Sameh H. Soror

Probing the N-terminal β-sheet conversion in the crystal structure of the human prion protein bound to a nanobody.

Prions are fatal neurodegenerative transmissible agents causing several incurable illnesses in humans and animals. Prion diseases are caused by the structural conversion of the cellular prion protein, PrP(C), into its misfolded oligomeric form, known as prion or PrP(Sc). The canonical human PrP(C) (HuPrP) fold features an unstructured N-terminal part (residues 23-124) and a well-defined C-terminal globular domain (residues 125-231). Compelling evidence indicates that an evolutionary N-terminal conserved motif AGAAAAGA (residues 113-120) plays an important role in the conversion to PrP(Sc). The intrinsic flexibility of the N-terminal has hampered efforts to obtain detailed atomic information on the structural features of this palindromic region. In this study, we crystallized the full-length HuPrP in complex with a nanobody (Nb484) that inhibits prion propagation. In the complex, the prion protein is unstructured from residue 23 to 116. The palindromic motif adopts a stable and fully extended configuration to form a three-stranded antiparallel β-sheet with the β1 and β2 strands, demonstrating that the full-length HuPrP(C) can adopt a more elaborate β0-β1-α1-β2-α2-α3 structural organization than the canonical β1-α1-β2-α2-α3 prion-like fold. From this structure, it appears that the palindromic motif mediates β-enrichment in the PrP(C) monomer as one of the early events in the conversion of PrP(C) into PrP(Sc).

Ethionamide Boosters. 2. Combining Bioisosteric Replacement and Structure-Based Drug Design To Solve Pharmacokinetic Issues in a Series of Potent 1,2,4-Oxadiazole EthR Inhibitors

Mycobacterial transcriptional repressor EthR controls the expression of EthA, the bacterial monooxygenase activating ethionamide, and is thus largely responsible for the low sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. We recently reported structure-activity relationships of a series of 1,2,4-oxadiazole EthR inhibitors leading to the discovery of potent ethionamide boosters. Despite high metabolic stability, pharmacokinetic evaluation revealed poor mice exposure; therefore, a second phase of optimization was required. Herein a structure-property relationship study is reported according to the replacement of the two aromatic heterocycles: 2-thienyl and 1,2,4-oxadiazolyl moieties. This work was done using a combination of structure-based drug design and in vitro/ex vivo evaluations of ethionamide boosters on the targeted protein EthR and on the human pathogen Mycobacterium tuberculosis. Thanks to this process, we identified compound 42 (BDM41906), which displays improved efficacy in addition to high exposure to mice after oral administration.

Ligand Efficiency Driven Design of New Inhibitors of Mycobacterium tuberculosis Transcriptional Repressor EthR Using Fragment Growing, Merging, and Linking Approaches.

Tuberculosis remains a major cause of mortality and morbidity, killing each year more than one million people. Although the combined use of first line antibiotics (isoniazid, rifampicin, pyrazinamide, and ethambutol) is efficient to treat most patients, the rapid emergence of multidrug resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. Mycobacterial transcriptional repressor EthR is a key player in the control of second-line drugs bioactivation such as ethionamide and has been shown to impair the sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. As a way to identify new potent ligands of this protein, we have developed fragment-based approaches. In the current study, we combined surface plasmon resonance assay, X-ray crystallography, and ligand efficiency driven design for the rapid discovery and optimization of new chemotypes of EthR ligands starting from a fragment. The design, synthesis, and in vitro and ex vivo activities of these compounds will be discussed.

A novel expression system for production of soluble prion proteins in E. coli

Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies.

Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors.

Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes

Prion proteins (PrPs) are difficult to crystallize, probably due to their inherent flexibility. Several PrPs structures have been solved by nuclear magnetic resonance (NMR) techniques; however, only three structures were solved by X-ray crystallography. Here we combined in-situ proteolysis with automated microseed matrix screening (MMS) to crystallize two different PrP(C)-nanobody (Nb) complexes. Nanobodies are single-domain antibodies derived from heavy-chain-only antibodies of camelids. Initial crystallization screening conditions using in-situ proteolysis of mouse prion (23-230) in complex with a nanobody (Nb_PrP_01) gave thin needle aggregates, which were of poor diffraction quality. Next, we used these microcrystals as nucleants for automated MMS. Good-quality crystals were obtained from mouse PrP (89-230)/Nb_PrP_01, belonged to the monoclinic space group P 1 21 1, with unit-cell parameters a = 59.13, b = 63.80, c = 69.79 Å, β = 101.96° and diffracted to 2.1 Å resolution using synchrotron radiation. Human PrP (90-231)/Nb_PrP_01 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 131.86, b = 45.78, c = 45.09 Å, β = 96.23° and diffracted to 1.5 Å resolution. This combined strategy benefits from the power of the MMS technique without suffering from the drawbacks of the in-situ proteolysis. It proved to be a successful strategy to crystallize PrP-nanobodies complexes and could be exploited for the crystallization of other difficult antigen-antibody complexes.

Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.

Here we present the preparation, biophysical characterization, and nuclear magnetic resonance (NMR) spectroscopy study of yeast cytochrome c peroxidase (CcP) constructs with enhanced solubility. Using a high-yield Escherichia coli expression system, we routinely produced uniformly labeled [(2)H,(13)C,(15)N]CcP samples with high levels of deuterium incorporation (96-99%) and good yields (30-60 mg of pure protein from 1 L of bacterial culture). In addition to simplifying the purification procedure, introduction of a His tag at either protein terminus dramatically increases its solubility, allowing preparation of concentrated, stable CcP samples required for multidimensional NMR spectroscopy. Using a range of biophysical techniques and X-ray crystallography, we demonstrate that the engineered His tags neither perturb the structure of the enzyme nor alter the heme environment or its reactivity toward known ligands. The His-tagged CcP constructs remain catalytically active yet exhibit differences in the interaction with cytochrome c, the physiological binding partner, most likely because of steric occlusion of the high-affinity binding site by the C-terminal His tag. We show that protein perdeuteration greatly increases the quality of the double- and triple-resonance NMR spectra, allowing nearly complete backbone resonance assignments and subsequent study of the CcP by heteronuclear NMR spectroscopy.

High-resolution structure of a papaya plant-defence barwin-like protein solved by in-house sulfur-SAD phasing

The first crystal structure of a barwin-like protein, named carwin, has been determined at high resolution by single-wavelength anomalous diffraction (SAD) phasing using the six intrinsic S atoms present in the protein. The barwin-like protein was purified from Carica papaya latex and crystallized in the orthorhombic space group P212121. Using in-house Cu Kα X-ray radiation, 16 cumulative diffraction data sets were acquired to increase the signal-to-noise level and thereby the anomalous scattering signal. A sequence-database search on the papaya genome identified two carwin isoforms of 122 residues in length, both containing six S atoms that yield an estimated Bijvoet ratio of 0.93% at 1.54 Å wavelength. A systematic analysis of data quality and redundancy was performed to assess the capacity to locate the S atoms and to phase the data. It was observed that the crystal decay was low during data collection and that successful S-SAD phasing could be obtained with a relatively low data multiplicity of about 7. Using a synchrotron source, high-resolution data (1 Å) were collected from two different crystal forms of the papaya latex carwin. The refined structures showed a central β-barrel of six strands surrounded by several α-helices and loops. The β-barrel of carwin appears to be a common structural module that is shared within several other unrelated proteins. Finally, the possible biological function of the protein is discussed.

Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein.

Prion disorders are infectious diseases that are characterized by the conversion of the cellular prion protein PrPC into the pathogenic isoform PrPSc. Specific antibodies that interact with the cellular prion protein have been shown to inhibit this transition. Recombinant VHHs (variable domain of dromedary heavy-chain antibodies) or nanobodies are single-domain antibodies, making them the smallest antigen-binding fragments. A specific nanobody (Nb_PrP_01) was raised against mouse PrPC. A crystallization condition for this recombinant nanobody was identified using high-throughput screening. The crystals were optimized using streak-seeding and the hanging-drop method. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=30.04, b=37.15, c=83.00 Å, and diffracted to 1.23 Å resolution using synchrotron radiation. The crystal structure of this specific nanobody against PrPC together with the known PrPC structure may help in understanding the PrPC/PrPSc transition mechanism.

Discovery, Structure–Activity Relationship, and Binding Mode of an Imidazo[1,2-a]pyridine Series of Autotaxin Inhibitors

Autotaxin (ATX) is a secreted enzyme playing a major role in the production of lysophosphatidic acid (LPA) in blood through hydrolysis of lysophosphatidyl choline (LPC). The ATX-LPA signaling axis arouses a high interest in the drug discovery industry as it has been implicated in several diseases including cancer, fibrotic diseases, and inflammation, among others. An imidazo[1,2-a]pyridine series of ATX inhibitors was identified out of a high-throughput screening (HTS). A cocrystal structure with one of these compounds and ATX revealed a novel binding mode with occupancy of the hydrophobic pocket and channel of ATX but no interaction with zinc ions of the catalytic site. Exploration of the structure-activity relationship led to compounds displaying high activity in biochemical and plasma assays, exemplified by compound 40. Compound 40 was also able to decrease the plasma LPA levels upon oral administration to rats.