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A method is described to deconstruct the network of hydropathic interactions within and between a protein's sidechain and its environment into residue-based three-dimensional maps. These maps encode favorable and unfavorable hydrophobic and polar interactions, in terms of spatial positions for optimal interactions, relative interaction strength, as well as character. In addition, these maps are backbone angle-dependent. After map calculation and clustering, a finite number of unique residue sidechain interaction maps exist for each backbone conformation, with the number related to the residue's size and interaction complexity. Structures for soluble proteins (~749,000 residues) and membrane proteins (~387,000 residues) were analyzed, with the latter group being subdivided into three subsets related to the residue's position in the membrane protein: soluble domain, core-facing transmembrane domain, and lipid-facing transmembrane domain. This work suggests that maps representing residue types and their backbone conformation can be reassembled to optimize the medium-to-high resolution details of a protein structure. In particular, the information encoded in maps constructed from the lipid-facing transmembrane residues appears to paint a clear picture of the protein-lipid interactions that are difficult to obtain experimentally.
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Proteínas de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Proteica , Lipídeos/química , Ligação ProteicaRESUMO
Sphingolipids are a diverse class of lipids with essential functions as determinants of membrane physical properties and as intra- and intercellular signaling agents. Disruption of the normal biochemical processes that establish the levels of individual sphingolipids is associated with a variety of human diseases including cancer, cardiovascular disease, metabolic disease, skin diseases, and lysosomal storage diseases. A unique aspect of this metabolic network is that there is a single enzymatic step that initiates the biosynthetic pathway for all sphingolipids. This step is catalyzed by the enzyme serine palmitoyltranserase (SPT). Under most circumstances SPT condenses serine and the 16-carbon acyl-CoA, palmitoyl-CoA to produce the precursor of all sphingolipids. SPT, a four-subunit protein complex, is subject to classic feedback regulation: when cellular sphingolipids are elevated, SPT activity is inhibited. Ceramide is the sphingolipid sensed by this system and it regulates SPT by directly binding to the complex. The ceramide binding site in the SPT complex, and how ceramide binding results in SPT inhibition, has now been determined in vertebrates, plants, and yeast using molecular modeling and cryo-electron microscopy. Here we discuss the similarities and differences revealed by these resolved structures and the surprising result that ceramide binds at almost identical positions in the SPT complex of these divergent organisms, but accomplishes SPT regulation in very different ways.
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Ceramidas , Serina C-Palmitoiltransferase , Animais , Humanos , Ceramidas/genética , Ceramidas/metabolismo , Microscopia Crioeletrônica , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , SerinaRESUMO
Human sirtuins play important roles in various cellular events including DNA repair, gene silencing, mitochondrial biogenesis, insulin secretion and apoptosis. They regulate a wide array of protein and enzyme targets through their NAD+ -dependent deacetylase activities. Sirtuins are also thought to mediate the beneficial effects of low-calorie intake to extend longevity in diverse organisms from yeast to mammals. Small molecules mimicking calorie restriction to stimulate sirtuin activity are attractive therapeutics against age-related disorders such as cardiovascular diseases, diabetes and neurodegeneration. Little is known about one of the mitochondrial sirtuins, SIRT5. SIRT5 has emerged as a critical player in maintaining cardiac health and neuronal viability upon stress and functions as a tumour suppressor in a context-specific manner. Much has been debated about whether SIRT5 has evolved away from being a deacetylase because of its weak catalytic activity, especially in the in vitro testing. We have, for the first time, identified a SIRT5-selective allosteric activator, nicotinamide riboside (NR). It can increase SIRT5 catalytic efficiency with different synthetic peptide substrates. The mechanism of action was further explored using a combination of molecular biology and biochemical strategies. Based on the existing structural biology information, the NR binding site was also mapped out. These activators are powerful chemical probes for the elucidation of cellular regulations and biological functions of SIRT5. The knowledge gained in this study can be used to guide the design and synthesis of more potent, isotype-selective SIRT5 activators and to develop them into therapeutics for metabolic disorders and age-related diseases.
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Sirtuínas , Animais , Humanos , Sirtuínas/genética , Niacinamida/farmacologia , Peptídeos/química , Compostos de Piridínio/farmacologia , Mamíferos/metabolismoRESUMO
A long-lasting goal of computational biochemists, medicinal chemists, and structural biologists has been the development of tools capable of deciphering the molecule-molecule interaction code that produces a rich variety of complex biomolecular assemblies comprised of the many different simple and biological molecules of life: water, small metabolites, cofactors, substrates, proteins, DNAs, and RNAs. Software applications that can mimic the interactions amongst all of these species, taking account of the laws of thermodynamics, would help gain information for understanding qualitatively and quantitatively key determinants contributing to the energetics of the bimolecular recognition process. This, in turn, would allow the design of novel compounds that might bind at the intermolecular interface by either preventing or reinforcing the recognition. HINT, hydropathic interaction, was a model and software code developed from a deceptively simple idea of Donald Abraham with the close collaboration with Glen Kellogg at Virginia Commonwealth University. HINT is based on a function that scores atom-atom interaction using LogP, the partition coefficient of any molecule between two phases; here, the solvents are water that mimics the cytoplasm milieu and octanol that mimics the protein internal hydropathic environment. This review summarizes the results of the extensive and successful collaboration between Abraham and Kellogg at VCU and the group at the University of Parma for testing HINT in a variety of different biomolecular interactions, from proteins with ligands to proteins with DNA.
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The aliphatic hydrophobic amino acid residues-alanine, isoleucine, leucine, proline and valine-are among the most common found in proteins. Their structural role in proteins is seemingly obvious: engage in hydrophobic interactions to stabilize secondary, and to a lesser extent, tertiary and quaternary structure. However, favorable hydrophobic interactions involving the sidechains of these residue types are generally less significant than the unfavorable set arising from interactions with polar atoms. Importantly, the constellation of interactions between residue sidechains and their environments can be recorded as three-dimensional maps that, in turn, can be clustered. The clustered average map sets compose a library of interaction profiles encoding interaction strengths, interaction types and the optimal 3D position for the interacting partners. This library is backbone angle-dependent and suggests solvent and lipid accessibility for each unique interaction profile. In this work, in addition to analysis of soluble proteins, a large set of membrane proteins that contained optimized artificial lipids were evaluated by parsing the structures into three distinct components: soluble extramembrane domain, lipid facing transmembrane domain, core transmembrane domain. The aliphatic residues were extracted from each of these sets and passed through our calculation protocol. Notable observations include: the roles of aliphatic residues in soluble proteins and in the membrane protein's soluble domains are nearly identical, although the latter are slightly more solvent accessible; by comparing maps calculated with sidechain-lipid interactions to maps ignoring those interactions, the potential extent of residue-lipid and residue-interactions can be assessed and likely exploited in structure prediction and modeling; amongst these residue types, the levels of lipid engagement show isoleucine as the most engaged, while the other residues are largely interacting with neighboring helical residues.
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The Department of Medicinal Chemistry, together with the Institute for Structural Biology, Drug Discovery and Development, at Virginia Commonwealth University (VCU) has evolved, organically with quite a bit of bootstrapping, into a unique drug discovery ecosystem in response to the environment and culture of the university and the wider research enterprise. Each faculty member that joined the department and/or institute added a layer of expertise, technology and most importantly, innovation, that fertilized numerous collaborations within the University and with outside partners. Despite moderate institutional support with respect to a typical drug discovery enterprise, the VCU drug discovery ecosystem has built and maintained an impressive array of facilities and instrumentation for drug synthesis, drug characterization, biomolecular structural analysis and biophysical analysis, and pharmacological studies. Altogether, this ecosystem has had major impacts on numerous therapeutic areas, such as neurology, psychiatry, drugs of abuse, cancer, sickle cell disease, coagulopathy, inflammation, aging disorders and others. Novel tools and strategies for drug discovery, design and development have been developed at VCU in the last five decades; e.g., fundamental rational structure-activity relationship (SAR)-based drug design, structure-based drug design, orthosteric and allosteric drug design, design of multi-functional agents towards polypharmacy outcomes, principles on designing glycosaminoglycans as drugs, and computational tools and algorithms for quantitative SAR (QSAR) and understanding the roles of water and the hydrophobic effect.
Assuntos
Química Farmacêutica , Química Computacional , Humanos , Ecossistema , Universidades , Virginia , Descoberta de Drogas/métodos , Relação Quantitativa Estrutura-Atividade , Biologia MolecularRESUMO
Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65-S401) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phospho-mimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Both S401A and the S401D cells showed impaired DSB repair (nonhomologous end joining and homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Furthermore, S401D cells displayed increased ERK and AKT signaling resulting in enhanced growth rate further underscoring the multiple roles ATM-PP2A signaling plays in regulating prosurvival responses. Time-lapse video and cellular localization experiments showed that PR65 was exported to the cytoplasm after radiation by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects observed. A putative nuclear export sequence (NES) close to S401 was identified and when mutated resulted in aberrant PR65 shuttling. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear-cytoplasmic shuttling mediated by the nuclear export receptor CRM1.
Assuntos
Ataxia Telangiectasia , Animais , Camundongos , Ataxia Telangiectasia/genética , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Proteínas Nucleares/metabolismo , Dano ao DNARESUMO
Evaluation of the intramolecular stability of proteins plays a key role in the comprehension of their biological behavior and mechanism of action. Small structural alterations such as mutations induced by single nucleotide polymorphism can impact biological activity and pharmacological modulation. Covid-19 mutations, that affect viral replication and the susceptibility to antibody neutralization, and the action of antiviral drugs, are just one example. In this work, the intramolecular stability of mutated proteins, like Spike glycoprotein and its complexes with the human target, is evaluated through hydropathic intramolecular energy scoring originally conceived by Abraham and Kellogg based on the "Extension of the fragment method to calculate amino acid zwitterion and side-chain partition coefficients" by Abraham and Leo in Proteins: Struct. Funct. Genet. 1987, 2:130 - 52. HINT is proposed as a fast and reliable tool for the stability evaluation of any mutated system. This work has been written in honor of Prof. Donald J. Abraham (1936-2021).
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Proteínas Oncogênicas , Glicoproteína da Espícula de Coronavírus , Humanos , Proteínas Oncogênicas/química , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
BACKGROUND: Deregulated translation initiation is implicated extensively in cancer initiation and progression. It is actively pursued as a viable target that circumvents the dependency on oncogenic signaling, a significant factor in current strategies. Eukaryotic translation initiation factor (eIF) 4A plays an essential role in translation initiation by unwinding the secondary structure of messenger RNA (mRNA) upstream of the start codon, enabling active ribosomal recruitment on the downstream genes. Several natural product molecules with similar scaffolds, such as Rocaglamide A (RocA), targeting eIF4A have been reported in the last decade. However, their clinical utilization is still elusive due to several pharmacological limitations. In this study we identified new eIF4A1 inhibitors and their possible mechanisms. METHODS: In this report, we conducted a pharmacophore-based virtual screen of RocA complexed with eIF4A and a polypurine RNA strand for novel eIF4A inhibitors from commercially available compounds in the MolPort Database. We performed target-based screening and optimization of active pharmacophores. We assessed the effects of novel compounds on biochemical and cell-based assays for efficacy and mechanistic evaluation. RESULTS: We validated three new potent eIF4A inhibitors, RBF197, RBF 203, and RBF 208, which decreased diffuse large B-cell lymphoma (DLBCL) cell viability. Biochemical and cellular studies, molecular docking, and functional assays revealed that thosenovel compounds clamp eIF4A into mRNA in an ATP-independent manner. Moreover, we found that RBF197 and RBF208 significantly depressed eIF4A-dependent oncogene expression as well as the colony formation capacity of DLBCL. Interestingly, exposure of these compounds to non-malignant cells had only minimal impact on their growth and viability. CONCLUSIONS: Identified compounds suggest a new strategy for designing novel eIF4A inhibitors.
Assuntos
Linfoma , Neoplasias , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Humanos , Linfoma/tratamento farmacológico , Simulação de Acoplamento Molecular , RNA Mensageiro/metabolismoRESUMO
Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 µM and 2.59 ± 0.11 µM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.
Assuntos
Piridoxaminafosfato Oxidase , Piridoxina , Piridoxaminafosfato Oxidase/metabolismo , Dopa Descarboxilase , Fosfato de Piridoxal/metabolismo , Oxirredutases , Piridoxal Quinase/metabolismoRESUMO
Knowledge of three-dimensional protein structure is integral to most modern drug discovery efforts. Recent advancements have highlighted new techniques for 3D protein structure determination and, where structural data cannot be collected experimentally, prediction of protein structure. We have undertaken a major effort to use existing protein structures to collect, characterize, and catalogue the inter-atomic interactions that define and compose 3D structure by mapping hydropathic interaction environments as maps in 3D space. This work has been performed on a residue-by-residue basis, where we have seen evidence for relationships between environment character, residue solvent-accessible surface areas and their secondary structures. In this graphical review, we apply principles from our earlier studies and expand the scope to all common amino acid residue types in both soluble and membrane proteins. Key to this analysis is parsing the Ramachandran plot to an 8-by-8 chessboard to define secondary structure bins. Our analysis yielded a number of quantitative discoveries: 1) increased fraction of hydrophobic residues (alanine, isoleucine, leucine, phenylalanine and valine) in membrane proteins compared to their fractions in soluble proteins; 2) less burial coupled with significant increases in favorable hydrophobic interactions for hydrophobic residues in membrane proteins compared to soluble proteins; and 3) higher burial and more favorable polar interactions for polar residues now preferring the interior of membrane proteins. These observations and the supporting data should provide benchmarks for current studies of protein residues in different environments and may be able to guide future protein structure prediction efforts.
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Aspartic acid, glutamic acid and histidine are ionizable residues occupying various protein environments and perform many different functions in structures. Their roles are tied to their acid/base equilibria, solvent exposure, and backbone conformations. We propose that the number of unique environments for ASP, GLU and HIS is quite limited. We generated maps of these residue's environments using a hydropathic scoring function to record the type and magnitude of interactions for each residue in a 2703-protein structural dataset. These maps are backbone-dependent and suggest the existence of new structural motifs for each residue type. Additionally, we developed an algorithm for tuning these maps to any pH, a potentially useful element for protein design and structure building. Here, we elucidate the complex interplay between secondary structure, relative solvent accessibility, and residue ionization states: the degree of protonation for ionizable residues increases with solvent accessibility, which in turn is notably dependent on backbone structure.
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Atomic-resolution protein structural models are prerequisites for many downstream activities like structure-function studies or structure-based drug discovery. Unfortunately, this data is often unavailable for some of the most interesting and therapeutically important proteins. Thus, computational tools for building native-like structural models from less-than-ideal experimental data are needed. To this end, interaction homology exploits the character, strength and loci of the sets of interactions that define a structure. Each residue type has its own limited set of backbone angle-dependent interaction motifs, as defined by their environments. In this work, we characterize the interactions of serine, cysteine and S-bridged cysteine in terms of 3D hydropathic environment maps. As a result, we explore several intriguing questions. Are the environments different between the isosteric serine and cysteine residues? Do some environments promote the formation of cystine S-S bonds? With the increasing availability of structural data for water-insoluble membrane proteins, are there environmental differences for these residues between soluble and membrane proteins? The environments surrounding serine and cysteine residues are dramatically different: serine residues are about 50% solvent exposed, while cysteines are only 10% exposed; the latter are more involved in hydrophobic interactions although there are backbone angle-dependent differences. Our analysis suggests that one driving force for -S-S- bond formation is a rather substantial increase in burial and hydrophobic interactions in cystines. Serine and cysteine become less and more, respectively, solvent-exposed in membrane proteins. 3D hydropathic environment maps are an evolving structure analysis tool showing promise as elements in a new protein structure prediction paradigm.
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Three-dimensional (3D) maps of the hydropathic environments of protein amino acid residues are information-rich descriptors of preferred conformations, interaction types and energetics, and solvent accessibility. The interactions made by each residue are the primary factor for rotamer selection and the secondary, tertiary, and even quaternary protein structure. Our evolving basis set of environmental data for each residue type can be used to understand the protein structure. This work focuses on the aromatic residues phenylalanine, tyrosine, and tryptophan and their structural roles. We calculated and analyzed side chain-to-environment 3D maps for over 70,000 residues of these three types that reveal, with respect to hydrophobic and polar interactions, the environment around each. After binning with backbone Ï/ψ and side chain χ1, we clustered each bin by 3D similarities between map-map pairs. For each of the three residue types, four bins were examined in detail: one in the ß-pleat, two in the right-hand α-helix, and one in the left-hand α-helix regions of the Ramachandran plot. For high degrees of side chain burial, encapsulation of the side chain by hydrophobic interactions is ubiquitous. The more solvent-exposed side chains are more likely to be involved in polar interactions with their environments. Evidence for π-π interactions was observed in about half of the residues surveyed [phenylalanine (PHE): 53.3%, tyrosine (TYR): 34.1%, and tryptophan (TRP): 55.7%], but on an energy basis, this contributed to only â¼4% of the total. Evidence for π-cation interactions was observed in 14.1% of PHE, 8.3% of TYR, and 26.8% of TRP residues, but on an energy basis, this contributed to only â¼1%. This recognition of even these subtle interactions in the 3D hydropathic environment maps is key support for our interaction homology paradigm of protein structure elucidation and possibly prediction.
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Fenilalanina , Tirosina , Cátions , Proteínas , TriptofanoRESUMO
In our continuing efforts to develop novel neuroprotectants for Alzheimer's disease (AD), a series of analogs based on a lead compound that was recently shown to target the mitochondrial complex I were designed, synthesized and biologically characterized to understand the structure features that are important for neuroprotective activities. The results from a cellular AD model highlighted the important roles of the 4-OH on the phenyl ring and the 5-OCH3 on the indole ring of the lead compound. The results also demonstrated that the ß-keto moiety can be modified to retain or improve the neuroprotective activity. Docking studies of selected analogs to the FMN site of mitochondrial complex I also supported the observed neuroprotective activities. Collectively, the results provide further information to guide optimization and development of analogs based on this chemical scaffold as neuroprotectants with a novel mechanism of action for AD.
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Doença de Alzheimer/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Relação Estrutura-AtividadeRESUMO
Previously, we discovered that FOSL1 facilitates the metastasis of head and neck squamous cell carcinoma (HNSCC) cancer stem cells in a spontaneous mouse model. However, the molecular mechanisms remained unclear. Here, we demonstrated that FOSL1 serves as the dominant activating protein 1 (AP1) family member and is significantly upregulated in HNSCC tumor tissues and correlated with metastasis of HNSCC. Mechanistically, FOSL1 exerts its function in promoting tumorigenicity and metastasis predominantly via selective association with Mediators to establish super-enhancers (SEs) at a cohort of cancer stemness and pro-metastatic genes, such as SNAI2 and FOSL1 itself. Depletion of FOSL1 led to disruption of SEs and expression inhibition of these key oncogenes, which resulted in the suppression of tumor initiation and metastasis. We also revealed that the abundance of FOSL1 is positively associated with the abundance of SNAI2 in HNSCC and the high expression levels of FOSL1 and SNAI2 are associated with short overall disease-free survival. Finally, the administration of the FOSL1 inhibitor SR11302 significantly suppressed tumor growth and lymph node metastasis of HNSCC in a patient-derived xenograft model. These findings indicate that FOSL1 is a master regulator that promotes the metastasis of HNSCC through a SE-driven transcription program that may represent an attractive target for therapeutic interventions.
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Elementos Facilitadores Genéticos , Neoplasias de Cabeça e Pescoço/patologia , Proteínas Proto-Oncogênicas c-fos/genética , Fatores de Transcrição da Família Snail/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Retinoides/farmacologia , Retinoides/uso terapêutico , Fatores de Transcrição da Família Snail/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Mitochondrial dysfunction has been recognized as an essential contributor to many human diseases including neurodegenerative disorders. However, the exact pathological role of mitochondrial dysfunction, especially in mitochondrial reactive oxygen species-associated oxidative stress, remains elusive, partially due to the lack of chemical probes with well-defined mechanisms of action. Herein, we describe the characterization and discovery of a rationally designed small molecule ZCM-I-1 as a selective modulator of the production of reactive oxygen species from mitochondrial complex I that does not alter mitochondrial membrane potential and bioenergetics. Chemical biology studies employing photoaffinity probes derived from ZCM-I-1 demonstrated its novel mechanism of action of modulating complex I via interactions with the flavin mononucleotide site, proximal in the reaction pathway within complex I.
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Descoberta de Drogas , Complexo I de Transporte de Elétrons/metabolismo , Indóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Indóis/síntese química , Indóis/química , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Estrutura Molecular , Espécies Reativas de Oxigênio/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
The serotonin 5-HT7 G protein-coupled receptor (GPCR) is a proposed pharmacotherapeutic target for a variety of central and peripheral indications, albeit, there are no approved drugs selective for binding 5-HT7. We previously reported that a lead analog based on the 5-substituted-N,N-disubstituted-1,2,3,4-tetrahydronaphthalen-2-amine (5-substituted-2-aminotetralin, 5-SAT) scaffold binds with high affinity at the 5-HT7 GPCR, and can treat symptoms of autism in mouse models; subsequently, the lead was found to have high affinity at the 5-HT1A GPCR. Herein, we report the synthesis of novel 5-SAT analogs to develop a 3-dimensional quantitative structure-affinity relationship (3D-QSAR) at the human 5-HT7 receptor for comparison with similar studies at the highly homologous 5-HT1A receptor. We report 35 new 5-SAT ligands, some with very high affinity (Ki ≤ 1 nM) and stereoselectivity at 5-HT7 + or 5-HT1A receptors, several with modest selectivity (up to 12-fold) for binding at 5-HT7, and, several ligands with high selectivity (up to 40-fold) at the 5-HT1A receptor. 3D-QSAR results indicate that steric extensions at the C(5)-position improve selectivity for the 5-HT7 over 5-HT1A receptor, while steric and hydrophobic extensions at the chiral C(2)-amino position impart 5-HT1A selectivity. In silico receptor homology modeling studies, supplemented with molecular dynamics simulations and binding free energy calculations, were used to rationalize experimentally-determined receptor selectivity and stereoselective affinity results. The data from these studies indicate that the 5-SAT chemotype, previously shown to be safe and efficacious in rodent paradigms of neurodevelopmental and neuropsychiatric disorders, is amenable to structural modification to optimize affinity at serotonin 5-HT7 vs. 5-HT1A GPCRs, as may be required for successful clinical translation.
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Relação Quantitativa Estrutura-Atividade , Receptor 5-HT1A de Serotonina/metabolismo , Receptores de Serotonina/metabolismo , Tetra-Hidronaftalenos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo , Tetra-Hidronaftalenos/síntese química , Tetra-Hidronaftalenos/químicaRESUMO
Phosphatidylinositol-transfer proteins (PITPs) are key regulators of lipid signaling in eukaryotic cells. These proteins both potentiate the activities of phosphatidylinositol (PtdIns) 4-OH kinases and help channel production of specific pools of phosphatidylinositol 4-phosphate (PtdIns(4)P) dedicated to specific biological outcomes. In this manner, PITPs represent a major contributor to the mechanisms by which the biological outcomes of phosphoinositide are diversified. The two-ligand priming model proposes that the engine by which Sec14-like PITPs potentiate PtdIns kinase activities is a heterotypic lipid-exchange cycle where PtdIns is a common exchange substrate among the Sec14-like PITP family, but the second exchange ligand varies with the PITP. A major prediction of this model is that second-exchangeable ligand identity will vary from PITP to PITP. To address the heterogeneity in the second exchange ligand for Sec14-like PITPs, we used structural, computational, and biochemical approaches to probe the diversities of the lipid-binding cavity microenvironments of the yeast Sec14-like PITPs. The collective data report that yeast Sec14-like PITP lipid-binding pockets indeed define diverse chemical microenvironments that translate into differential ligand-binding specificities across this protein family.