Fragment-Based Drug Discovery

Erlanson, Daniel A.; McDowell, Robert S.; O’Brien, Tom

doi:10.1021/jm040031v

Cited by 599 publications

(458 citation statements)

References 96 publications

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“…Although proteinprotein interfaces were originally thought to be difficult targets for drug development, recent advances in the understanding of structural constraints at interacting surfaces in combination with structural and mutational analysis has allowed for the development of small-molecule mimics of hot spots that have been found to inhibit protein-protein interactions. [45][46][47][48] In addition, effective modulation of protein-protein interactions has been described for smallmolecule binding to clefts or grooves at interface regions. 49, 50 It appears promising that the NHR2 hot spot is located around the largest indentation on the dimer surface.…”

Section: Discussionmentioning

confidence: 99%

Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity

Wichmann

Becker

Chen-Wichmann

et al. 2010

Blood

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RUNX1/ETO, the fusion protein resulting from the chromosomal translocation t(8;21), is one of the most frequent translocation products in acute myeloid leukemia. Several in vitro and in vivo studies have shown that the homo-tetramerization domain of ETO, the nervy homology region 2 (NHR2), is essential for RUNX1/ETO oncogenic activity. We analyzed the energetic contribution of individual amino acids within the NHR2 to RUNX1/ETO dimertetramer transition and found a clustered area of 5 distinct amino acids with strong contribution to the stability of tetramers. Substitution of these amino acids abolishes tetramer formation without affecting dimer formation. Similar to RUNX1/ ETO monomers, dimers failed to bind efficiently to DNA and to alter expression of RUNX1-dependent genes. RUNX1/ETO dimers do not block myeloid differentiation, are unable to enhance the selfrenewal capacity of hematopoietic progenitors, and fail to induce leukemia in a murine transplantation model. Our data reveal the existence of an essential structural motif (hot spot) at the NHR2 dimertetramer interface, suitable for a molecular intervention in t(8;21) leukemias. IntroductionChromosomal translocations are frequent events during malignant cell transformation, particularly during leukemogenesis. 1 The translocation t(8;21), one of the most frequent chromosomal anomalies in acute myeloid leukemia (AML), involves the RUNX1 gene (also known as AML1, CBF␣2, or PEBP2␣B) on chromosome 21 and the ETO gene (also known as MTG8 or RUNX1T1) on chromosome 8. The ubiquitously expressed RUNX1 is a transcription factor and belongs to the key regulators of hematopoietic cell differentiation. 2 The fusion protein RUNX1/ETO contains the DNA-binding domain (Runt, RHD) of the RUNX1 transcription factor but lacks the C-terminal transactivation sequence that is replaced by almost the entire ETO protein. 2 forms of RUNX1/ETO coexist in AML-leukemia samples: the originally discovered full-length RUNX1/ETO and a splice variant called RUNX1/ETO9a, which lacks 178 amino acids at the C-terminus. Only RUNX1/ETO9a does not require cooperative events for inducing leukemia development in mice. 3,4 We and others have shown that RUNX1/ETO has a modular structure. Besides the Runt domain, RUNX1/ETO contains 4 functional domains, which are generally referred to as nervy homology region (NHR1 to NHR4). The NHR domains serve as docking interface for a variety of different proteins, including the E-protein HEB, 5,6 the apoptosis-related protein SON, 7 and nuclear corepressor proteins, such as N-CoR, SMRT, mSIN3A, and MTGR1, as well as histone deacetylases (HDACs). [8][9][10][11] In addition, the NHR2 domain mediates tetramer formation through hydrophobic and ionic/polar interactions. Two ␣-helices align in a head-to-tail fashion to form an antiparallel dimer. Two dimers subsequently are positioned on top of each other in a sandwich-like fashion. The total interaction area composing all contact points of the 4 ␣-helices is approximately 10 000 Å. 2 Substitution of 7 leucines withi...

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Section: Discussionmentioning

confidence: 99%

Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity

Wichmann

Becker

Chen-Wichmann

et al. 2010

Blood

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“…F ragment-based development of protein ligands has been recognized over the last years as a powerful strategy to identify molecules with an optimized fit to protein-binding pockets, providing ligands with reduced molecular weight and with higher ligand efficiency, thereby possibly improving the bioavailability, cell permeability and metabolic stability of drug molecules [1][2][3][4][5][6][7][8] . Scrutinizing the chemical space of small molecule fragments-instead of screening large libraries of drug-like molecules-requires considerably smaller chemical libraries for covering the chemical space and thus enables to identify and optimize hit ligands with less effort for testing and chemical synthesis.…”

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confidence: 99%

Catalytic activation of pre-substrates via dynamic fragment assembly on protein templates

Burda

Rademann

2014

Nat Commun

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Sensitive detection of small molecule fragments binding to defined sites of biomacromolecules is still a considerable challenge. Here we demonstrate that protein-binding fragments are able to induce enzymatic reactions on the protein surface via dynamic fragment ligation. Fragments binding to the S1 pocket of serine proteases containing a nitrogen, oxygen or sulphur nucleophile are found to activate electrophilic pre-substrates through a reversible, covalent ligation reaction. The dynamic ligation reaction positions the pre-substrate molecule at the active site of the protein thereby inducing its enzymatic cleavage. Catalytic activation of pre-substrates is confirmed by fluorescence spectroscopy and by high-performance liquid chromatography. The approach is investigated with 3 pre-substrates and 14 protein-binding fragments and the specific activation and the templating effect exerted by the enzyme is quantified for each protease-fragment-pre-substrate combination. The described approach enables the site-specific identification of protein-binding fragments, the functional characterization of enzymatic sites and the quantitative analysis of protein template-assisted ligation reactions.

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“…For the former, biophysical methods such as X-ray crystallography 26 , NMR spectroscopy 25, 27, 30 , and surface plasmon resonance 38 have been used to design and synthesize high-affinity ligands, based on fragments with good binding properties 25,31,32,[35][36][37] . Some compounds identified using fragment-based approaches have entered clinical trials 36 , and fragment based discovery can identify quality leads for targets where HTS has not succeeded 31, 32, 39 .…”

Section: Introductionmentioning

confidence: 99%

Novel inhibitors of anthrax edema factor

Chen

Misra

Sower

et al. 2008

Bioorganic & Medicinal Chemistry

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Several pathogenic bacteria produce adenylyl cyclase toxins, such as the edema factor (EF) of Bacillus anthracis. These disturb cellular metabolism by catalyzing production of excessive amounts of the regulatory molecule cAMP. Here, a structure-based method, where a 3D-pharmacophore that fit the active site of EF was constructed from fragments, was used to identify non-nucleotide inhibitors of EF. A library of small molecule fragments was docked to the EF-active site in existing crystal structures and those with the highest HINT scores were assembled into a 3D-pharmacophore. About 10,000 compounds, from over 2.7 million compounds in the ZINC database, had a similar molecular framework. These were ranked according to their docking scores, using methodology that was shown to achieve maximum accuracy (i.e., how well the docked position matched the experimentally determined site for ATP analogues in crystal structures of the complex). Finally, 19 diverse compounds with the best AutoDock binding/docking scores were assayed in a cell based assay for their ability to reduce cAMP secretion induced by EF. Four of the test compounds, from different structural groups, inhibited in the low micromolar range. One of these has a core structure common to phosphatase inhibitors previously identified by high-throughput assays of a diversity library. Thus, the fragment based pharmacophore identified a small number of diverse compounds for assay, and greatly enhanced the selection process of advanced lead compounds for combinatorial design.

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Fragment-Based Drug Discovery

Cited by 599 publications

References 96 publications

Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity

Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity

Catalytic activation of pre-substrates via dynamic fragment assembly on protein templates

Novel inhibitors of anthrax edema factor

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