ABPP) and mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays.ResultsWe developed a novel clickable, photo-affinity chloroquine analog probe (CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photo-crosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods.ConclusionsWe found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled (ABPP) and label-free (MS-CETSA) methods.

Apoptolidin family glycomacrolides target leukemia through inhibition of ATP synthase.

Cancer cells have long been recognized to exhibit unique bioenergetic requirements. The apoptolidin family of glycomacrolides are distinguished by their selective cytotoxicity towards oncogene transformed cells, yet their molecular mechanism remains uncertain. We used photoaffinity analogs of the apoptolidins to identify the F1 subcomplex of mitochondrial ATP synthase as the target of apoptolidin A. CryoEM of apoptolidin and ammocidin-ATP synthase complexes revealed a novel shared mode of inhibition that was confirmed by deep mutational scanning of the binding interface to reveal resistance mutations which were confirmed using CRISPR-Cas9. Ammocidin A was found to suppress leukemia progression in vivo at doses that were tolerated with minimal toxicity. The combination of cellular, structural, mutagenesis, and in vivo evidence define the mechanism of action of apoptolidin family glycomacrolides and establish a path to address OXPHOS-dependent cancers.

Antibiotic Adjuvant Activity Revealed in a Photoaffinity Approach to Determine the Molecular Target of Antipyocyanin Compounds.

Infections with Pseudomonas aeruginosa are a looming threat to public health. New treatment strategies are needed to combat this pathogen, for example, by blocking the production of virulence factors like pyocyanin. A photoaffinity analogue of an antipyocyanin compound was developed to interrogate the inhibitor's molecular mechanism of action. While we sought to develop antivirulence inhibitors, the proteomics results suggested that the compounds had antibiotic adjuvant activity. Unexpectedly, we found that these compounds amplify the bactericidal activity of colistin, a well-characterized antibiotic, suggesting they may represent a first-in-class antibiotic adjuvant therapy. Analogues have the potential not only to widen the therapeutic index of cationic antimicrobial peptides like colistin, but also to be effective against colistin-resistant strains, strengthening our arsenal to combat P. aeruginosa infections.

Mechanistic Insights into Volatile Anesthetic Modulation of K2P Channels

K2P potassium channels are known to be modulated by volatile anesthetic (VA) drugs and play important roles in clinically relevant effects that accompany general anesthesia. By utilizing a photoaffinity analog of the VA isoflurane, we have identified a VA binding site in the TREK1 K2P channel. The functional importance of the identified site was validated by mutagenesis and biochemical modification. Molecular dynamic simulations of TREK1 in the presence of VA found multiple neighboring residues on TREK1 TM2, TM3 and TM4 that contribute to anesthetic binding. Residues within the identified VA binding region play roles in the mechanisms by which heat, mechanical stretch, and other pharmacological modulators alter TREK1 channel activity and overlap with positions found to modulate TASK K2P channel VA sensitivity. Our findings define molecular contacts that mediate VA binding to TREK1 channels and suggest a mechanistic basis to explain how K2P channels are modulated by VAs.

Mechanistic insights into volatile anesthetic modulation of K2P channels.

K2P potassium channels are known to be modulated by volatile anesthetic (VA) drugs and play important roles in clinically relevant effects that accompany general anesthesia. Here, we utilize a photoaffinity analog of the VA isoflurane to identify a VA-binding site in the TREK1 K2P channel. The functional importance of the identified site was validated by mutagenesis and biochemical modification. Molecular dynamics simulations of TREK1 in the presence of VA found multiple neighboring residues on TREK1 TM2, TM3, and TM4 that contribute to anesthetic binding. The identified VA-binding region contains residues that play roles in the mechanisms by which heat, mechanical stretch, and pharmacological modulators alter TREK1 channel activity and overlaps with positions found to modulate TASK K2P channel VA sensitivity. Our findings define molecular contacts that mediate VA binding to TREK1 channels and suggest a mechanistic basis to explain how K2P channels are modulated by VAs.

Photoaffinity Approach Reveals Antibiotic Adjuvant Activity toward Pseudomonas aeruginosa

Pseudomonas aeruginosa is an emerging public health threat. A common Gram‐negative source of secondary infections, it has few effective treatment options, which are currently being undermined by the rise of resistance. New therapies are urgently needed. We had previously pursued an antivirulence strategy to block the production of pyocyanin, a redox‐active virulence factor. A photoaffinity analog of an antipyocyanin compound was developed to interrogate the inhibitor’s molecular mechanism of action. In the process, antibiotic adjuvant activity was suggested by the proteomics results. Using susceptibility assays, we found that these compounds amplify the bactericidal activity of colistin, a well‐characterized antibiotic, suggesting they may represent a first‐in‐class antibiotic adjuvant therapy. Analogs have the potential to not only widen the therapeutic index of cationic antibiotic peptides like colistin, but to be effective against colistin‐resistant strains. Multidrug‐resistant infections with P. aeruginosa are currently treated with colistin and the related polymyxin b, however, resistance to these drugs is also increasingly encountered. The adjuvants have the potential to preserve the life‐saving therapy of colistin when used in a combination therapy.Support or Funding InformationNational Institutes of Health 5SC3GM118199California State University Program for Education & Research in Biotechnology New Investigator Grant

Inhibitable photolabeling by neurosteroid diazirine analog in the β3-Subunit of human hetereopentameric type A GABA receptors

Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxiolytic effects due to their positive modulatory interactions with the GABAA receptors in the brain. Binding sites for these neurosteroids have been recently identified at subunit interfaces in the transmembrane domain (TMD) of homomeric β3 GABAA receptors using photoaffinity labeling techniques, and in homomeric chimeric receptors containing GABAA receptor α subunit TMDs by crystallography. Steroid binding sites have yet to be determined in human, heteromeric, functionally reconstituted, full-length, glycosylated GABAA receptors. Here, we report on the synthesis and pharmacological characterization of several photoaffinity analogs of pregnanolone and allopregnanolone, of which 21-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoxy]allopregnanolone (21-pTFDBzox-AP) was the most potent ligand. It is a partial positive modulator of the human α1β3 and α1β3γ2L GABAA receptors at sub-micromolar concentrations. [3H]21-pTFDBzox-AP photoincorporated in a pharmacologically specific manner into the α and β subunits of those receptors, with the β3 subunit photolabeled most efficiently. Importantly, photolabeling by [3H]21-pTFDBzox-AP was inhibited by the positive steroid modulators alphaxalone, pregnanolone and allopregnanolone, but not by inhibitory neurosteroid pregnenolone sulfate or by two potent general anesthetics and GABAAR positive allosteric modulators, etomidate and an anesthetic barbiturate. The latter two ligands bind to sites at subunit interfaces in the GABAAR that are different from those interacting with neurosteroids. 21-pTFDBzox-AP's potency and pharmacological specificity of photolabeling indicate its suitability for characterizing neurosteroid binding sites in native GABAA receptors.

Identification of the benztropine analog [125I]GA II 34 binding site on the human dopamine transporter

The dopamine transporter (DAT) is a neuronal membrane protein that is responsible for reuptake of dopamine (DA) from the synapse and functions as a major determinant in control of DA neurotransmission. Cocaine and many psychostimulant drugs bind to DAT and block reuptake, inducing DA overflow that forms the neurochemical basis for euphoria and addiction. Paradoxically, however, some ligands such as benztropine (BZT) bind to DAT and inhibit reuptake but do not produce these effects, and it has been hypothesized that differential mechanisms of binding may stabilize specific transporter conformations that affect downstream neurochemical or behavioral outcomes. To investigate the binding mechanisms of BZT on DAT we used the photoaffinity BZT analog [125I]N-[n-butyl-4-(4‴-azido-3‴-iodophenyl)]-4′,4″-difluoro-3α-(diphenylmethoxy)tropane ([125I]GA II 34) to identify the site of cross-linking and predict the binding pose relative to that of previously-examined cocaine photoaffinity analogs. Biochemical findings show that adduction of [125I]GA II 34 occurs at residues Asp79 or Leu80 in TM1, with molecular modeling supporting adduction to Leu80 and a pharmacophore pose in the central S1 site similar to that of cocaine and cocaine analogs. Substituted cysteine accessibility method protection analyses verified these findings, but identified some differences in structural stabilization relative to cocaine that may relate to BZT neurochemical outcomes.

Identification of a Modulatory Site of Action for the Volatile Anesthetic Isoflurane in TREK1 Tandem Pore Potassium Channels

Many members of the K2P family of potassium channels are modulated by volatile anesthetic (VA) agents. While individual residues within a subset of K2P channels are known to be important for anesthetic modulation, those actually contributing to the VA binding site have not yet been revealed. Using a validated photoaffinity analog of the haloether VA isoflurane (azi-isoflurane), we identified a VA binding site within the TREK1 K2P channel. Azi-isoflurane was reacted with recombinantly expressed, purified and reconstituted TREK1 protein, and modified residues were detected by liquid chromatography-tandem mass spectrometry. Two TREK1 residues are modified by Azi-isoflurane: a glycine in the inner helix bundle crossing region of TM2 and a membrane exposed residue in the TM2/TM3 loop. Protection from azi-isoflurane modification by photolabeling in the presence of 100-fold excess isoflurane was only observed for the inner helix bundle glycine, suggesting that the parent VA isoflurane specifically binds in this region. Two electrode voltage clamp studies on TREK1-expressing Xenopus leavis oocytes demonstrate that mutagenesis of the identified inner helix glycine to tryptophan causes a marked increase in channel activity and eliminates TREK1 anesthetic sensitivity. In molecular dynamics simulations, an azi-isoflurane molecule placed in the TREK1 inner helix bundle region makes stable contacts with this glycine as well as a TM3 residue known to modulate anesthetic responsiveness of TASK K2P channels. Mutating the glycine to tryptophan substantially decreases azi-isoflurane binding affinity, as calculated using free energy perturbation. Future work combining photoaffinity labeling, functional characterization, and MD simulation will aim to understand the mechanism by which anesthetic binding at this region leads to channel activation, whether all VAs share this modulatory site, and whether the identified modulatory site is shared by all anesthetic sensitive K2P channels.

Use of In Silico Docking and Peptide Mapping Studies with the Cocaine Analog MFZ 2 24 to Elucidate the Dopamine Transporter Cocaine Binding Site

Background Cocaine inhibits the dopamine transporter (DAT), resulting in altered dopaminergic homeostasis, that leads to psychostimulant effects. However, the molecular mechanisms underlying this phenomenon are not fully understood. Objective: Identification of the adduction site of the photoactive cocaine analog MFZ 2 24 through computational docking and biochemical peptide mapping to elucidate the cocaine-binding site on DAT. Results: Computational docking methods RosettaLigand and Induced Fit Docking independently predicted the azide adduction of MFZ 2 24 azide group occurs on residue Asp79 or Leu80. Peptide mapping of CNBr digested fragments obtained through introduction of site-directed methionine substitution mutants between Ile67 and Leu80 of transmembrane domain one (TM1) confirmed the computational results. These findings place the tropane core of MFZ 2 24 in the central binding pocket similar to our results with the cocaine photoaffinity analog RTI 82 which adducts to TM6. Conclusion Computational and biochemical docking analysis reveals that MFZ 2 24 adducts to Asp79 or Leu80 in TM1 of DAT with the tropane core positioned in the central binding site. Grant Support: INBRE Program of the NCRR and NIDA-IRP (AHN), DA027845 (LKH & RAV), P20 RR017699 from the COBRE Program of the NCRR, and P20 RR016741 from ND EPSCoR IIG (RAV & JDF).

Cocaine Photo‐affinity Analogs Bind in the S1 Binding Pocket of the Dopamine Transporter Providing a Mechanism for Competitive Inhibition of Dopamine Uptake

Background Cocaine inhibition of dopamine (DA) uptake through the dopamine transporter (DAT) has been proposed to occur by both competitive and non-competitive mechanisms. Current pharmacological and computational data support the existence of two independent cocaine binding sites on DAT, a centrally located site (S1) and a more extracellularly positioned site (S2), which may underlie the complexity of cocaine inhibition of DAT. Objective: Identify the binding site(s) of the high-affinity cocaine photo-affinity analogs RTI 82, MFZ 2 24, and JHC 2 48. Results: Computational docking of the analogs to DAT as well as biochemical peptide mapping analyses of crosslinked DAT complexes revealed independent sites of adduction of the 4ʹ-azido-3ʹ-iodophenylethyl ester moiety on each analog. Our computational and crosslinking data place the core tropane pharmacophore in the S1 binding pocket, which was verified biochemically using the substituted cysteine accessibility method. Cocaine and the analogs could protect S1, but not S2 residues from reacting with methanethiosulfonate reagents. Conclusion The tropane pharmacophore binds to the S1 site, which overlaps the putative DA binding site, supporting a competitive mechanism for cocaine inhibition of DA uptake. Grant Support: INBRE Program of the NCRR and NIDA-IRP (AHN), DA027845 (LKH & RAV), P20 RR017699 from the COBRE Program of the NCRR, and P20 RR016741 from ND EPSCoR IIG (RAV & JDF).

Molecular characterization and tissue localization of sensory neuron membrane protein from Chinese honey bee, Apis cerana cerana (Hymenoptera: Apidae)

Sensory neuron membrane protein (SNMP) is an olfactory receptor with photoaffinity analogs, capable of binding the pheromone membrane protein receptor deduced from receptor membrane protein with the pheromone–pheromone binding protein complex. However, this hypothesis has not yet been experimentally verified. In this experiment, the cDNA sequence encoding an open reading frame (ORF) of the SNMP gene AccSNMP1 (GenBank, KC012595) was cloned from Chinese honey bee, Apis cerana cerana Fabricius. Results from sequence analysis showed that this gene is 1,563 bp long, and that the ORF encodes 520 amino acids with a predicted molecular weight of 58.02 kDa, and has a theoretical isoelectric point of 5.83. Furthermore, there are two putative transmembrane domains. Multiple sequence alignment indicated that the AccSNMP1 gene from A. cerana cerana had different degrees of identity with the corresponding genes in nineteen other insects at the amino acid level. Phylogenetic analysis of the aligned sequences showed that A. cerana cerana is closely related to Apis mellifera Linnaeus and Bombus impatiens Cresson. Its distribution in tissues, as quantified using real-time RT-PCR, indicated that AccSNMP1 is highly expressed in the antennae and legs of A. cerana cerana, and there was a significant difference (p < 0.05) in gene expression between those tissues and tissues in the thorax, abdomen, snout, and head (not including antennae). Western blotting also confirmed the existence in the antennae of AccSNMP1 with an M W of 58.0 kDa, which is the same as the expected value of 58.02 kDa. An immunohistochemistry study showed that AccSNMP1 is expressed in the trichoid sensilla of A. cerana cerana antenna. Therefore, the results of this study provide the basis for further studies of the function of SNMP from A. cerana cerana.

Allosteric nature of P2X receptor activation probed by photoaffinity labelling

In P2X receptors, agonist binding at the interface between neighbouring subunits is efficiently transduced to ion channel gating. However, the relationship between binding and gating is difficult to study because agonists continuously bind and unbind. Here, we covalently incorporated agonists in the binding pocket of P2X receptors and examined how binding site occupancy affects the ability of the channel to gate. We used a strategy for tethering agonists to their ATP-binding pocket, while simultaneously probing ion channel gating using electrophysiology. The agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), a photoaffinity analogue of ATP, enabled us to trap rat homomeric P2X2 receptor and a P2X2/1 receptor chimera in different agonist-bound states. UV light was used to control the degree of covalent occupancy of the receptors. Irradiation of the P2X2/1 receptor chimera - BzATP complex resulted in a persistent current that lasted even after extensive washout, consistent with photochemical tethering of the agonist BzATP and trapping of the receptors in an open state. Partial labelling with BzATP primed subsequent agonist binding and modulated gating efficiency for both full and partial agonists. Our photolabelling strategy provides new molecular insights into the activation mechanism of the P2X receptor. We show here that priming with full agonist molecules leads to an increase in gating efficiency after subsequent agonist binding.

G-protein-coupled receptor dynamics: dimerization and activation models compared with experiment

Our previously derived models of the active state of the β2-adrenergic receptor are compared with recently published X-ray crystallographic structures of activated GPCRs (G-protein-coupled receptors). These molecular dynamics-based models using experimental data derived from biophysical experiments on activation were used to restrain the receptor to an active state that gave high enrichment for agonists in virtual screening. The β2-adrenergic receptor active model and X-ray structures are in good agreement over both the transmembrane region and the orthosteric binding site, although in some regions the active model is more similar to the active rhodopsin X-ray structures. The general features of the microswitches were well reproduced, but with minor differences, partly because of the unexpected X-ray results for the rotamer toggle switch. In addition, most of the interacting residues between the receptor and the G-protein were identified. This analysis of the modelling has also given important additional insight into GPCR dimerization: re-analysis of results on photoaffinity analogues of rhodopsin provided additional evidence that TM4 (transmembrane helix 4) resides at the dimer interface and that ligands such as bivalent ligands may pass between the mobile helices. A comparison, and discussion, is also carried out between the use of implicit and explicit solvent for active-state modelling.

The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles

BackgroundBevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1.ResultsIn this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly.ConclusionsThis is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

Synthesis of sparsomycin derivatives, addressing its binding to the large ribosomal subunit

The syntheses of compounds 14–19, 6-methyluracilacryloyl derivatives, and 27–29, photoreactive aryl azide analogs of the antitumor antibiotic sparsomycin (1) are described. All of the modifications in the 6-methyluracilacryloyl part of 1 were achieved by selective reduction or by selective N- and O-methylation of 6 (Scheme 2). The key step in the synthetic scheme to the photo-affinity analogs is the reaction of the chloromethyl sulfoxides 20 and 21 with the properly protected cystamine 22 in liquid ammonia, to give the sparsomycin N-protected amines 23 and 24, respectively (Scheme 4). Subsequently, the photoreactive groups, viz. a p-azidosalicylyl and a 4-azido-2-nitrophenyl group, were coupled with these amines. For an evaluation of the biological activity of the compounds prepared, inhibition of protein biosynthesis in two cell-free assays, cell-growth inhibition of E. coli in liquid medium and inhibition of murine leukemia L1210 colony formation in soft agar were studied. All variations introduced in the “western A” fragment of 1 render the drug biologically inactive. Apparently, the uracil ring plays an important role in the interaction of sparsomycin with the ribosomal peptidyl transferase and its unmodified presence is crucial for displaying activity. The photoprobes, carrying modifications in the “eastern C” fragment of 1, do interact with the ribosome, but less effectively than sparsomycin itself.

ChemInform Abstract: Synthesis of Phytyl- and Chroman-Derivatized Photoaffinity Labels Based on α-Tocopherol.

Photoaffinity analogues of α-tocopherol have been prepared by substituting photosensitive functional groups at either the terminus of an alkyl chain of varying length mimicking the phytyl tail or on C-3 of the chroman portion of tocopherol. The alkyl chain-modified compounds 2a−d contain a hexyl to nonyl alkyl chain extending from C-2 of the chroman, terminating in a tetrafluoroazidobenzyloxy group. These compounds were prepared starting from the commercially available Trolox acid 4, followed by esterification, protection, and reduction to the silyl-protected Trolox aldehyde 7, which was coupled using Wittig chemistry to different ω-hydroxyphosphonium bromides. Reduction of the alkene product, coupling with p-azidotetrafluorobenzyl bromide, and deprotection of the phenolic silyl group gave compounds 2a−d in excellent yields. Chroman-functionalized photoaffinity labels were synthesized starting from the protected tocopherol chromene 16b which was a key intermediate for preparation of a 3-hydroxy derivative...

The ND2 subunit is labeled by a photoaffinity analogue of asimicin, a potent complex I inhibitor

NADH:ubiquinone oxidoreductase (complex I) is the entry enzyme of mitochondrial oxidative phosphorylation. To obtain the structural information on inhibitor/quinone binding sites, we synthesized [ 3H]benzophenone-asimicin ([ 3H]BPA), a photoaffinity analogue of asimicin, which belongs to the acetogenin family known as the most potent complex I inhibitor. We found that [ 3H]BPA was photo-crosslinked to ND2, ND1 and ND5 subunits, by the three dimensional separation (blue-native/doubled SDS–PAGE) of [ 3H]BPA-treated bovine heart submitochondrial particles. The cross-linking was blocked by rotenone. This is the first finding that ND2 was photo-crosslinked with a potent complex I inhibitor, suggesting its involvement in the inhibitor/quinone-binding.

Synthesis and application of a photoaffinity analog of dehydroepiandrosterone (DHEA)

We have synthesized an analog of dehydroepiandrosterone (DHEA, 1) containing both a benzophenone (BP) and a biotin (Bt) group (DHEA–BP–Bt, 8). Compound 8 was prepared by functionalization on C-17 of 1. Biocytin was reacted with 4-benzoylbenzoic acid and the product was condensed with 1 containing a diamine–hexane linker. We detected specific protein bands of approximately 55, 80, and 150 kDa by SDS–PAGE analysis of vascular endothelial cell plasma membranes which had been photoirradiated in the presence of 8.