-
Milan Bruncko,
Stephen K Tahir,
Xiaohong Song,
Jun Chen,
Hong Ding, Jeffrey R Huth,
Sha Jin,
Russell A Judge,
David J Madar,
Chang H Park,
Cheol-Min Park,
Andrew M Petros,
Christin Tse,
Saul H Rosenberg,
Steven W Elmore
[show abstract]
[hide abstract]
ABSTRACT: We describe the development of a novel series of N-aryl-benzimidazolone HSP90 inhibitors (9) targeting the N-terminal ATP-ase site. SAR development was influenced by structure-based design based around X-ray structures of ligand bound HSP90 complexes. Lead compounds exhibited high binding affinities, ATP-ase inhibition and cellular client protein degradation.
Bioorganic & medicinal chemistry letters 12/2010; 20(24):7503-6. · 2.65 Impact Factor
-
Andrew M Petros, Jeffrey R Huth,
Thorsten Oost,
Cheol-Min Park,
Hong Ding,
Xilu Wang,
Haichao Zhang,
Paul Nimmer,
Renaldo Mendoza,
Chaohong Sun,
Jamey Mack,
Karl Walter,
Sarah Dorwin,
Emily Gramling,
Uri Ladror,
Saul H Rosenberg,
Steven W Elmore,
Stephen W Fesik,
Philip J Hajduk
[show abstract]
[hide abstract]
ABSTRACT: The Bcl-2 family of proteins plays a major role in the regulation of apoptosis, or programmed cell death. Overexpression of the anti-apoptotic members of this family (Bcl-2, Bcl-x(L), and Mcl-1) can render cancer cells resistant to chemotherapeutic agents and therefore these proteins are important targets for the development of new anti-cancer agents. Here we describe the discovery of a potent, highly selective, Bcl-2 inhibitor using SAR by NMR and structure-based drug design which could serve as a starting point for the development of a Bcl-2 selective anti-cancer agent. Such an agent would potentially overcome the Bcl-x(L) mediated thrombocytopenia observed with ABT-263.
Bioorganic & medicinal chemistry letters 11/2010; 20(22):6587-91. · 2.65 Impact Factor
-
Kent D Stewart, Jeffrey R Huth,
Teresa I Ng,
Keith McDaniel,
Rebecca Newlin Hutchinson,
Vincent S Stoll,
Renaldo R Mendoza,
Edmund D Matayoshi,
Robert Carrick,
Hongmei Mo, [......],
Karl Walter,
Paul L Richardson,
Leo W Barrett,
Robert Meadows,
Steve Anderson,
William Kohlbrenner,
Clarence Maring,
Dale J Kempf,
Akhter Molla,
Edward T Olejniczak
[show abstract]
[hide abstract]
ABSTRACT: The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide(1) is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3-41microM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.
Bioorganic & medicinal chemistry letters 11/2009; 20(2):612-7. · 2.65 Impact Factor
-
Jun Chen,
Xu-Feng Zhang,
Michael E Kort, Jeffrey R Huth,
Chaohong Sun,
Laura J Miesbauer,
Steven C Cassar,
Torben Neelands,
Victoria E Scott,
Robert B Moreland,
Regina M Reilly,
Philip J Hajduk,
Philip R Kym,
Charles W Hutchins,
Connie R Faltynek
[show abstract]
[hide abstract]
ABSTRACT: TRPA1 is an excitatory, nonselective cation channel implicated in somatosensory function, pain, and neurogenic inflammation. Through covalent modification of cysteine and lysine residues, TRPA1 can be activated by electrophilic compounds, including active ingredients of pungent natural products (e.g., allyl isothiocyanate), environmental irritants (e.g., acrolein), and endogenous ligands (4-hydroxynonenal). However, how covalent modification leads to channel opening is not understood. Here, we report that electrophilic, thioaminal-containing compounds [e.g., CMP1 (4-methyl-N-[2,2,2-trichloro-1-(4-nitro-phenylsulfanyl)-ethyl]-benzamide)] covalently modify cysteine residues but produce striking species-specific effects [i.e., activation of rat TRPA1 (rTRPA1) and blockade of human TRPA1 (hTRPA1) activation by reactive and nonreactive agonists]. Through characterizing rTRPA1 and hTRPA1 chimeric channels and point mutations, we identified several residues in the upper portion of the S6 transmembrane domains as critical determinants of the opposite channel gating: Ala-946 and Met-949 of rTRPA1 determine channel activation, whereas equivalent residues of hTRPA1 (Ser-943 and Ile-946) determine channel block. Furthermore, side-chain replacements at these critical residues profoundly affect channel function. Therefore, our findings reveal a molecular basis of species-specific channel gating and provide novel insights into how TRPA1 respond to stimuli.
Journal of Neuroscience 06/2008; 28(19):5063-71. · 7.11 Impact Factor
-
Vijaya Gracias,
Zhiqin Ji,
Irini Akritopoulou-Zanze,
Cele Abad-Zapatero, Jeffrey R Huth,
Danying Song,
Philip J Hajduk,
Eric F Johnson,
Keith B Glaser,
Patrick A Marcotte,
Lori Pease,
Nirupama B Soni,
Kent D Stewart,
Steven K Davidsen,
Michael R Michaelides,
Stevan W Djuric
[show abstract]
[hide abstract]
ABSTRACT: We report the discovery of the pyrimido-diazepine scaffolds as novel adenine mimics. Structure-based design led to the discovery of analogs with potent inhibitory activity against receptor tyrosine kinases, such as KDR, Flt3 and c-Kit. Compound 14 exhibited low nanomolar KDR enzymatic and cellular potencies (IC(50)=9 and 52 nM, respectively).
Bioorganic & medicinal chemistry letters 05/2008; 18(8):2691-5. · 2.65 Impact Factor
-
Jeffrey R Huth,
Danying Song,
Renaldo R Mendoza,
Candice L Black-Schaefer,
Jamey C Mack,
Sarah A Dorwin,
Uri S Ladror,
Jean M Severin,
Karl A Walter,
Diane M Bartley,
Philip J Hajduk
[show abstract]
[hide abstract]
ABSTRACT: We have recently reported on the development of a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) to detect reactive false positive hits from high-throughput screening, in which we observed a surprisingly large number of compounds that can oxidize or form covalent adducts with protein thiols groups. In the vast majority of these cases, the covalent interactions are largely nonspecific (e.g., affect many protein targets) and therefore unsuitable for drug development. However, certain thiol-reactive species do appear to inhibit the target of interest in a specific manner. The question then arises as to the potential toxicology risks of developing a drug that can react with protein thiol groups. Here, we report on the evaluation of a large set of ALARM-reactive and -nonreactive compounds against a panel of additional proteins (aldehyde dehydrogenase, superoxide dismutase, and three cytochrome P450 enzymes). It was observed that ALARM-reactive compounds have significantly increased risks of interacting with one or more of these enzymes in vitro. Thus, ALARM NMR seems to be a sensitive tool to rapidly identify compounds with an enhanced risk of producing side effects in humans, including alcohol intolerance, the formation of reactive oxygen species, and drug-drug interactions. In conjunction with other toxicology assays, ALARM NMR should be a valuable tool for prioritizing compounds for lead optimization and animal testing.
Chemical Research in Toxicology 01/2008; 20(12):1752-9. · 3.78 Impact Factor
-
Jeffrey R Huth,
Chang Park,
Andrew M Petros,
Aaron R Kunzer,
Michael D Wendt,
Xilu Wang,
Christopher L Lynch,
Jamey C Mack,
Kerry M Swift,
Russell A Judge, [......],
Stephen K Tahir,
Edward D Matayoshi,
Sarah A Dorwin,
Uri S Ladror,
Jean M Severin,
Karl A Walter,
Diane M Bartley,
Stephen W Fesik,
Steven W Elmore,
Philip J Hajduk
[show abstract]
[hide abstract]
ABSTRACT: The molecular chaperone HSP90 has been shown to facilitate cancer cell survival by stabilizing key proteins responsible for a malignant phenotype. We report here the results of parallel fragment-based drug design approaches in the design of novel HSP90 inhibitors. Initial aminopyrimidine leads were elaborated using high-throughput organic synthesis to yield nanomolar inhibitors of the enzyme. Second site leads were also identified which bound to HSP90 in two distinct conformations, an 'open' and 'closed' form. Intriguingly, linked fragment approaches targeting both of these conformations were successful in producing novel, micromolar inhibitors. Overall, this study shows that, with only a few fragment hits, multiple lead series can be generated for HSP90 due to the inherent flexibility of the active site. Thus, ample opportunities exist to use these lead series in the development of clinically useful HSP90 inhibitors for the treatment of cancers.
Chemical Biology & Drug Design 08/2007; 70(1):1-12. · 2.28 Impact Factor
-
08/2006: pages 181 - 192; , ISBN: 9783527608768
-
[show abstract]
[hide abstract]
ABSTRACT: We report the synthesis of kinase targeted libraries based on the thienopyrazole scaffold. Several thienopyrazole analogs have been identified as submicromolar inhibitors of KDR.
Bioorganic & Medicinal Chemistry Letters 02/2006; 16(1):96-9. · 2.55 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The ability to predict whether a particular protein can bind with high affinity and specificity to small, drug-like compounds based solely on its 3D structure has been a longstanding goal of structural biologists and computational scientists. The promise is that an accurate prediction of protein druggability can capitalize on the huge investments already made in structural genomics initiatives by identifying highly druggable proteins and using this information in target identification and validation campaigns. Here we discuss the potential utility of tools that characterize protein targets and describe strategies for the optimal integration of protein druggability data with bioinformatic approaches to target selection.
Drug Discovery Today 01/2006; 10(23-24):1675-82. · 6.83 Impact Factor
-
ChemBioChem 10/2005; 6(9):1592-600. · 3.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An analysis of heteronuclear-NMR-based screening data is used to derive relationships between the ability of small molecules to bind to a protein and various parameters that describe the protein binding site. It is found that a simple model including terms for polar and apolar surface area, surface complexity, and pocket dimensions accurately predicts the experimental screening hit rates with an R(2) of 0.72, an adjusted R(2) of 0.65, and a leave-one-out Q(2) of 0.56. Application of the model to predict the druggability of protein targets not used in the training set correctly classified 94% of the proteins for which high-affinity, noncovalent, druglike leads have been reported. In addition to understanding the pocket characteristics that contribute to high-affinity binding, the relationships that have been defined allow for quantitative comparative analyses of protein binding sites for use in target assessment and validation, virtual ligand screening, and structure-based drug design.
Journal of Medicinal Chemistry 05/2005; 48(7):2518-25. · 5.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: High-throughput screening (HTS) of large compound collections typically results in numerous small molecule hits that must be carefully evaluated to identify valid drug leads. Although several filtering mechanisms and other tools exist that can assist the chemist in this process, it is often the case that costly synthetic resources are expended in pursuing false positives. We report here a rapid and reliable NMR-based method for identifying reactive false positives including those that oxidize or alkylate a protein target. Importantly, the reactive species need not be the parent compound, as both reactive impurities and breakdown products can be detected. The assay is called ALARM NMR (a La assay to detect reactive molecules by nuclear magnetic resonance) and is based on monitoring DTT-dependent (13)C chemical shift changes of the human La antigen in the presence of a test compound or mixture. Extensive validation has been performed to demonstrate the reliability and utility of using ALARM NMR to assess thiol reactivity. This included comparing ALARM NMR to a glutathione-based fluorescence assay, as well as testing a collection of more than 3500 compounds containing HTS hits from 23 drug targets. The data show that current in silico filtering tools fail to identify more than half of the compounds that can act via reactive mechanisms. Significantly, we show how ALARM NMR data has been critical in identifying reactive compounds that would otherwise have been prioritized for lead optimization. In addition, a new filtering tool has been developed on the basis of the ALARM NMR data that can augment current in silico programs for identifying nuisance compounds and improving the process of hit triage.
Journal of the American Chemical Society 02/2005; 127(1):217-24. · 9.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The advent of large-scale NMR-based screening has enabled new strategies for the design of novel, potent inhibitors of therapeutic targets. In particular, fragment-based strategies, in which molecular portions of the final high-affinity ligand are experimentally identified prior to chemical synthesis, have found widespread utility. This chapter will discuss some of the practical considerations for identifying and utilizing these fragment leads in drug design, with special emphasis on some of the lessons learned from more than a decade of industry experience.
Methods in Enzymology 02/2005; 394:549-71. · 2.04 Impact Factor
-
Jeffrey R Huth,
Liping Yu,
Irene Collins,
Jamey Mack,
Renaldo Mendoza,
Binumol Isaac,
Demetrios T Braddock,
Steven W Muchmore,
Kenneth M Comess,
Stephen W Fesik,
G Marius Clore,
David Levens,
Philip J Hajduk
[show abstract]
[hide abstract]
ABSTRACT: Reversal of aberrant gene expression that is induced by the proto-oncogene c-myc is likely to be effective for treating a variety of tumors that rely on this pathway for growth. One strategy to down-regulate the c-myc pathway is to target transcription factors that regulate its own expression. A host of proteins act in coordination to regulate c-myc expression and any one of them are theoretical targets for small-molecule therapy. Experimentally, it has been shown that the far upstream element (FUSE) binding protein (FBP) is essential for c-myc expression, and reductions in FBP levels both reduce c-myc expression and correlate with slower cell growth. FBP binds to ssDNA by capturing exposed DNA bases in a hydrophobic pocket. This suggests that a small molecule could be designed to occupy this pocket and inhibit FBP function. Using a variety of screening methodologies, we have identified ligands that bind to the DNA binding pockets of the KH domains of FBP. Gel shift analyses using full length FBP and a related transcription factor confirm that a small-molecule lead compound inhibits DNA binding in a specific manner. The benzoylanthranilic acid compounds described here represent leads in the design of FBP inhibitors that can serve as useful tools in the study of c-myc regulation and in the development of therapeutics that target the c-myc pathway.
Journal of Medicinal Chemistry 10/2004; 47(20):4851-7. · 5.25 Impact Factor
-
Jie Qian,
Martin J Voorbach, Jeffrey R Huth,
Michael L Coen,
Haichao Zhang,
Shi-Chung Ng,
Kenneth M Comess,
Andrew M Petros,
Saul H Rosenberg,
Usha Warrior,
David J Burns
[show abstract]
[hide abstract]
ABSTRACT: Bcl-xL is a member of the Bcl-2 family of proteins that are implicated to play a vital role in several diseases including cancer. Bcl-xL suppresses apoptosis; thus the inhibition of Bcl-xL function could restore the apoptotic process. To identify antagonists of Bcl-xL function, two ultra-high-throughput screens were implemented. An activity assay utilized fluorescence polarization, based on the binding of fluorescein-labeled peptide [the BH3 domain of BAD protein (F-Bad 6)] to Bcl-xL. A 384-well plate assay with mixtures of 10 drug compounds per well, combined with a fast plate reader, resulted in a throughput of 46,080 data points/day. Utilizing this screening format, 370,400 compounds were screened in duplicate and 425 inhibitors with an IC(50) below 100 microM were identified. The second assay format, affinity selection/mass spectrometry (ASMS), used ultrafiltration to separate Bcl-xL binders from nonbinders in mixtures of 2400 compounds. The bound species were subsequently separated from the protein and analyzed by flow injection electrospray mass spectrometry. Utilizing the ASMS format, 263,382 compounds were screened in duplicate and 29 binders with affinities below 100 microM were identified. Two novel classes of Bcl-xL inhibitors were identified by both methods and confirmed to bind (13)C-labeled Bcl-xL using heteronuclear magnetic resonance spectroscopy.
Analytical Biochemistry 06/2004; 328(2):131-8. · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: NMR has proven to be a valuable tool for identifying small molecule drug leads that serve as starting points for lead optimization programs. In addition, NMR screening can also be applied during lead optimization in order to improve the pharmacokinetic properties of a compound. In this paper we review the NMR methods that can be used for this purpose. Several examples are then summarized to demonstrate the usefulness of fragment-based approaches in optimizing the physical properties of potential drug candidates.
Combinatorial Chemistry & High Throughput Screening 01/2003; 5(8):631-43. · 1.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling
in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state
that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1
(CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal α-helix. We
report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1.
We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a
cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding.
A second cluster of residues distal to the MIDAS that included the C-terminal α-helix of the CD11a I domain was also affected.
Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1.
Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding
interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate
that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked
to the MIDAS.
Proceedings of the National Academy of Sciences 05/2000; 97(10):5231-5236. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic analysis of the fusion protein by 1H-15N correlation spectroscopy. Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, 15-labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using 15N-labeled GB1-peptide fusions and unlabeled target.
Protein Science 10/1997; 6(11):2359 - 2364. · 2.80 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mammalian male sex determination is controlled by a complex hierarchyof gene regulatory proteins and hormones, which promote male gonadal development and regression of the female primordia. At the core of this pathway lies the SRY protein, the master developmental switch for testicular differentiation and hence, the male sex. The three-dimensional structure of the SRY-DNA complex suggests a model of developmental gene regulation in which proteins that alter DNA structure and promote the assembly of higher-order nucleoprotein complexes play an essential role in the timing of cell specialization events.
Trends in Biochemical Sciences.
