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ABSTRACT: To aid in the pursuit of selective kinase inhibitors, we have developed a unique ATP site binder tool for the detection of binders outside the ATP site by nuclear magnetic resonance (NMR). We report here the novel synthesis that led to this paramagnetic spin-labeled pyrazolopyrimidine probe (1), which exhibits nanomolar inhibitory activity against multiple kinases. We demonstrate the application of this probe by performing NMR binding experiments with Lck and Src kinases and utilize it to detect the binding of two compounds proximal to the ATP site. The complex structure of the probe with Lck is also presented, revealing how the probe fits in the ATP site and the specific interactions it has with the protein. We believe that this spin-labeled probe is a valuable tool that holds broad applicability in a screen for non-ATP site binders.
Journal of Medicinal Chemistry 02/2010; 53(3):1238-49. · 4.80 Impact Factor
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Alessandro Mascioni, Franklin J Moy,
Lisa K McNeil,
Ellen Murphy,
Breagh E Bentley,
Rosaria Camarda,
Deborah A Dilts,
Pamela S Fink,
Viktoria Gusarova,
Susan K Hoiseth,
Karl Malakian,
Terri Mininni,
Elena Novikova,
Shuo Lin,
Scott Sigethy,
Gary W Zlotnick,
Désirée H H Tsao
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ABSTRACT: Neisseria meningitidis is a major cause of meningitis. Although protective vaccination is available against some pathogenic serogroups, serogroup B meningococci have been a challenge for vaccinologists. A family of outer membrane lipoproteins, LP2086 (or factor H binding proteins, fHbp), has been shown to elicit bactericidal antibodies and is currently part of a cocktail vaccine candidate. The NMR structure of the variant LP2086-B01 in micellar solution provided insights on the topology of this family of proteins on the biological membrane. Based on flow cytometry experiments on whole meningococcal cells, binding experiments with monoclonal antibodies, and the NMR structure in micellar solution, we previously proposed that LP2086-B01 anchors the outer bacterial membrane through its lipidated N-terminal cysteine, while a flexible 20 residue linker positions the protein above the layer of lipo-oligosaccharides that surrounds the bacteria. This topology was suggested to increase the antigen exposure to the immune system. In the present work, using micellar solution as a membrane mimicking system, we characterized the backbone dynamics of the variant LP2086-B01 in both its lipidated and unlipidated forms. In addition, binding experiments with a Fab fragment derived from the monoclonal MN86-1042-2 were also performed. Our data suggests that due to the length and flexibility of the N-terminal linker, the antigen is not in contact with the micelle, thus making both N- and C-domains highly available to the host immune system. This dynamic model, combined with the binding data obtained with MN86-1042-2, supports our previously proposed arrangement that LP2086-B01 exposes one face to the extracellular space. Binding of MN86-1042-2 antibody shows that the N-domain is the primary target of this monoclonal, providing further indication that this domain is immunologically important for this family of proteins.
Biochimica et Biophysica Acta 10/2009; 1798(2):87-93. · 4.66 Impact Factor
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Guang-Yi Xu,
Wah-Tung Hum,
Steven F Sukits,
Chu-Lai Hsiao,
Yan Liu,
Karl Malakian,
Karen Monteiro,
Scott Wolfrom,
Yuren Wang,
Kathleen H Young, Franklin J Moy
Journal of Biomolecular NMR 05/2004; 28(4):409-10. · 3.61 Impact Factor
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Ariamala Gopalsamy,
Kitae Lim,
John W Ellingboe,
Boris Mitsner,
Antonia Nikitenko,
Janis Upeslacis,
Tarek S Mansour,
Matthew W Olson,
Geraldine A Bebernitz,
Diane Grinberg,
Boris Feld, Franklin J Moy,
John O'Connell
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ABSTRACT: Through high throughput screening of various libraries, substituted styryl naphthalene 6 was identified as an HCMV protease inhibitor. Optimization of various regions of the lead molecule using parallel synthesis resulted in 1,6-substituted naphthalenes 19d-i. These compounds displayed good potency and were selective over elastase, trypsin, and chymotrypsin. The optimization approach on lead compound 6 in three different regions of the molecule using parallel solution-phase synthesis and the corresponding SAR are discussed in detail.
Journal of Medicinal Chemistry 05/2004; 47(8):1893-9. · 5.25 Impact Factor
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Guang-Yi Xu,
Wah-Tung Hum,
Steven F. Sukits,
Chu-Lai Hsiao,
Yan Liu,
Karl Malakian,
Karen Monteiro,
Scott Wolfrom,
Yuren Wang,
Kathleen H. Young, Franklin J. Moy
Journal of Biomolecular NMR 03/2004; 28(4):409-410. · 3.61 Impact Factor
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ABSTRACT: Structure-based approaches for drug design generally do not incorporate solvent effects and dynamic information to predict inhibitor-binding affinity because of practical limitations. The matrix metalloproteinases (MMPs) have previously been demonstrated to exhibit significant mobility in their active sites. This dynamic characteristic significantly complicates the drug design process based on static structures, which was clearly observed for a class of hydroxamic acids containing a butynyl moiety. Compound 1 was expected to be selective against MMP-1 based on predicted steric clashes between the butynyl P1' group and the S1' pocket, but the observation of complex inhibitor dynamics in the NMR structure of MMP-1:1 provides an explanation for the low nanomolar binding to MMP-1.
Journal of the American Chemical Society 11/2002; 124(43):12658-9. · 9.91 Impact Factor
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Journal of Biomolecular NMR 04/1999; 15(4):339-340. · 3.61 Impact Factor
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Alessandro Mascioni, Franklin J. Moy,
Lisa K. McNeil,
Ellen Murphy,
Breagh E. Bentley,
Rosaria Camarda,
Deborah A. Dilts,
Pamela S. Fink,
Viktoria Gusarova,
Susan K. Hoiseth,
Karl Malakian,
Terri Mininni,
Elena Novikova,
Shuo Lin,
Scott Sigethy,
Gary W. Zlotnick,
Désirée H.H. Tsao
[show abstract]
[hide abstract]
ABSTRACT: Neisseria meningitidis is a major cause of meningitis. Although protective vaccination is available against some pathogenic serogroups, serogroup B meningococci have been a challenge for vaccinologists. A family of outer membrane lipoproteins, LP2086 (or factor H binding proteins, fHbp), has been shown to elicit bactericidal antibodies and is currently part of a cocktail vaccine candidate. The NMR structure of the variant LP2086-B01 in micellar solution provided insights on the topology of this family of proteins on the biological membrane. Based on flow cytometry experiments on whole meningococcal cells, binding experiments with monoclonal antibodies, and the NMR structure in micellar solution, we previously proposed that LP2086-B01 anchors the outer bacterial membrane through its lipidated N-terminal cysteine, while a flexible 20 residue linker positions the protein above the layer of lipo-oligosaccharides that surrounds the bacteria. This topology was suggested to increase the antigen exposure to the immune system. In the present work, using micellar solution as a membrane mimicking system, we characterized the backbone dynamics of the variant LP2086-B01 in both its lipidated and unlipidated forms. In addition, binding experiments with a Fab fragment derived from the monoclonal MN86-1042-2 were also performed. Our data suggests that due to the length and flexibility of the N-terminal linker, the antigen is not in contact with the micelle, thus making both N- and C-domains highly available to the host immune system. This dynamic model, combined with the binding data obtained with MN86-1042-2, supports our previously proposed arrangement that LP2086-B01 exposes one face to the extracellular space. Binding of MN86-1042-2 antibody shows that the N-domain is the primary target of this monoclonal, providing further indication that this domain is immunologically important for this family of proteins.
Biochimica et Biophysica Acta (BBA) - Biomembranes.
