Botulinum neurotoxin inhibitor binding dynamics and kinetics relevant for drug design.

BACKGROUND: A natural product analog, 3-(4-nitrophenyl)-7H-furo[3,2-g]chromen-7-one, which is a nitrophenyl psoralen (NPP) was found to be an effective inhibitor of botulinum neurotoxin type A (BoNT/A). METHODS: In this work, we performed enzyme inhibition kinetics and employed biochemical techniques such as isothermal calorimetry (ITC) and fluorescence spectroscopy as well as molecular modeling to examine the kinetics and binding mechanism of NPP inhibitor with BoNT/A LC. RESULTS: Studies of inhibition mechanism and binding dynamics of NPP to BoNT/A light chain (BoNT/A LC) showed that NPP is a mixed type inhibitor for the zinc endopeptidase activity, implying that at least part of the inhibitor-enzyme binding site may be different from the substrate-enzyme binding site. By using biochemical techniques, we demonstrated NPP forms a stable complex with BoNT/A LC. These observations were confirmed by Molecular Dynamics (MD) simulation, which demonstrates that NPP binds to the site near the active site. CONCLUSION: The NPP binding interferes with BoNT/A LC binding to the SNAP-25, hence, inhibits its cleavage. Based on these results, we propose a modified strategy for designing a molecule to enhance the efficiency of the inhibition against the neurotoxic effect of BoNT. GENERAL SIGNIFICANCE: Insights into the interactions of NPP with BoNT/A LC using biochemical and computational approaches will aid in the future development of effective countermeasures and better pharmacological strategies against botulism.

Selective inhibition of carbonic anhydrase IX and XII by coumarin and psoralen derivatives.

A small library of coumarin and their psoralen analogues EMAC10157a-b-d-g and EMAC10160a-b-d-g has been designed and synthesised to investigate the effect of structural modifications on their inhibition ability and selectivity profile towards carbonic anhydrase isoforms I, II, IX, and XII. None of the new compounds exhibited activity towards hCA I and II isozymes. Conversely, both coumarin and psoralen derivatives were active against tumour associated isoforms IX and XII in the low micromolar or nanomolar range of concentration. These data further corroborate our previous findings on analogous derivatives, confirming that both coumarins and psoralens are interesting scaffolds for the design of isozyme selective hCA inhibitors.

Psoralen as an interstrand DNA crosslinker in the selection of DNA-Encoded dynamic chemical library.

DNA-encoded chemical library (DEL) has emerged as a powerful technology for ligand discovery in biomedical research. Recently, we have developed a DNA-encoded dynamic library (DEDL) approach by incorporating the concept of dynamic combinatorial library (DCL) with DELs. DEDL has shown excellent potential in ligand discovery towards a variety of protein targets. However, the requirement of having a pair of unnatural p-stilbazoles as the interstrand DNA crosslinker has limited the chemical diversity of DEDLs. Here, we replaced p-stilbazole with psoralen (PS) and tested the feasibility of psoralen as the crosslinker in DEDL selection. Since psoralen is commercially available and does not require any special crosslinking partner, existing DELs may be directly used to create high-diversity DEDLs. This study is expected to greatly facilitate the development of DEDLs as a versatile tool in drug discovery.

Novel benzopsoralen analogues: synthesis, biological activity and molecular docking studies.

New benzopsoralen analogues were synthesized and their inhibitory effect on the growth of tumourtumour cell lines (MDA MB231 and TCC-SUP) was evaluated. The in vitro antitumour activity of the new benzopsoralen analogues was discussed in terms of structure-activity relationship. Molecular docking studies with human-CYP2A6 enzymes were also carried out with the synthesized compounds to evaluate the potential of these molecules to interact with the haem group of the enzymes. The results demonstrated that the compounds that are able to interact with the iron ion of the haem cofactor and at the same time with active site Asn297 are those that have better anti-proliferative activity.

Novel porphyrin-psoralen conjugates: synthesis, DNA interaction and cytotoxicity studies.

A Cu(i)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) has been utilized to prepare novel triazole-linked cationic porphyrin-psoralen conjugates that exhibited significant photocytotoxicity against A549 cancer cells (IC50 = 84 nM).

Synthesis of novel psoralen analogues and their in vitro antitumor activity.

New tetracyclic benzofurocoumarin (benzopsoralen) analogues were synthesized and their inhibitory effect on the growth of tumor cell lines was evaluated. The human tumor cell lines used were MDA MB231 (breast adenocarcinoma), HeLa (cervix adenocarcinoma) and TCC-SUP (bladder transitional cell carcinoma). The in vitro antitumor activity of the new benzopsoralens was discussed in terms of structure-activity relationship. Molecular docking studies with human-CYP2A6 enzymes were also carried out with the synthesized compounds in order to evaluate the potential of these compounds to interact with the heme group of the enzymes. The results have demonstrated that the linear compounds have the most pronounced activity against tumor cell lines and this might be related to the better accessibility that these compounds have to the active site in relation to the angular ones that have shown in the majority of the cases multiple binding poses in the active site of CYP2A6.

Immobilization of double-stranded DNA onto glass beads by psolaren.

We demonstrated the immobilization of double-stranded DNA onto the glass beads by psoralen, one of the DNA-intercalators in nature. As a result, DNA-immobilized glass beads (DNA-P-beads) were prepared by the intercalation of psoralen, which was immobilized onto the glass surface, onto the double-stranded DNA. These DNA-P-beads formed covalent bondings between psoralen and the nucleic acid base by 365 nm UV irradiation. The amount of immobilized-DNA was 0.24 mg per gram of glass beads. These DNA-P-beads were stable in water, and the DNA on the bead surface maintained its double-stranded structure. These DNA-P-beads selectively removed the planar-structure containing harmful compounds, such as dioxin and polychlorinated biphenyl (PCB) derivatives, from an aqueous multi-component solution. Additionally, a DNA-P-bead column effectively removed harmful compounds. Furthermore, the DNA-P-bead column could be reused by the addition of common organic solvents, such as ethanol.

A new psoralen derivative with enlarged antiproliferative properties.

Following our results with benzopsoralens as potent photochemotherapeutic agents, we report the antiproliferative evaluation of nitrogenated isoster upon and without UVA irradiation. The evaluated pyridazinopsoralen showed a higher photochemotherapeutic activity with respect to the well-known drug, 8-MOP, and a significant cytotoxicity, also in the dark. This result enlarges the interest in this tetracyclic psoralen derivative skeleton in the search of new anticancer agents.

Synthesis of psoralen derivatives and their blocking effect of hKv1.5 channel.

Previously, we found that a furocoumarin derivative, psoralen (7H-furo[3,2-g][1]benzopyran-7-one), blocked a human Kv1.5 potassium channel (hKv1.5) and has a potential antiarrhythmic effect. In the present study, to develop more potent hKv1.5 blockers or antiarrhythmic drugs, we synthesized ten psoralen derivatives and examined their blocking effects on hKv1.5 stably expressed in Ltk cells. Among the newly synthesized psoralen derivatives, three derivatives (Compounds 5, 9 and 10) showed the open channel-blocking effect. Compound 9 among them was the most potent in blocking hKv1.5. We found that compound 9, one of the psoralen derivatives, inhibited the hKv1.5 current in a concentration-, use- and voltage-dependent manner with an IC50 value of 27.4 +/- 5.1 nM at +60 mV. Compound 9 accelerated the inactivation kinetics of the hKv1.5 channel, slowed the deactivation kinetics of hKv1.5 current resulting in a tail crossover phenomenon. Compound 9 inhibited hKv1.5 current in a use-dependent manner. These results indicate that compound 9, one of psoralen derivatives, acts on hKv1.5 channel as an open channel blocker and is much more potent than psoralen in blocking hKv1.5 channel. If further studies were done, compound 9 might be an ideal antiarrhythmic drug for atrial fibrillation.

Synthesis of 2,3-dihydro-3-hydroxy-2-hydroxylalkylbenzofurans from epoxy aldehydes. One-step syntheses of brosimacutin G, vaginidiol, vaginol, smyrindiol, xanthoarnol, and Avicenol A. Biomimetic syntheses of angelicin and psoralen.

We have developed two practical one-step syntheses of 2,3-dihydro-3-hydroxy-2-hydroxyalkylbenzofurans from readily available optically pure alpha,beta-epoxy aldehydes. Electron-deficient resorcinols react with epoxy aldehydes using either Cs2CO3 in DMF or KOH/CaCl2 in MeOH to give adducts 13, 16, 18, 20, 21, and brosimacutin G (6t). Grignard reagents prepared by low-temperature halogen-metal exchange of acetoxy iodocoumarins 35d and 40 and acetoxy bromonaphthalene 41 add to epoxy aldehyde (S)-26 to complete the first syntheses of vaginidiol (7c), vaginol (7t), smyrindiol (8c), xanthoarnol (8t), and avicenol A (9t). Acid-catalyzed fragmentation of vaginidiol or vaginol provides angelicin, while that of smyrindiol or xanthoarnol affords psoralen. In both cases, the trans isomers fragment only twice as fast as the cis isomers, possibly through the intermediacy of a common benzylic cation. This may have implications for the biosynthesis of angelicin and psoralen.

Synthesis and photocytotoxic activity of new alpha-methylene-gamma-butyrolactone-psoralen heterodimers.

The synthesis of a new alpha-methylene-gamma-butyrolactone-psoralen heterodimer 2 is reported. Its photoantiproliferative activity and skin phototoxicity were compared with that of 5-methoxypsoralen (5-MOP) and another heterodimer 1. Both derivatives show a significant phototoxicity toward malignant cell lines including melanoma cells A375 compared to their intrinsic cytotoxicity in the dark. Both compounds were found to be nonphototoxic on mice skin and therefore could be active potential drugs in photochemotherapy.

Direct attachment of conjugate groups to the 5' terminus of oligodeoxyribonucleotides.

This unit gives protocols for the attachment of intercalating and photoreactive conjugate groups to oligodeoxyribonucleotides. Protocols are given for acridine- and psoralen-conjugated oligonucleotides, and include attachment of the linker, preparation of the phosphoramidite, coupling to the oligonucleotide, deprotection, purification, and characterization.

Protein orientation in the Tat-TAR complex determined by psoralen photocross-linking.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. We have used a new method based on psoralen photochemistry to identify a specific contact between a fragment of Tat protein (residues 38-72) and TAR RNA. We synthesized a 35-amino acid fragment containing arginine-rich RNA-binding domain of Tat (38-72), and replaced Arg57 with Cys to introduce a unique thiol group (-SH) in our model peptide. A psoralen derivative, which can react with thiol groups, was synthesized and used for specific chemical modification of Cys57-Tat-(38-72). We used this psoralen-Tat conjugate (psoralen-Cys57-Tat-(38-72)) to form a specific complex with TAR RNA. Upon near-ultraviolet irradiation (360 nm), this synthetic psoralen-peptide cross-linked to a single site in the TAR RNA sequence. The RNA-protein complex was purified and the cross-link site on TAR RNA was determined by RNA sequencing, which revealed that Cys57 of Tat is close to U31 of TAR RNA. Our results provide high-resolution proximity and orientation information about Tat-TAR complex. Such psoralen-peptide conjugates provide a new class of probes for sequence-specific protein-nucleic acid interactions and could be used to selectively control gene expression or to induce site-directed mutations.

Preparation and skin-photosensitizing activity of substituted psoralens.

8-Monosubstituted and 5,8-disubstituted psoralen derivatives were prepared, and their skin-photosensitizing activity was evaluated. The results were correlated in terms of molecular configuration, and 8-allyloxypsoralen can be considered was a new agent of potent photodynamic activity.

Synthesis and characterization of new psoralen derivatives with superior photoreactivity with DNA and RNA.

The synthesis of five new psoralen derivatives is described. Three of these, 4'-hydroxymethyl-4,5',8-trimethylpsoralen, 4'-methoxymethyl-4,5',8-trimethylpsoralen, and 4'-aminomethyl-4,5',8-trimethylpsoralen hydrochloride, and characterized with respect to their photoreactivity with DNA and RNA. They are found to be greatly superior to 4,5',8-trimethylpsoralen and 8-methoxypsoralen, the two commonly used psoralens, in their abilities to saturate the photoreactive sites on DNA and RNA without repeated addition of reagent. A simplified mechanism for the photoreaction of psoralens with nucleic acids is presented and provides a basis for understanding the superior properties of these compounds. The compounds have superior reactivity not only with isolated DNA and RNA but also in viruses and in cells. Psoralens are shown for the first time to cross-link RNA double helices.