RESUMO
Secondary structures tend to be recognizable because they have repeating structural motifs, but mimicry of these does not have to follow such well-defined patterns. Bioinformatics studies to match side-chain orientations of a novel hydantoin triazole chemotype (1) to protein-protein interfaces revealed it tends to align well across parallel and antiparallel sheets, like rungs on a ladder. One set of these overlays was observed for the protein-protein interaction uPAâ uPAR. Consequently, chemotype 1 was made with appropriate side-chains to mimic uPA at this interface. Biophysical assays indicate these compounds did in fact bind uPAR, and elicit cellular responses that affected invasion, migration, and wound healing.
Assuntos
Materiais Biomiméticos/química , Proteínas/química , Materiais Biomiméticos/farmacologia , Estrutura Secundária de Proteína , Triazóis/química , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Free cysteine residues react with QuatCy 1, by simply mixing the protein and dye in aqueous buffer at 37 °C. Another dye, MHI-148, can be used for a similar labelling protocol, but QuatCy reacts faster with all proteins studied, except albumin; it emerges here that this is because MHI-148 instantly forms of a non-covalent complex with albumin, but QuatCy does not. Labelling with QuatCy has advantages insofar as it is over five times brighter, and much more photostable, than MHI-148, and combination labelling with this dye pair will allow multiplexing in the near-IR region.
Assuntos
Carbocianinas/química , Corantes Fluorescentes/química , Albumina Sérica Humana/química , Sítios de Ligação , Carbocianinas/síntese química , Corantes Fluorescentes/síntese química , Humanos , Raios Infravermelhos , Estrutura Molecular , Imagem ÓpticaRESUMO
An active segment of the research community designing small molecules ("minimalist mimics" of peptide fragments) to interfere with protein-protein interactions have based their studies on an implicit hypothesis. Here we refer to this as the Secondary Structure Hypothesis, that might be defined as, "If a small molecule can orient amino acid side-chains in directions that resemble side-chains of the parent secondary structure at the interface, then that small molecule is a candidate to perturb the protein-protein interaction". Rigorous tests of this hypothesis require co-crystallization of minimalist mimics with protein receptors, and comparison of the bound conformations with the interface secondary structures they were designed to resemble. Unfortunately, to the best of our knowledge, there is no such analysis in the literature, and it is unlikely that enough examples will emerge in the near future to test the hypothesis. Research described here was designed to challenge this hypothesis from a different perspective. In a previous study, preferred conformations of a series of novel minimalist mimics were simulated then systematically overlaid on >240 000 crystallographically characterized protein-protein interfaces. Select data from that overlay procedure revealed chemotypes that overlay side chains on various PPI interfaces with a relatively high frequency of occurrence. The first aim of this work was to determine if good secondary structure mimics overlay frequently on PPI interfaces. The second aim of this work was to determine if overlays of preferred conformers at interface regions involve secondary structures. Thus situations where these conformations overlaid extremely well on PPI interfaces were analyzed to determine if secondary structures featured the PPI regions where these molecules overlaid in the previous study. Combining conclusions from these two studies enabled us to formulate a hypothesis that is complementary to the Secondary Structure Hypothesis, but, unlike this, is supported by abundant data. We call this the Interface Mimicry Hypothesis.
Assuntos
Modelos Químicos , Mimetismo Molecular , Proteínas/química , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
Small molecules that can interrupt or inhibit protein-protein interactions (PPIs) are valuable as probes in chemical biology and medicinal chemistry, but they are also notoriously difficult to develop. Design of non-peptidic small molecules that mimic amino acid side-chain interactions in PPIs ("minimalist mimics") is seen as a way to fast track discovery of PPI inhibitors. However, there has been little comment on general design criteria for minimalist mimics, even though such guidelines could steer construction of libraries to screen against multiple PPI targets. We hypothesized insight into general design criteria for minimalist mimics could be gained by comparing preferred conformations of typical minimalist mimic designs against side-chain orientations on a huge number of PPI interfaces. That thought led to this work which features nine minimalist mimic designs: one from the literature, and eight new "hypothetical" ones conceived by us. Simulated preferred conformers of these were systematically aligned with >240 000 PPI interfaces from the Protein Data Bank. Conclusions from those analyses did indeed reveal various design considerations that are discussed here. Surprisingly, this study also showed one of the minimalist mimic designs aligned on PPI interface segments more than 15 times more frequently than any other in the series (according to uniform standards described herein); reasons for this are also discussed.
Assuntos
Quinase 2 Dependente de Ciclina/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Bases de Dados de Proteínas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , EstereoisomerismoRESUMO
Molecular entities that localize in tumor tissue are clinically important for targeted delivery of diagnostic, imaging, and therapeutic reagents. Often these targeting entities are designed for specific receptors (e.g., EGFR or integrin receptors). However, there is a subset of cyanine-7 dyes that apparently localize in every type of solid tumor tissue (at least, no exceptions have been reported so far), and they persist there for several days. Consequently, these dyes can be used for near-IR optical imaging of tumors in animal studies, they can be conjugated with cytotoxic species to give experimental theranostics, and there is potential for expanding their use into the development of clinically useful derivatives. Data presented in the literature and in this work indicate that the half-lives of these compounds in serum at 37 °C is on the order of minutes to a few hours, so what accounts for the persistent fluorescence of these dyes in tumor tissue over periods of several days? Literature, solely based on tissue culture experiments featuring a particular receptor blocker, indicates that uptake of these dyes is mediated by the organic anion transporter proteins (OATPs). Data presented in this paper agrees with that conclusion for short-term uptake, but significantly expands understanding of the likely reasons for long-term uptake and persistent tumor localization in vivo.
Assuntos
Benzotiazóis/metabolismo , Carbocianinas/metabolismo , Corantes Fluorescentes/metabolismo , Neoplasias/metabolismo , Benzotiazóis/química , Benzotiazóis/farmacocinética , Carbocianinas/química , Carbocianinas/farmacocinética , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Humanos , Modelos Moleculares , Neoplasias/diagnóstico por imagem , Imagem Óptica/métodos , Transportadores de Ânions Orgânicos/metabolismo , Albumina Sérica Humana/metabolismoRESUMO
Convenient labeling of proteins is important for observing its function under physiological conditions. In tissues particularly, heptamethine cyanine dyes (Cy-7) are valuable because they absorb in the near-infrared (NIR) region (750â»900 nm) where light penetration is maximal. In this work, we found Cy-7 dyes with a meso-Cl functionality covalently binding to proteins with free Cys residues under physiological conditions (aqueous environments, at near neutral pH, and 37 °C). It transpired that the meso-Cl of the dye was displaced by free thiols in protein, while nucleophilic side-chains from amino acids like Tyr, Lys, and Ser did not react. This finding shows a new possibility for convenient and selective labeling of proteins with NIR fluorescent probes.
Assuntos
Carbocianinas/química , Corantes Fluorescentes/química , Proteínas/química , Espectroscopia de Luz Próxima ao Infravermelho , Coloração e Rotulagem , Aminoácidos/química , Estrutura Molecular , Compostos de SulfidrilaRESUMO
Targeting tumor vasculature represents an intriguing therapeutic strategy in the treatment of cancer. In an effort to discover new vascular disrupting agents with improved water solubility and potentially greater bioavailability, various amino acid prodrug conjugates (AAPCs) of potent amino combretastatin, amino dihydronaphthalene, and amino benzosuberene analogs were synthesized along with their corresponding water-soluble hydrochloride salts. These compounds were evaluated for their ability to inhibit tubulin polymerization and for their cytotoxicity against selected human cancer cell lines. The amino-based parent anticancer agents 7, 8, 32 (also referred to as KGP05) and 33 (also referred to as KGP156) demonstrated potent cytotoxicity (GI50=0.11-40nM) across all evaluated cell lines, and they were strong inhibitors of tubulin polymerization (IC50=0.62-1.5µM). The various prodrug conjugates and their corresponding salts were investigated for cleavage by the enzyme leucine aminopeptidase (LAP). Four of the glycine water-soluble AAPCs (16, 18, 44 and 45) showed quantitative cleavage by LAP, resulting in the release of the highly cytotoxic parent drug, whereas partial cleavage (<10-90%) was observed for other prodrugs (15, 17, 24, 38 and 39). Eight of the nineteen AAPCs (13-16, 42-45) showed significant cytotoxicity against selected human cancer cell lines. The previously reported CA1-diamine analog and its corresponding hydrochloride salt (8 and 10, respectively) caused extensive disruption (at a concentration of 1.0µM) of human umbilical vein endothelial cells growing in a two-dimensional tubular network on matrigel. In addition, compound 10 exhibited pronounced reduction in bioluminescence (greater than 95% compared to saline control) in a tumor bearing (MDA-MB-231-luc) SCID mouse model 2h post treatment (80mg/kg), with similar results observed upon treatment (15mg/kg) with the glycine amino-dihydronaphthalene AAPC (compound 44). Collectively, these results support the further pre-clinical development of the most active members of this structurally diverse collection of water-soluble prodrugs as promising anticancer agents functioning through a mechanism involving vascular disruption.
Assuntos
Antineoplásicos/uso terapêutico , Bibenzilas/uso terapêutico , Cumarínicos/uso terapêutico , Naftalenos/uso terapêutico , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Pró-Fármacos/uso terapêutico , Aminoácidos/química , Aminoácidos/uso terapêutico , Animais , Antineoplásicos/química , Bibenzilas/química , Mama/irrigação sanguínea , Mama/efeitos dos fármacos , Mama/patologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cumarínicos/química , Desenho de Fármacos , Feminino , Humanos , Camundongos , Camundongos SCID , Naftalenos/química , Neoplasias/patologia , Imagem Óptica , Pró-Fármacos/química , Solubilidade , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Água/químicaRESUMO
The discovery of 3-methoxy-9-(30,40,50-trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-ol (a benzosuberene-based analogue referred to as KGP18) was originally inspired by the natural products colchicine and combretastatin A-4 (CA4). The relative structural simplicity and ease of synthesis of KGP18, coupled with its potent biological activity as an inhibitor of tubulin polymerization and its cytotoxicity (in vitro) against human cancer cell lines, has resulted in studies focused on new analogue design and synthesis. Our goal was to probe the relationship of structure to function in this class of anticancer agents. A series of twenty-two new benzosuberene-based analogues of KGP18 was designed and synthesized. These compounds vary in their methoxylation pattern and separately incorporate trifluoromethyl groups around the pendant aryl ring for the evaluation of the effect of functional group modifications on the fused six-membered aromatic ring. In addition, the 8,9-saturated congener of KGP18 has been synthesized to assess the necessity of unsaturation at the carbon atom bearing the pendant aryl ring. Six of the molecules from this benzosuberene-series of compounds were active (IC50 < 5 lM) as inhibitors of tubulin polymerization while four analogues were comparable (IC50 approximately 1 lM) in their tubulin inhibitory activity to CA4 and KGP18. The potency of a bis-trifluoromethyl analogue 74 and the unsaturated KGP18 derivative 73 as inhibitors of tubulin assembly along with their moderate cytotoxicity suggested the potential utility of these compounds as vascular disrupting agents (VDAs) to selectively target microvessels feeding tumors. Accordingly, water-soluble and DMSO-soluble phosphate prodrug salts of each were synthesized for preliminary in vivo studies to assess their potential efficacy as VDAs.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Cumarínicos/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cumarínicos/uso terapêutico , Humanos , Camundongos SCID , Simulação de Acoplamento Molecular , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Polimerização/efeitos dos fármacos , Moduladores de Tubulina/uso terapêuticoRESUMO
Diversely functionalized, fused aryl-alkyl ring systems hold a prominent position as well-established molecular frameworks for a variety of anti-cancer agents. The benzosuberene (6,7 fused, also referred to as dihydro-5H-benzo[7]annulene and benzocycloheptene) ring system has emerged as a valuable molecular core component for the development of inhibitors of tubulin assembly, which function as antiproliferative anti-cancer agents and, in certain cases, as vascular disrupting agents (VDAs). Both a phenolic-based analogue (known as KGP18, compound 39) and its corresponding amine-based congener (referred to as KGP156, compound 45), which demonstrate strong inhibition of tubulin assembly (low micromolar range) and potent cytotoxicity (picomolar range for KGP18 and nanomolar range for KGP156) are noteworthy examples of such benzosuberene-based compounds. In order to extend the structure-activity relationship (SAR) knowledge base related to benzosuberene anti-cancer agents, a series of eleven analogues (including KGP18) were prepared in which the methoxylation pattern on the pendant aryl ring as well as functional group incorporation on the fused aryl ring were varied. The synthetic approach to these compounds featured a sequential Wittig olefination, reduction, Eaton's reagent-mediated cyclization strategy to achieve the core benzosuberone intermediate, and represented a higher-yielding synthesis of KGP18 (which we prepared previously through a ring-expansion strategy). Incorporation of a fluorine or chlorine atom at the 1-position of the fused aryl ring or replacement of one of the methoxy groups with hydrogen (on the pendant aryl ring of KGP18) led to benzosuberene analogues that were both strongly inhibitory against tubulin assembly (IC50 approximately 1.0 µM) and strongly cytotoxic against selected human cancer cell lines (for example, GI50=5.47 nM against NCI-H460 cells with fluoro-benzosuberene analogue 37). A water-soluble phosphate prodrug salt of KGP18 (referred to as KGP265, compound 44) and a water-soluble serinamide salt (compound 48) of KGP156 were also synthesized and evaluated in this study.
Assuntos
Antineoplásicos/farmacologia , Benzocicloeptenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Benzocicloeptenos/síntese química , Benzocicloeptenos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Polimerização/efeitos dos fármacos , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismoRESUMO
Only a few small molecules that disrupt the uPA and uPA receptor (uPAR) interaction have been discovered despite decades of research in the area, and none have been approved in clinical trials. Research reported here features two new ways of considering the problem of discovering small molecules to disrupt uPAâ¢uPAR, specifically in terms of chemotype design and method of evaluation. Chemotypes used in this work are piptides (Arancillo . Angew. Chem., Int. Ed., 2021, 60, 6653-6659) with side chains corresponding to the uPA loop that binds uPAR. Further, hybrids of 1 and another uPAR ligand developed in these labs (2), i.e., 3 and 4, were also designed and tested. All the piptide chemotypes bound uPAR at concentrations of 50 µM or less. Members of this series had Ki values <3 µM and showed favorable responses in cellular assays; these data are comparable with the best small molecule uPAâ¢uPAR disruptors in the literature (from conventional screening).
Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase , Ligantes , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Ubiquitination is a major controller of protein homeostasis in cells. Some ubiquitination pathways are modulated by a NEDDylation cascade, that also features E1 - 3 enzymes. The E1 enzyme in the NEDDylation cascade involves a protein-protein interaction (PPI) between NEDD8 (similar to ubiquitin) and NAE (NEDD8 Activating Enzyme). A small molecule inhibitor of the ATP binding site in NAE is in clinical trials. We hypothesized a similar effect could be induced by disrupting the NEDD8·NAE PPI, though, to the best of our knowledge, no small molecules have been reported to disrupt this to date. In the research described here, Exploring Key Orientations (EKO) was used to evaluate several chemotype designs for their potential to disrupt NEDD8·NAE; specifically, for their biases towards orientation of side-chains in similar ways to protein segments at the interface. One chemotype design was selected, and a targeted library of 24 compounds was made around this theme via solid phase synthesis. An entry level hit for disrupting NEDD8·NAE was identified from this library on the basis of its ability to bind NAE (K i of 6.4 ± 0.3 µM from fluorescence polarization), inhibit NEDDylation, suppress formation of the corresponding E1 - 3 complexes as monitored by cell-based immunoblotting, and cytotoxicity to K562 leukemia cells via early stage apoptosis. The cell-based immunoblot assay also showed the compound caused NEDD8 to accumulate in cells, presumably due to inhibition of the downstream pathways involving the E1 enzyme. The affinity and cellular activities of the hit compound are modest, but is interesting as first in class for this mode of inhibition of NEDDylation, and as another illustration of the way EKO can be used to evaluate user-defined chemotypes as potential inhibitors of PPIs.