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1.
Angew Chem Int Ed Engl ; 56(7): 1908-1913, 2017 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-28097765

RESUMO

With the rising popularity of fragment-based approaches in drug development, more and more attention has to be devoted to the detection of false-positive screening results. In particular, the small size and low affinity of fragments drives screening techniques to their limit. The pursuit of a false-positive hit can cause significant loss of time and resources. Here, we present an instructive and intriguing investigation into the origin of misleading assay results for a fragment that emerged as the most potent binder for the aspartic protease endothiapepsin (EP) across multiple screening assays. This molecule shows its biological effect mainly after conversion into another entity through a reaction cascade that involves major rearrangements of its heterocyclic scaffold. The formed ligand binds EP through an induced-fit mechanism involving remarkable electrostatic interactions. Structural information in the initial screening proved to be crucial for the identification of this false-positive hit.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Desenho de Fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sordariales/enzimologia , Ácido Aspártico Endopeptidases/química , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica
2.
J Med Chem ; 59(21): 9743-9759, 2016 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-27726357

RESUMO

Crystallography is frequently used as follow-up method to validate hits identified by biophysical screening cascades. The capacity of crystallography to directly screen fragment libraries is often underestimated, due to its supposed low-throughput and need for high-quality crystals. We applied crystallographic fragment screening to map the protein-binding site of the aspartic protease endothiapepsin by individual soaking experiments. Here, we report on 41 fragments binding to the catalytic dyad and adjacent specificity pockets. The analysis identifies already known warheads but also reveals hydrazide, pyrazole, or carboxylic acid fragments as novel functional groups binding to the dyad. A remarkable swapping of the S1 and S1' pocket between structurally related fragments is explained by either steric demand, required displacement of a well-bound water molecule, or changes of trigonal-planar to tetrahedral geometry of an oxygen functional group in a side chain. Some warheads simultaneously occupying both S1 and S1' are promising starting points for fragment-growing strategies.


Assuntos
Ácido Aspártico Proteases/química , Ácido Aspártico Proteases/metabolismo , Biocatálise , Ácidos Carboxílicos/metabolismo , Hidrazinas/metabolismo , Pirazóis/metabolismo , Calorimetria , Ácidos Carboxílicos/química , Domínio Catalítico , Cristalografia por Raios X , Hidrazinas/química , Modelos Moleculares , Pirazóis/química
3.
Angew Chem Int Ed Engl ; 55(32): 9422-6, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27400756

RESUMO

Fragment-based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit-identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X-ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis-acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240-fold improvement in potency compared to the parent hits. Subsequent X-ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit-identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit-to-lead optimization.


Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Técnicas de Química Combinatória , Inibidores de Proteases/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Desenho de Fármacos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-Atividade
4.
Structure ; 24(8): 1398-1409, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27452405

RESUMO

Today the identification of lead structures for drug development often starts from small fragment-like molecules raising the chances to find compounds that successfully pass clinical trials. At the heart of the screening for fragments binding to a specific target, crystallography delivers structural information essential for subsequent drug design. While it is common to search for bound ligands in electron densities calculated directly after an initial refinement cycle, we raise the important question whether this strategy is viable for fragments characterized by low affinities. Here, we describe and provide a collection of high-quality diffraction data obtained from 364 protein crystals treated with diverse fragments. Subsequent data analysis showed that ∼25% of all hits would have been missed without further refining the resulting structures. To enable fast and reliable hit identification, we have designed an automated refinement pipeline that will inspire the development of optimized tools facilitating the successful application of fragment-based methods.


Assuntos
Cristalografia por Raios X/estatística & dados numéricos , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas/química , Água/química , Cristalografia por Raios X/métodos , Conjuntos de Dados como Assunto , Desenho de Fármacos , Humanos , Difração de Raios X
5.
J Med Chem ; 59(16): 7561-75, 2016 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-27463859

RESUMO

Successful optimization of a given lead scaffold requires thorough binding-site mapping of the target protein particular in regions remote from the catalytic center where high conservation across protein families is given. We screened a 361-entry fragment library for binding to the aspartic protease endothiapepsin by crystallography. This enzyme is frequently used as a surrogate for the design of renin and ß-secretase inhibitors. A hit rate of 20% was achieved, providing 71 crystal structures. Here, we discuss 45 binding poses of fragments accommodated in pockets remote from the catalytic dyad. Three major hot spots are discovered in remote binding areas: Asp81, Asp119, and Phe291. Compared to the dyad binders, bulkier fragments occupy these regions. Many of the discovered fragments suggest an optimization concept on how to grow them into larger ligands occupying adjacent binding pockets that will possibly endow them with the desired selectivity for one given member of a protein family.


Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Biocatálise , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Inibidores de Proteases/química , Relação Estrutura-Atividade
6.
ACS Chem Biol ; 11(6): 1693-701, 2016 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-27028906

RESUMO

Fragment-based lead discovery (FBLD) has become a pillar in drug development. Typical applications of this method comprise at least two biophysical screens as prefilter and a follow-up crystallographic experiment on a subset of fragments. Clearly, structural information is pivotal in FBLD, but a key question is whether such a screening cascade strategy will retrieve the majority of fragment-bound structures. We therefore set out to screen 361 fragments for binding to endothiapepsin, a representative of the challenging group of aspartic proteases, employing six screening techniques and crystallography in parallel. Crystallography resulted in the very high number of 71 structures. Yet alarmingly, 44% of these hits were not detected by any biophysical screening approach. Moreover, any screening cascade, building on the results from two or more screening methods, would have failed to predict at least 73% of these hits. We thus conclude that, at least in the present case, the frequently applied biophysical prescreening filters deteriorate the number of possible X-ray hits while only the immediate use of crystallography enables exhaustive retrieval of a maximum of fragment structures, which represent a rich source guiding hit-to-lead-to-drug evolution.


Assuntos
Descoberta de Drogas/métodos , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Biofísica , Calorimetria , Cristalografia por Raios X , Bases de Dados de Compostos Químicos , Modelos Moleculares , Inibidores de Proteases/química , Ligação Proteica , Relação Estrutura-Atividade
7.
Int J Mol Sci ; 16(8): 19184-94, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26287174

RESUMO

Aspartic proteases are a class of enzymes that play a causative role in numerous diseases such as malaria (plasmepsins), Alzheimer's disease (ß-secretase), fungal infections (secreted aspartic proteases), and hypertension (renin). We have chosen endothiapepsin as a model enzyme of this class of enzymes, for the design, preparation and biochemical evaluation of a new series of inhibitors of endothiapepsin. Here, we have optimized a hit, identified by de novo structure-based drug design (SBDD) and DCC, by using structure-based design approaches focusing on the optimization of an amide-π interaction. Biochemical results are in agreement with SBDD. These results will provide useful insights for future structure-based optimization of inhibitors for the real drug targets as well as insights into molecular recognition.


Assuntos
Ascomicetos/enzimologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Desenho de Fármacos , Hidrazonas/química , Hidrazonas/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Acilação , Ascomicetos/efeitos dos fármacos , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Micoses/tratamento farmacológico , Micoses/microbiologia , Relação Estrutura-Atividade
8.
ChemMedChem ; 10(9): 1511-21, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26259992

RESUMO

Fragment-based lead discovery is gaining momentum in drug development. Typically, a hierarchical cascade of several screening techniques is consulted to identify fragment hits which are then analyzed by crystallography. Because crystal structures with bound fragments are essential for the subsequent hit-to-lead-to-drug optimization, the screening process should distinguish reliably between binders and non-binders. We therefore investigated whether different screening methods would reveal similar collections of putative binders. First we used a biochemical assay to identify fragments that bind to endothiapepsin, a surrogate for disease-relevant aspartic proteases. In a comprehensive screening approach, we then evaluated our 361-entry library by using a reporter-displacement assay, saturation-transfer difference NMR, native mass spectrometry, thermophoresis, and a thermal shift assay. While the combined results of these screening methods retrieve 10 of the 11 crystal structures originally predicted by the biochemical assay, the mutual overlap of individual hit lists is surprisingly low, highlighting that each technique operates on different biophysical principles and conditions.


Assuntos
Bioquímica/métodos , Biofísica/métodos , Ensaios de Triagem em Larga Escala/métodos , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Descoberta de Drogas/métodos , Espectroscopia de Ressonância Magnética , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Espectrometria de Massas por Ionização por Electrospray/métodos
9.
Angew Chem Int Ed Engl ; 53(12): 3259-63, 2014 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-24532096

RESUMO

Structure-based design (SBD) can be used for the design and/or optimization of new inhibitors for a biological target. Whereas de novo SBD is rarely used, most reports on SBD are dealing with the optimization of an initial hit. Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify bioactive ligands given that it enables the target to direct the synthesis of its strongest binder. We have designed a library of potential inhibitors (acylhydrazones) generated from five aldehydes and five hydrazides and used DCC to identify the best binder(s). After addition of the aspartic protease endothiapepsin, we characterized the protein-bound library member(s) by saturation-transfer difference NMR spectroscopy. Cocrystallization experiments validated the predicted binding mode of the two most potent inhibitors, thus demonstrating that the combination of de novo SBD and DCC constitutes an efficient starting point for hit identification and optimization.


Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/síntese química , Técnicas de Química Combinatória/instrumentação , Técnicas de Química Combinatória/métodos , Desenho de Fármacos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Difração de Raios X
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