Analysis Of Pyronaridine And Pyronaridine Tetraphosphate Using High Performance Liquid Chromatography And High Resolution Nuclear Magnetic Resonance Spectroscopy

Pironaridina (PND), 2-metoksi-7 -kloro-1 0-[3' ,5'-bis-(pyrrolidinil-1-metil)-4'- hidroksianinilo]benzo[b]-1,5-naftiridina (formula molekul: C29H32CIN502) yang disintesiskan oleh Zheng et al pada awal 1970an telah digunakan sebagai ubat antimalaria selama 20 tahun. la berwarna merah bata, dengan rasa pahit dan tidak berbau dengan takat lebur pada suhu 174-176 °C. Bagi tujuan formulasi ubat, PND diubah kepada terbitan fosfatnya iaitu pironaridina tetrafosfat (PNDT) yang terlarut dalam air (formula molekul: C29H32CIN502.4H3P04) dan mempunyai takat lebur pada 227-230 °C. Kajian yang dilakukan ke atas PNDT sebelum ini hanya menunjukkan satu puncak pad a kromatogram apabila dianalisis dengan menggunakan kromatografi cecair prestasi tinggi (KGPT). Tetapi, dengan menggunakan kaedah terubahsuai Jayaraman yang dikembangkan oleh Karupiah (2003), tiga puncak diperhatikan pada kromatogram. Tiga puncak ini dilabel sebagai komponen B pad a 10.0 min, X pada 21.2 min dan Y pad a 24.3 min. Sebatian B diekstrak dan pencirian struktur sebatian tersebut dilakukan. Akan tetapi, untuk memastikan identiti komponen B, informasi struktur PND dan PNDT perlu ditentukan. Sehingga sekarang, data NMR yang wujud hanya daripada spektrum yang dirakam pada 2.11 Tesla (T) (90 MHz untuk 1H). Pyronaridine (PND), 2-methoxy-7 -chloro-1 0-[3' ,S'-bis-(pyrrolidinyl-1-methyl)-4'hydroxyanilino] benzo[b]-1 ,S-napthyridines (molecular formula: C29H32CINs02), synthesized by Zheng et al. in the early 1970s, has been used in China for over 20 years as an antimalarial drug. It is an odourless, brick red powder with a bitter taste and decomposes at 174-176 °c. For drug formulation purposes, PND is converted to its water-soluble tetraphosphate derivative, PNDT (molecular formula: C29H32CINs02.4H3P04) that has a melting point of 227-230 °c. All previous studies conducted on PND and PNDT using HPLC showed only one peak present in the chromatogram. However, the modified version of Jayaraman's HPLC method developed by Karupiah (2003) showed the presence of three peaks. These were labelled as component B at 10.0 min, X at 21.2 min and Y at 24.3 min. Component B was then extracted and characterized. However, in order to ascertain its identity, detailed structural information of both PND and PNDT must first be established. To date, the only NMR data available in literature on PND was deduced from a proton spectrum recorded at 2.11 Tesla (T) (90 MHz in lH)

Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1

Translation termination in eukaryotes is catalyzed by two release factors eRF1 and eRF3 in a cooperative manner. The precise mechanism of stop codon discrimination by eRF1 remains obscure, hindering drug development targeting aberrations at translation termination. By solving the solution structures of the wild-type N-domain of human eRF1 exhibited omnipotent specificity, i.e. recognition of all three stop codons, and its unipotent mutant with UGA-only specificity, we found the conserved GTS loop adopting alternate conformations. We propose that structural variability in the GTS loop may underline the switching between omnipotency and unipotency of eRF1, implying the direct access of the GTS loop to the stop codon. To explore such feasibility, we positioned N-domain in a pre-termination ribosomal complex using the binding interface between N-domain and model RNA oligonucleotides mimicking Helix 44 of 18S rRNA. NMR analysis revealed that those duplex RNA containing 2-nt internal loops interact specifically with helix α1 of N-domain, and displace C-domain from a non-covalent complex of N-domain and C-domain, suggesting domain rearrangement in eRF1 that accompanies N-domain accommodation into the ribosomal A site

NMR studies of protein interactions with nucleic acids : translation termination and transcription

My thesis includes three subprojects related to NMR-based studies of three protein classes involved in DNA/RNA recognition. My focus is to use the advanced solution NMR methods to answer critical biological questions complementing collaborative efforts within each individual subproject. In the first subproject I studied structural changes in a stop-codon recognizing protein induced by mutations altering stop-codon recognition specificity. Ribosome is one of the most sophisticated supramolecular complexes in the cell. Some of the processes that occur within this machinery include translation initiation, elongation, and termination, all of which involve coding mRNA, a set of aminoacyl-tRNAs, and various protein factors acting in a concerted manner. Translation termination requires class I release factors: RF1 and RF2 in Bacteria, and eRF1 in Eukarya. eRF1 is able to decode all three stop codons. Until now, the mechanism of stop codon recognition by eRF1 leading to hydrolysis of the ester bond on the peptidyl tRNA remains obscure. We working as a team at Prof. Pervushin’s lab (Li Yan and Leo Wong) proposed a model of the binding mode of eRF1 to pre-translation termination ribosomal complex based on our solution structures of wild-type and several mutants of eRF1’s N-domain as well as its interactions with model RNA and M- and C- domains of the same protein. Due to the global character of structural perturbation induced by mutations, Residual Dipolar Coupling (RDC) data were obtained using 15N labeled sample aligned with Pf1 phages. The RDC measurements obtained were then applied in the structure calculation and refinement of the eRF1 mutant Q122FM(Y)F126 using structure calculation software. The most interesting mutant, Q122FM(Y)F126 restricts the decoding capability of eRF1 to UGA codon only. From the 3D structures, we established that the mutations alter conformation and dynamics of the GTS loop distant from the sites of mutations. Based on published biochemical and mutagenesis studies, we propose that the GTS loop forms a switch that allows reading of the multiple codons. NMR analysis revealed that helix α1 of N-domain interacts specifically with double-stranded RNA with a bulge or internal loop resembling a mismatch of H44 in 18S rRNA of Eukaryotic ribosome. From these results, a 3D model of eRF1 interactions with ribosome in pre-termination state is proposed. In my second project we collaborated with Dr. Ralf Jauch in order to provide structural basis for activity of novel drug candidates interfering with transcription factor/cognate DNA interactions. The DNA binding domains of transcription factors have so far been considered too impervious to be tackled as drug targets although upregulated transcription factors are a major cause of cancer and other diseases. Here we identified a Dawson-POM as an unconventional but potent compound to inhibit the DNA binding activity of Sox2. We used NMR to locate binding site of the drug candidates on Sox2. The mode of interaction of the Dawson-POM with the Sox2-HMG domain involves predominantly electrostatic interactions at the pocket just outside of the DNA binding region, but still adequately positioned to compete with the negatively charged DNA backbone. In summary, the inhibitory mechanism demonstrated here could eventually spawn the development of modified classes of POM based drugs to specifically combat aberrant gene expression. The objective of the third project is to assess the structural flexibility of N-terminal tail domain of Histone 4 when it is in the compact nucleosome array using deuterium/protons exchange experiment in NCP. 15N labeled Histone 4 was prepared and 2D [1H-15N]-TROSY series of experiments were carried out for protein recovered from reconstituted NCPs in H2O followed by controlled exposure to D2O. The sequential assignment of the backbone H4 was used to identify the dynamics of the NCP as well as arrays in the compact form. We observed that that although in compact states, the magnitude of protection is not as pronounced as expected. This suggests that the NCP is still flexible, albeit being in a compact state.The first two projects are either prepared or published. The last project is in active development.Doctor of Philosophy (SBS

Structural characterization of eRF1 mutants indicate a complex mechanism of stop codon recognition

Eukarya translation termination requires the stop codon recognizing protein eRF1. In contrast to the multiple proteins required for translation termination in Bacteria, eRF1 retains the ability to recognize all three of the stop codons. The details of the mechanism that eRF1 uses to recognize stop codons has remained elusive. This study describes the structural effects of mutations in the eRF1 N-domain that have previously been shown to alter stop codon recognition specificity. Here, we propose a model of eRF1 binding to the pre-translation termination ribosomal complex that is based in part on our solution NMR structures of the wild-type and mutant eRF1 N-domains. Since structural perturbations induced by these mutations were spread throughout the protein structure, residual dipolar coupling (RDC) data were recorded to establish the long-range effects of the specific mutations, E55Q, Y125F, Q122FM(Y)F126. RDCs were recorded on 15N-labeled eRF1 N-domain weakly aligned in either 5% w/v n-octyl-penta (ethylene glycol)/octanol (C8E5) or the filamentous phage Pf1. These data indicate that the mutations alter the conformation and dynamics of the GTS loop that is distant from the mutation sites. We propose that the GTS loop forms a switch that is key for the multiple codon recognition capability of eRF1.Published versio

NMR structure and localization of a large fragment of the SARS-CoV fusion protein : implications in viral cell fusion

The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. 15N{1H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV.MOE (Min. of Education, S’pore)Accepted versio

Identification of a polyoxometalate inhibitor of the DNA binding activity of sox2

10.1021/cb100432xACS Chemical Biology66573-58