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[This corrects the article DOI: 10.1371/journal.pbio.2001882.].
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Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.
Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Animais , Linhagem Celular , Sistema Livre de Células , Colesterol/sangue , Escherichia coli/genética , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Masculino , Espectrometria de Massas , Terapia de Alvo Molecular , Pró-Proteína Convertase 9/sangue , Pró-Proteína Convertase 9/genética , Biossíntese de Proteínas/fisiologia , Coelhos , Ratos , Ratos Sprague-Dawley , Ribossomos/metabolismo , Ribossomos/fisiologiaRESUMO
CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.
Assuntos
Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes , Linhagem Celular Tumoral , Endonucleases/genética , Células Hep G2 , Humanos , Estrutura Molecular , Engenharia de ProteínasRESUMO
Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22-27) directing the binding of Phe(22) into a hydrophobic pocket on the GLP-1R.
Assuntos
Conotoxinas/química , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeos/química , Proteínas Recombinantes de Fusão/química , Peçonhas/química , Animais , Células CHO , Conotoxinas/genética , Cricetinae , Cricetulus , Exenatida , Peptídeo 1 Semelhante ao Glucagon/genética , Humanos , Peptídeos/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Peçonhas/genéticaRESUMO
A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.
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Receptor de Asialoglicoproteína/metabolismo , Compostos Bicíclicos com Pontes/química , Hepatócitos/metabolismo , Cetonas/química , Fígado/metabolismo , Polímeros/química , Compostos Bicíclicos com Pontes/metabolismo , Cristalografia por Raios X , Portadores de Fármacos/química , Humanos , Cetonas/metabolismo , Fígado/citologia , Modelos Moleculares , Estrutura Molecular , Polímeros/metabolismoRESUMO
The synthesis and in vivo pharmacokinetic profile of an analogue of cyclosporine is disclosed. An acyclic congener was also profiled in in vitro assays to compare cell permeability. The compounds possess similar calculated and measured molecular descriptors however have different behaviors in an RRCK assay to assess cell permeability.
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Ciclosporina/farmacocinética , Oligopeptídeos/farmacocinética , Animais , Ciclosporina/administração & dosagem , Ciclosporina/química , Masculino , Conformação Molecular , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Ratos , Ratos Wistar , EstereoisomerismoRESUMO
Enhancing the oral bioavailability of peptide drug leads is a major challenge in drug design. As such, methods to address this challenge are highly sought after by the pharmaceutical industry. Here, we propose a strategy to identify appropriate amides for N-methylation using temperature coefficients measured by NMR to identify exposed amides in cyclic peptides. N-methylation effectively caps these amides, modifying the overall solvation properties of the peptides and making them more membrane permeable. The approach for identifying sites for N-methylation is a rapid alternative to the elucidation of 3D structures of peptide drug leads, which has been a commonly used structure-guided approach in the past. Five leucine-rich peptide scaffolds are reported with selectively designed N-methylated derivatives. In vitro membrane permeability was assessed by parallel artificial membrane permeability assay and Caco-2 assay. The most promising N-methylated peptide was then tested in vivo. Here we report a novel peptide (15), which displayed an oral bioavailability of 33% in a rat model, thus validating the design approach. We show that this approach can also be used to explain the notable increase in oral bioavailability of a somatostatin analog.
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Amidas/química , Desenho de Fármacos , Espectroscopia de Ressonância Magnética , Peptídeos/farmacocinética , Administração Oral , Animais , Disponibilidade Biológica , Células CACO-2 , Cromatografia Líquida , Humanos , Ligação de Hidrogênio , Espectrometria de Massas , Metilação , Peptídeos/síntese química , Permeabilidade , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Ratos , Ratos Wistar , Solventes/química , Tecnologia Farmacêutica/métodos , TemperaturaRESUMO
Targeting of the human ribosome is an unprecedented therapeutic modality with a genome-wide selectivity challenge. A liver-targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesteraseâ 1. The synthesis of select tetrazole prodrugs was crucial. A cell-free inâ vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. Inâ vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography 18 F-isotopologue validated our liver-targeting approach.
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Fígado/efeitos dos fármacos , Inibidores de PCSK9 , Pró-Proteína Convertase 9/biossíntese , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/enzimologia , Fígado/metabolismo , Estrutura Molecular , Pró-Proteína Convertase 9/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Disulfide-rich cyclic peptides have exciting potential as leads or frameworks in drug discovery; however, their use is faced with some synthetic challenges, mainly associated with construction of the circular backbone and formation of the correct disulfides. Here we describe a simple and efficient Fmoc solid-phase peptide synthesis (SPPS)-based method for synthesizing disulfide-rich cyclic peptides. This approach involves SPPS on 2-chlorotrityl resin, cyclization of the partially protected peptide in solution, cleavage of the side-chain protecting groups, and oxidization of cysteines to yield the desired product. We illustrate this method with the synthesis of peptides from three different classes of cyclic cystine knot motif-containing cyclotides: Möbius (M), trypsin inhibitor (T), and bracelet (B). We show that the method is broadly applicable to peptide engineering, illustrated by the synthesis of two mutants and three grafted analogues of kalata B1. The method reduces the use of highly caustic and toxic reagents and is better suited for high-throughput synthesis than previously reported methods for producing disulfide-rich cyclic peptides, thus offering great potential to facilitate pharmaceutical optimization of these scaffolds.
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Ciclotídeos/síntese química , Cisteína/química , Dissulfetos/química , Fluorenos/química , Peptídeos Cíclicos/síntese química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Ciclização , Ciclotídeos/química , Oxirredução , Peptídeos Cíclicos/química , Técnicas de Síntese em Fase SólidaRESUMO
The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR-derived structures, amide H-D exchange rates, and temperature-dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N-methylation.
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Oligopeptídeos/farmacocinética , Peptídeos Cíclicos/farmacocinética , Administração Oral , Sequência de Aminoácidos , Disponibilidade Biológica , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/química , Conformação ProteicaRESUMO
Helix-constrained polypeptides have attracted great interest for modulating protein-protein interactions (PPI). It is not known which are the most effective helix-inducing strategies for designing PPI agonists/antagonists. Cyclization linkers (X1-X5) were compared here, using circular dichroism and 2D NMR spectroscopy, for α-helix induction in simple model pentapeptides, Ac-cyclo(1,5)-[X1-Ala-Ala-Ala-X5]-NH2, in water. In this very stringent test of helix induction, a Lys1âAsp5 lactam linker conferred greatest α-helicity, hydrocarbon and triazole linkers induced a mix of α- and 310-helicity, while thio- and dithioether linkers produced less helicity. The lactam-linked cyclic pentapeptide was also the most effective α-helix nucleator attached to a 13-residue model peptide.
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Oligopeptídeos/química , Peptídeos Cíclicos/química , Água/química , Sequência de Aminoácidos , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Estrutura Secundária de Proteína , TemperaturaRESUMO
Backbone N-methylation is common among peptide natural products and has a substantial impact on both the physical properties and the conformational states of cyclic peptides. However, the specific impact of N-methylation on passive membrane diffusion in cyclic peptides has not been investigated systematically. Here we report a method for the selective, on-resin N-methylation of cyclic peptides to generate compounds with drug-like membrane permeability and oral bioavailability. The selectivity and degree of N-methylation of the cyclic peptide was dependent on backbone stereochemistry, suggesting that conformation dictates the regiochemistry of the N-methylation reaction. The permeabilities of the N-methyl variants were corroborated by computational studies on a 1,024-member virtual library of N-methyl cyclic peptides. One of the most permeable compounds, a cyclic hexapeptide (molecular mass = 755 Da) with three N-methyl groups, showed an oral bioavailability of 28% in rat.
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Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacocinética , Animais , Disponibilidade Biológica , Química Farmacêutica , Técnicas de Química Combinatória , Simulação por Computador , Descoberta de Drogas/métodos , Masculino , Metilação , Estrutura Molecular , Peptídeos Cíclicos/química , Ratos , Relação Estrutura-AtividadeRESUMO
Conditions have been identified for the efficient Ullmann macrocyclization of phenol and imidazole nucleophiles with aryl iodides at high reaction concentrations of up to 100 mM and using 5-10 mol % loading of an inexpensive copper catalyst. A range of substitution patterns and ring sizes are tolerated, and the method has been exemplified by the synthesis of a set of druglike macrocycles.
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Desenho de Fármacos , Compostos Macrocíclicos/química , Compostos Macrocíclicos/síntese química , Técnicas de Química Sintética , Química Farmacêutica , Ciclização , Histidina/química , Imidazóis/química , Fenóis/química , Tirosina/químicaRESUMO
A general method for constraining peptide conformations via linkage of aromatic sidechains has been developed. Macrocyclization of suitably functionalized tri-, tetra- and pentapeptides via Suzuki-Miyaura cross-coupling has been used to generate side chain to side chain, biaryl-bridged 14- to 21-membered macrocyclic peptides. Biaryl bridges possessing three different configurations, meta-meta, meta-ortho, and ortho-meta, were systematically explored through regiochemical variation of the aryl halide and aryl boronate coupling partners, allowing fine-tuning of the resultant macrocycle conformation. Suzuki-Miyaura macrocyclizations were successfully achieved both in solution and on solid phase for all three sizes of peptide. This approach constitutes a means of constraining peptide conformation via direct carbogenic fusion of side chains of naturally occurring amino acids such as phenylalanine and tyrosine, and so is complementary to strategies involving non-natural, for example, hydrocarbon, bridges.
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Aminoácidos/química , Peptídeos Cíclicos/química , Peptídeos/química , Fenilalanina/química , Tirosina/química , Estrutura Molecular , Soluções , EstereoisomerismoRESUMO
The application of conformationally dependent measures of size and polarity to characterize beyond rule-of-5 (Ro5) space for passive permeation was investigated. Specifically, radius of gyration, an alternative to molecular weight, and three-dimensional polar surface area and the generalized Born/surface area dehydration free energy, alternatives to hydrogen-bond donor and acceptor counts, were computed on models of the permeating conformations of over 35 000 molecules. The resulting guidelines for size and polarity, described by the 3D properties, should aid the design of Ro5 violators with passive permeability.
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Membrana Celular/metabolismo , Modelos Químicos , Medicamentos sob Prescrição/química , Animais , Permeabilidade da Membrana Celular , Cães , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Células Madin Darby de Rim Canino , Conformação Molecular , Estrutura Molecular , Peso Molecular , Eletricidade Estática , TermodinâmicaRESUMO
In this paper, we describe a lead transformation tool, NEAT (Novel and Electronically equivalent Aromatic Template), which can help identify novel aromatic rings that are estimated to have similar electrostatic potentials, dipoles, and hydrogen bonding capabilities to a query template; hence, they may offer similar bioactivity profiles. In this work, we built a comprehensive heteroaryl database, and precalculated high-level quantum mechanical (QM) properties, including electrostatic potential charges, hydrogen bonding ability, dipole moments, chemical reactivity, and othe properties. NEAT bioisosteric similarities are based on the electrostatic potential surface calculated by Brood, using the precalculated QM ESP charges and other QM properties. Compared with existing commercial lead transformation software, (1) NEAT is the only one that covers the comprehensive heteroaryl chemical space, and (2) NEAT offers a better characterization of novel aryl cores by using high-evel QM properties that are relevant to molecular interactions. NEAT provides unique value to medicinal chemists quickly exploring the largely uncharted aromatic chemical space, and one successful example of its application is discussed herein.
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Descoberta de Drogas , Hidrocarbonetos Aromáticos/química , Modelos Químicos , Teoria Quântica , Humanos , Piperazinas/química , Purinas/química , Citrato de Sildenafila , Sulfonas/químicaRESUMO
Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu â Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.
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Anemia Falciforme/tratamento farmacológico , Hemoglobina A/efeitos dos fármacos , Hemoglobina Falciforme/efeitos dos fármacos , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Animais , Eritrócitos/metabolismo , Camundongos , Oxigênio/metabolismo , Quinolinas/químicaRESUMO
The design and synthesis of novel opiates are reported. Based on the message-address principle a novel class of 4,4- and 3,3-biaryl piperidines was designed and synthesized. Biological evaluation confirmed that these compounds exhibit high affinity and selectivity for the delta opioid receptor. Key structure-activity relationships that influence affinity, selectivity, functional activity and clearance are reported.
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Ligantes , Piperidinas/química , Receptores Opioides delta/antagonistas & inibidores , Animais , Desenho de Fármacos , Humanos , Microssomos Hepáticos/metabolismo , Piperidinas/síntese química , Piperidinas/farmacologia , Ligação Proteica , Ratos , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/metabolismo , Relação Estrutura-AtividadeRESUMO
Preclinical and clinical data suggest that acetyl-CoA carboxylase (ACC) inhibitors have the potential to rebalance disordered lipid metabolism, leading to improvements in nonalcoholic steatohepatitis (NASH). Consistent with these observations, first-in-human clinical trials with our ACC inhibitor PF-05175157 led to robust reduction of de novo lipogenesis (DNL), albeit with concomitant reductions in platelet count, which were attributed to the inhibition of fatty acid synthesis within bone marrow. Herein, we describe the design, synthesis, and evaluation of carboxylic acid-based ACC inhibitors with organic anion transporting polypeptide (OATP) substrate properties, which facilitated selective distribution of the compounds at the therapeutic site of action (liver) relative to the periphery. These efforts led to the discovery of clinical candidate PF-05221304 (12), which selectively inhibits liver DNL in animals, while demonstrating considerable safety margins against platelet reduction in a nonhuman primate model.
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Acetil-CoA Carboxilase/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/farmacologia , Fígado/efeitos dos fármacos , Acetil-CoA Carboxilase/metabolismo , Animais , Inibidores Enzimáticos/uso terapêutico , Humanos , Lipogênese , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Especificidade por SubstratoRESUMO
Macrocycles have emerged as a viable approach for the modulation of tough targets in drug discovery. In this Innovations article we discuss recent progress toward the design of cell permeable and orally bioavailable peptide macrocycles and cyclotides and provide a perspective for their potential as therapeutics. We highlight design concepts that may be broadly relevant to drug discovery efforts beyond the rule of five.