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1.
SLAS Discov ; 29(1): 40-51, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37714432

RESUMO

Surface plasmon resonance (SPR) biosensor methods are ideally suited for fragment-based lead discovery.  However, generally applicable experimental procedures and detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are not available. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits. The range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded by adopting multiplexed strategies, using multiple complementary surfaces or experimental conditions. Here we illustrate principles and multiplexed approaches for using flow-based SPR biosensor systems for screening fragment libraries of different sizes (90 and 1056 compounds) against a selection of challenging targets. It shows strategies for the identification of fragments interacting with 1) large and structurally dynamic targets, represented by acetyl choline binding protein (AChBP), a Cys-loop receptor ligand gated ion channel homologue, 2) targets in multi protein complexes, represented by lysine demethylase 1 and a corepressor (LSD1/CoREST), 3) structurally variable or unstable targets, represented by farnesyl pyrophosphate synthase (FPPS), 4) targets containing intrinsically disordered regions, represented by protein tyrosine phosphatase 1B  (PTP1B), and 5) aggregation-prone proteins, represented by an engineered form of human tau  (tau K18M). Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted. The study shows that the challenges for addressing these types of targets is not identification of potentially useful fragments per se, but establishing methods for their validation and evolution into leads.


Assuntos
Técnicas Biossensoriais , Ressonância de Plasmônio de Superfície , Humanos , Ressonância de Plasmônio de Superfície/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas , Proteínas de Transporte
2.
Chembiochem ; 23(17): e202200178, 2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-35767695

RESUMO

The development of protein-protein interaction (PPI) inhibitors has been a successful strategy in drug development. However, the identification of PPI stabilizers has proven much more challenging. Here we report a fragment-based drug screening approach using the regulatory hub-protein 14-3-3 as a platform for identifying PPI stabilizers. A homogenous time-resolved FRET assay was used to monitor stabilization of 14-3-3/peptide binding using the known interaction partner estrogen receptor alpha. Screening of an in-house fragment library identified fragment 2 (VUF15640) as a putative PPI stabilizer capable of cooperatively stabilizing 14-3-3 PPIs in a cooperative fashion with Fusicoccin-A. Mechanistically, fragment 2 appears to enhance 14-3-3 dimerization leading to increased client-protein binding. Functionally, fragment 2 enhanced potency of 14-3-3 in a cell-free system inhibiting the enzyme activity of the nitrate reductase. In conclusion, we identified a general PPI stabilizer targeting 14-3-3, which could be used as a tool compound for investigating 14-3-3 client protein interactions.


Assuntos
Proteínas 14-3-3 , Proteínas 14-3-3/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligação Proteica
3.
J Med Chem ; 63(9): 4430-4444, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31913033

RESUMO

This Perspective, the fourth in an annual series, summarizes fragment-to-lead (F2L) success stories published during 2018. Topics such as target class, screening methods, physicochemical properties, and ligand efficiency are discussed for the 2018 examples as well as for the combined 111 F2L examples covering 2015-2018. While the overall properties of fragments and leads have remained constant, a number of new trends are noted, for example, broadening of target class coverage and application of FBDD to covalent inhibitors. Moreover, several studies make use of fragment hits that were previously described in the literature, illustrating that fragments are versatile starting points that can be optimized to structurally diverse leads. By focusing on success stories, the hope is that this Perspective will identify and inform best practices in fragment-based drug discovery.


Assuntos
Química Farmacêutica , Descoberta de Drogas/métodos , Química Farmacêutica/tendências , Descoberta de Drogas/tendências , Avaliação Pré-Clínica de Medicamentos/métodos , Publicações
4.
J Med Chem ; 62(8): 3857-3872, 2019 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-30462504

RESUMO

This Miniperspective is the third in a series reviewing fragment-to-lead publications from a given year. Following our reviews for 2015 and 2016, this Miniperspective provides tabulated summaries of relevant articles published in 2017 along with some general observations. In addition, we discuss insights obtained from analysis of the combined data set of 85 examples from all three years of publications.


Assuntos
Química Farmacêutica , Descoberta de Drogas/métodos , Química Farmacêutica/tendências , Descoberta de Drogas/tendências , Avaliação Pré-Clínica de Medicamentos/métodos
5.
Sci Rep ; 6: 28288, 2016 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-27339552

RESUMO

The ability of scoring functions to correctly select and rank docking poses of small molecules in protein binding sites is highly target dependent, which presents a challenge for structure-based drug discovery. Here we describe a virtual screening method that combines an energy-based docking scoring function with a molecular interaction fingerprint (IFP) to identify new ligands based on G protein-coupled receptor (GPCR) crystal structures. The consensus scoring method is prospectively evaluated by: 1) the discovery of chemically novel, fragment-like, high affinity histamine H1 receptor (H1R) antagonists/inverse agonists, 2) the selective structure-based identification of ß2-adrenoceptor (ß2R) agonists, and 3) the experimental validation and comparison of the combined and individual scoring approaches. Systematic retrospective virtual screening simulations allowed the definition of scoring cut-offs for the identification of H1R and ß2R ligands and the selection of an optimal ß-adrenoceptor crystal structure for the discrimination between ß2R agonists and antagonists. The consensus approach resulted in the experimental validation of 53% of the ß2R and 73% of the H1R virtual screening hits with up to nanomolar affinities and potencies. The selective identification of ß2R agonists shows the possibilities of structure-based prediction of GPCR ligand function by integrating protein-ligand binding mode information.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Receptores Acoplados a Proteínas G/metabolismo , Interface Usuário-Computador , Agonistas Adrenérgicos beta/química , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/química , Antagonistas Adrenérgicos beta/farmacologia , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Descoberta de Drogas , Células HEK293 , Agonistas dos Receptores Histamínicos/química , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos H1/química , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Ensaio Radioligante , Receptores Adrenérgicos beta/química , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Receptores Histamínicos H1/química , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
J Chem Inf Model ; 55(5): 1045-61, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25848966

RESUMO

The spectacular advances in G-protein-coupled receptor (GPCR) structure determination have opened up new possibilities for structure-based GPCR ligand discovery. The structure-based prediction of whether a ligand stimulates (full/partial agonist), blocks (antagonist), or reduces (inverse agonist) GPCR signaling activity is, however, still challenging. A total of 31 ß1 (ß1R) and ß2 (ß2R) adrenoceptor crystal structures, including antagonist, inverse agonist, and partial/full agonist-bound structures, allowed us to explore the possibilities and limitations of structure-based prediction of GPCR ligand function. We used all unique protein-ligand interaction fingerprints (IFPs) derived from all ligand-bound ß-adrenergic crystal structure monomers to post-process the docking poses of known ß1R/ß2R partial/full agonists, antagonists/inverse agonists, and physicochemically similar decoys in each of the ß1R/ß2R structures. The systematic analysis of these 1920 unique IFP-structure combinations offered new insights into the relative impact of protein conformation and IFP scoring on selective virtual screening (VS) for ligands with a specific functional effect. Our studies show that ligands with the same function can be efficiently classified on the basis of their protein-ligand interaction profile. Small differences between the receptor conformation (used for docking) and reference IFP (used for scoring of the docking poses) determine, however, the enrichment of specific ligand types in VS hit lists. Interestingly, the selective enrichment of partial/full agonists can be achieved by using agonist IFPs to post-process docking poses in agonist-bound as well as antagonist-bound structures. We have identified optimal structure-IFP combinations for the identification and discrimination of antagonists/inverse agonist and partial/full agonists, and defined a predicted IFP for the small full agonist norepinephrine that gave the highest retrieval rate of agonists over antagonists for all structures (with an enrichment factor of 46 for agonists and 8 for antagonists on average at a 1% false-positive rate). This ß-adrenoceptor case study provides new insights into the opportunities for selective structure-based discovery of GPCR ligands with a desired function and emphasizes the importance of IFPs in scoring docking poses.


Assuntos
Agonistas de Receptores Adrenérgicos beta 1/metabolismo , Antagonistas de Receptores Adrenérgicos beta 1/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/metabolismo , Biologia Computacional/métodos , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 1/farmacologia , Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Conformação Proteica , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 2/química
7.
J Chem Inf Model ; 55(5): 1030-44, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25815783

RESUMO

In the current study we have evaluated the applicability of ligand-based virtual screening (LBVS) methods for the identification of small fragment-like biologically active molecules using different similarity descriptors and different consensus scoring approaches. For this purpose, we have evaluated the performance of 14 chemical similarity descriptors in retrospective virtual screening studies to discriminate fragment-like ligands of three membrane-bound receptors from fragments that are experimentally determined to have no affinity for these proteins (true inactives). We used a complete fragment affinity data set of experimentally determined ligands and inactives for two G protein-coupled receptors (GPCRs), the histamine H1 receptor (H1R) and the histamine H4 receptor (H4R), and one ligand-gated ion channel (LGIC), the serotonin receptor (5-HT3AR), to validate our retrospective virtual screening studies. We have exhaustively tested consensus scoring strategies that combine the results of multiple actives (group fusion) or combine different similarity descriptors (similarity fusion), and for the first time systematically evaluated different combinations of group fusion and similarity fusion approaches. Our studies show that for these three case study protein targets both consensus scoring approaches can increase virtual screening enrichments compared to single chemical similarity search methods. Our cheminformatics analyses recommend to use a combination of both group fusion and similarity fusion for prospective ligand-based virtual fragment screening.


Assuntos
Técnicas de Química Combinatória/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Receptores Histamínicos H1/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Interface Usuário-Computador , Consenso , Ligantes
8.
J Biomol Screen ; 20(1): 131-40, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25231971

RESUMO

Methods to discover biologically active small molecules include target-based and phenotypic screening approaches. One of the main difficulties in drug discovery is elucidating and exploiting the relationship between drug activity at the protein target and disease modification, a phenotypic endpoint. Fragment-based drug discovery is a target-based approach that typically involves the screening of a relatively small number of fragment-like (molecular weight <300) molecules that efficiently cover chemical space. Here, we report a fragment screening on TbrPDEB1, an essential cyclic nucleotide phosphodiesterase (PDE) from Trypanosoma brucei, and human PDE4D, an off-target, in a workflow in which fragment hits and a series of close analogs are subsequently screened for antiparasitic activity in a phenotypic panel. The phenotypic panel contained T. brucei, Trypanosoma cruzi, Leishmania infantum, and Plasmodium falciparum, the causative agents of human African trypanosomiasis (sleeping sickness), Chagas disease, leishmaniasis, and malaria, respectively, as well as MRC-5 human lung cells. This hybrid screening workflow has resulted in the discovery of various benzhydryl ethers with antiprotozoal activity and low toxicity, representing interesting starting points for further antiparasitic optimization.


Assuntos
Antiparasitários/farmacologia , Descoberta de Drogas/métodos , Testes de Sensibilidade Parasitária/métodos , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Antiparasitários/química , Doença de Chagas/tratamento farmacológico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Doenças Negligenciadas/tratamento farmacológico , Proteínas de Protozoários/antagonistas & inibidores , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia
9.
J Chem Inf Model ; 52(12): 3308-24, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23140085

RESUMO

Virtual fragment screening (VFS) is a promising new method that uses computer models to identify small, fragment-like biologically active molecules as useful starting points for fragment-based drug discovery (FBDD). Training sets of true active and inactive fragment-like molecules to construct and validate target customized VFS methods are however lacking. We have for the first time explored the possibilities and challenges of VFS using molecular fingerprints derived from a unique set of fragment affinity data for the histamine H(3) receptor (H(3)R), a pharmaceutically relevant G protein-coupled receptor (GPCR). Optimized FLAP (Fingerprints of Ligands and Proteins) models containing essential molecular interaction fields that discriminate known H(3)R binders from inactive molecules were successfully used for the identification of new H(3)R ligands. Prospective virtual screening of 156,090 molecules yielded a high hit rate of 62% (18 of the 29 tested) experimentally confirmed novel fragment-like H(3)R ligands that offer new potential starting points for the design of H(3)R targeting drugs. The first construction and application of customized FLAP models for the discovery of fragment-like biologically active molecules demonstrates that VFS is an efficient way to explore protein-fragment interaction space in silico.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Receptores Histamínicos H3/química , Receptores Histamínicos H3/metabolismo , Interface Usuário-Computador , Biologia Computacional , Bases de Dados de Proteínas , Análise Discriminante , Ligantes , Simulação de Acoplamento Molecular , Conformação Proteica
10.
Exp Dermatol ; 21(1): 32-7, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22151388

RESUMO

The effects of the histamine H(4) receptor antagonist JNJ7777120 were evaluated in a model of acute skin inflammation induced by local application of croton oil. The influence of strain on the effect of JNJ7777120 was investigated in four different mouse strains (CD-1, NMRI, BALB/c and C57BL/6J). In CD-1 mice, JNJ777720 (30-100 mg/kg subcutaneously, s.c.) exerted a dose-dependent inhibition of croton oil-induced ear inflammation and polymorphonuclear leucocyte infiltration, as confirmed by histological evaluation of ear tissues. JNJ7777120 (30-100 mg/kg) did not reduce ear oedema in NMRI, BALB/c or C57BL/6J mice. The positive control, dexamethasone (2 mg/kg s.c.) induced significant anti-inflammatory effects only in CD-1 and NMRI mice. In these strains, also the histamine H(1) -receptor blocker pyrilamine (30 mg/kg s.c.) significantly reduced ear oedema at 2 h after croton oil challenge, being as effective as JNJ7777120 in CD-1 mice. Taken together, these data demonstrate that the H(4) receptor antagonist JNJ7777120 may reduce acute croton oil-induced skin inflammation as effectively as H(1) receptor blockade. However, present experiments evidenced for the first time marked strain-related differences in the JNJ7777120 pharmacological activity, which have to be carefully considered when using this ligand to characterize histamine H(4) receptor functions in murine models and translating preclinical data to clinical human settings.


Assuntos
Dermatite/tratamento farmacológico , Indóis/uso terapêutico , Piperazinas/uso terapêutico , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Animais , Anti-Inflamatórios/uso terapêutico , Óleo de Cróton , Dermatite/patologia , Fármacos Dermatológicos , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Orelha Externa/patologia , Antagonistas dos Receptores Histamínicos H1/uso terapêutico , Indóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Piperazinas/farmacologia , Pirilamina/uso terapêutico , Receptores Histamínicos , Receptores Histamínicos H4
11.
J Am Chem Soc ; 133(14): 5363-71, 2011 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-21322593

RESUMO

Optimization of fragment hits toward high-affinity lead compounds is a crucial aspect of fragment-based drug discovery (FBDD). In the current study, we have successfully optimized a fragment by growing into a ligand-inducible subpocket of the binding site of acetylcholine-binding protein (AChBP). This protein is a soluble homologue of the ligand binding domain (LBD) of Cys-loop receptors. The fragment optimization was monitored with X-ray structures of ligand complexes and systematic thermodynamic analyses using surface plasmon resonance (SPR) biosensor analysis and isothermal titration calorimetry (ITC). Using site-directed mutagenesis and AChBP from different species, we find that specific changes in thermodynamic binding profiles, are indicative of interactions with the ligand-inducible subpocket of AChBP. This study illustrates that thermodynamic analysis provides valuable information on ligand binding modes and is complementary to affinity data when guiding rational structure- and fragment-based discovery approaches.


Assuntos
Proteínas de Transporte/química , Descoberta de Drogas/métodos , Calorimetria , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Reprodutibilidade dos Testes , Especificidade da Espécie , Ressonância de Plasmônio de Superfície , Termodinâmica , Tirosina
12.
J Med Chem ; 48(6): 2100-7, 2005 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-15771452

RESUMO

In this study, we continue our efforts toward the development of potent and highly selective histamine H(3) receptor agonists. We introduced various alkyl or aryl alkyl groups on the piperidine nitrogen of the known H(3)/H(4) agonist immepip and its analogues (1-3a). We observed that N-methyl-substituted immepip (methimepip) exhibits high affinity and agonist activity at the human histamine H(3) receptor (pK(i) = 9.0 and pEC(50) = 9.5) with a 2000-fold selectivity at the human H(3) receptor over the human H(4) receptor and more than a 10000-fold selectivity over the human histamine H(1) and H(2) receptors. Methimepip was also very effective as an H(3) receptor agonist at the guinea pig ileum (pD(2) = 8.26). Moreover, in vivo microdialysis (in rat brain) showed that methimepip reduces the basal level of brain histamine to about 25% after a 5 mg/kg intraperitoneal administration.


Assuntos
Agonistas dos Receptores Histamínicos/síntese química , Imidazóis/síntese química , Piperidinas/síntese química , Receptores Histamínicos H3/efeitos dos fármacos , Animais , Ligação Competitiva , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Estimulação Elétrica , Cobaias , Histamina/metabolismo , Agonistas dos Receptores Histamínicos/química , Agonistas dos Receptores Histamínicos/farmacologia , Humanos , Hipotálamo/metabolismo , Íleo/efeitos dos fármacos , Íleo/fisiologia , Imidazóis/química , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Microdiálise , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Piperidinas/química , Piperidinas/farmacologia , Ensaio Radioligante , Ratos , Ratos Wistar
13.
Nat Rev Drug Discov ; 4(2): 107-20, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15665857

RESUMO

Since the cloning of the histamine H(3) receptor cDNA in 1999 by Lovenberg and co-workers, this histamine receptor has gained the interest of many pharmaceutical companies as a potential drug target for the treatment of various important disorders, including obesity, attention-deficit hyperactivity disorder, Alzheimer's disease, schizophrenia, as well as for myocardial ischaemia, migraine and inflammatory diseases. Here, we discuss relevant information on this target protein and describe the development of various H(3) receptor agonists and antagonists, and their effects in preclinical animal models.


Assuntos
Clonagem Molecular/métodos , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/uso terapêutico , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Agonistas dos Receptores Histamínicos/química , Agonistas dos Receptores Histamínicos/farmacologia , Agonistas dos Receptores Histamínicos/uso terapêutico , Humanos , Receptores Histamínicos H3/efeitos dos fármacos
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