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1.
J Mol Biol ; 436(16): 168640, 2024 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-38844044

RESUMEN

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how Protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study.

2.
bioRxiv ; 2024 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-38712280

RESUMEN

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study.

3.
Chembiochem ; 22(11): 1908-1914, 2021 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-33624332

RESUMEN

The PRMT5-MEP50 methyltransferase complex plays a key role in various cancers and is regulated by different protein-protein interactions. Several proteins have been reported to act as adaptor proteins that recruit substrate proteins to the active site of PRMT5 for the methylation of arginine residues. To define the interaction between these adaptor proteins and PRMT5, we employed peptide truncation and mutation studies and prepared truncated protein constructs. We report the characterisation of the interface between the TIM barrel of PRMT5 and the adaptor proteins pICln, RioK1 and COPR5, and identify the consensus amino acid sequence GQF[D/E]DA[E/D] involved in binding. Protein crystallography revealed that the RioK1 derived peptide interacts with a novel PPI site.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Factores de Transcripción/metabolismo , Humanos , Procesamiento Proteico-Postraduccional
4.
Angew Chem Int Ed Engl ; 60(4): 1813-1820, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33022847

RESUMEN

The scaffolding protein RbAp48 is part of several epigenetic regulation complexes and is overexpressed in a variety of cancers. In order to develop tool compounds for the study of RbAp48 function, we have developed peptide inhibitors targeting the protein-protein interaction interface between RbAp48 and the scaffold protein MTA1. Based on a MTA1-derived linear peptide with low micromolar affinity and informed by crystallographic analysis, a bicyclic peptide was developed that inhibits the RbAp48/MTA1 interaction with a very low nanomolar KD value of 8.56 nM, and which showed appreciable stability against cellular proteases. Design included exchange of a polar amide cyclization strategy to hydrophobic aromatic linkers enabling mono- and bicyclization by means of cysteine alkylation, which improved affinity by direct interaction of the linkers with a hydrophobic residue on RbAp48. Our results demonstrate that stepwise evolution of a structure-based design is a suitable strategy for inhibitor development targeting PPIs.


Asunto(s)
Diseño de Fármacos , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Proteína 4 de Unión a Retinoblastoma/antagonistas & inhibidores , Secuencia de Aminoácidos , Dicroismo Circular , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Mutación , Conformación Proteica , Termodinámica
5.
Nat Commun ; 11(1): 5425, 2020 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-33110077

RESUMEN

Transcription factors are key protein effectors in the regulation of gene transcription, and in many cases their activity is regulated via a complex network of protein-protein interactions (PPI). The chemical modulation of transcription factor activity is a long-standing goal in drug discovery but hampered by the difficulties associated with the targeting of PPIs, in particular when extended and flat protein interfaces are involved. Peptidomimetics have been applied to inhibit PPIs, however with variable success, as for certain interfaces the mimicry of a single secondary structure element is insufficient to obtain high binding affinities. Here, we describe the design and characterization of a stabilized protein tertiary structure that acts as an inhibitor of the interaction between the transcription factor TEAD and its co-repressor VGL4, both playing a central role in the Hippo signalling pathway. Modification of the inhibitor with a cell-penetrating entity yielded a cell-permeable proteomimetic that activates cell proliferation via regulation of the Hippo pathway, highlighting the potential of protein tertiary structure mimetics as an emerging class of PPI modulators.


Asunto(s)
Peptidomiméticos , Factores de Transcripción/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Vía de Señalización Hippo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
6.
J Med Chem ; 63(20): 11972-11989, 2020 10 22.
Artículo en Inglés | MEDLINE | ID: mdl-32907324

RESUMEN

Transcriptional enhanced associate domain (TEAD) transcription factors together with coactivators and corepressors modulate the expression of genes that regulate fundamental processes, such as organogenesis and cell growth, and elevated TEAD activity is associated with tumorigenesis. Hence, novel modulators of TEAD and methods for their identification are in high demand. We describe the development of a new "thiol conjugation assay" for identification of novel small molecules that bind to the TEAD central pocket. The assay monitors prevention of covalent binding of a fluorescence turn-on probe to a cysteine in the central pocket by small molecules. Screening of a collection of compounds revealed kojic acid analogues as TEAD inhibitors, which covalently target the cysteine in the central pocket, block the interaction with coactivator yes-associated protein with nanomolar apparent IC50 values, and reduce TEAD target gene expression. This methodology promises to enable new medicinal chemistry programs aimed at the modulation of TEAD activity.


Asunto(s)
Descubrimiento de Drogas , Pironas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Compuestos de Sulfhidrilo/farmacología , Factores de Transcripción/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Fluorescencia , Humanos , Modelos Moleculares , Estructura Molecular , Pironas/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/química , Factores de Transcripción/genética
7.
Angew Chem Int Ed Engl ; 59(46): 20338-20342, 2020 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-32537835

RESUMEN

DNA-encoded combinatorial synthesis provides efficient and dense coverage of chemical space around privileged molecular structures. The indole side chain of tryptophan plays a prominent role in key, or "hot spot", regions of protein-protein interactions. A DNA-encoded combinatorial peptoid library was designed based on the Ugi four-component reaction by employing tryptophan-mimetic indole side chains to probe the surface of target proteins. Several peptoids were synthesized on a chemically stable hexathymidine adapter oligonucleotide "hexT", encoded by DNA sequences, and substituted by azide-alkyne cycloaddition to yield a library of 8112 molecules. Selection experiments for the tumor-relevant proteins MDM2 and TEAD4 yielded MDM2 binders and a novel class of TEAD-YAP interaction inhibitors that perturbed the expression of a gene under the control of these Hippo pathway effectors.


Asunto(s)
ADN/metabolismo , Indoles/metabolismo , Peptidomiméticos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Factores de Transcripción/metabolismo , Humanos , Unión Proteica
8.
Chembiochem ; 20(24): 2987-2990, 2019 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-31680402

RESUMEN

The acyl-binding UNC119 proteins mediate the activation and transport of various N-myristoylated proteins. In particular, UNC119a plays a crucial role in the completion of cytokinesis. Herein, we report the use of a lipidated peptide originating from the UNC119 binding partner Gnat1 as the basis for the design of lipidated, stabilized α-helical peptides that target UNC119a. By using the hydrocarbon peptide-stapling approach, cell-permeable binders of UNC119a were generated that induced the accumulation of cytokinetic and binucleated cells; this suggests UNC119a as a potential target for the inhibition of cytokinesis.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Metabolismo de los Lípidos , Péptidos/metabolismo , Péptidos/farmacología , Proteínas Adaptadoras Transductoras de Señales/química , Secuencia de Aminoácidos , Células HeLa , Humanos , Modelos Moleculares , Terapia Molecular Dirigida , Péptidos/química , Unión Proteica , Conformación Proteica en Hélice alfa
9.
Chem Sci ; 10(25): 6341-6349, 2019 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-31341589

RESUMEN

Anthraquinones, a widely distributed class of aromatic natural products, are produced by a type II polyketide synthase system in the Gram-negative bacterium Photorhabdus luminescens. Heterologous expression of the antABCDEFGHI anthraquinone biosynthetic gene cluster in Escherichia coli identified AntI as an unusual lyase, catalysing terminal polyketide shortening prior to formation of the third aromatic ring. Functional in vitro and in vivo analysis of AntI using X-ray crystallography, structure-based mutagenesis, and molecular simulations revealed that AntI converts a defined octaketide to the tricyclic anthraquinone ring via retro-Claisen and Dieckmann reactions. Thus, AntI catalyses a so far unobserved multistep reaction in this PKS system.

10.
Beilstein J Org Chem ; 15: 535-541, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30873237

RESUMEN

The repeated and improper use of antibiotics had led to an increased number of multiresistant bacteria. Therefore, new lead structures are needed. Here, the synthesis and an expanded structure-activity relationship of the simple and antistaphylococcal amide nematophin from Xenorhabdus nematophila and synthetic derivatives are described. Moreover, the synthesis of intrinsic fluorescent derivatives, incorporating azaindole moieties was achieved for the first time.

11.
J Med Chem ; 61(9): 3930-3938, 2018 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-29660276

RESUMEN

Rhabdopeptides are a large class of nonribosomal peptides from the bacteria Xenorhabdus and Photorhabdus with low micromolar activity against different protozoa, which are the causative agents of several tropical diseases. The development of a facile and flexible synthesis combining backbone amide linking with on-resin peralkylation for the synthesis of permethylated rhabdopeptides is described. This strategy allows the fast generation of permethylated naturally occurring and artificial rhabdopeptides for a structure-activity study. Furthermore, in vitro experiments revealed their superior properties regarding their stability and passive membrane diffusion.


Asunto(s)
Amidas/química , Antiprotozoarios/química , Antiprotozoarios/farmacología , Péptidos/química , Péptidos/farmacología , Animales , Antiprotozoarios/metabolismo , Antiprotozoarios/farmacocinética , Técnicas de Química Sintética , Metilación , Nitrógeno/química , Péptidos/metabolismo , Péptidos/farmacocinética , Estabilidad Proteica , Ratas , Relación Estructura-Actividad , Xenorhabdus/química
12.
Angew Chem Int Ed Engl ; 56(35): 10294-10323, 2017 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-28186380

RESUMEN

Our ever-increasing understanding of biological systems is providing a range of exciting novel biological targets, whose modulation may enable novel therapeutic options for many diseases. These targets include protein-protein and protein-nucleic acid interactions, which are, however, often refractory to classical small-molecule approaches. Other types of molecules, or modalities, are therefore required to address these targets, which has led several academic research groups and pharmaceutical companies to increasingly use the concept of so-called "new modalities". This Review defines for the first time the scope of this term, which includes novel peptidic scaffolds, oligonucleotides, hybrids, molecular conjugates, as well as new uses of classical small molecules. We provide the most representative examples of these modalities to target large binding surface areas such as those found in protein-protein interactions and for biological processes at the center of cell regulation.


Asunto(s)
Descubrimiento de Drogas , Ácidos Nucleicos/antagonistas & inhibidores , Oligonucleótidos/farmacología , Proteínas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Humanos , Ácidos Nucleicos/química , Oligonucleótidos/química , Proteínas/química , Bibliotecas de Moléculas Pequeñas/química , Propiedades de Superficie
13.
Org Lett ; 19(4): 806-809, 2017 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-28134534

RESUMEN

Nematophin, a known antibiotic natural product against Staphylococcus aureus for almost 20 years, is produced by all strains of Xenorhabdus nematophila. Despite its simple structure, its biosynthesis was unknown. Its biosynthetic pathway is reported using heterologous production in Escherichia coli. Additionally, the identification, structure elucidation, and biosynthesis of six extended nematophin derivatives from Xenorhabdus PB62.4 carrying an additional valine are reported. Preliminary bioactivity studies suggest a biological role of these compounds in the bacteria-nematode-insect symbiosis.


Asunto(s)
Antibacterianos/biosíntesis , Animales , Antibacterianos/química , Antibacterianos/farmacología , Antiparasitarios/química , Antiparasitarios/metabolismo , Antiparasitarios/farmacología , Escherichia coli/metabolismo , Indoles/química , Indoles/metabolismo , Indoles/farmacología , Leishmania/efectos de los fármacos , Mioblastos/efectos de los fármacos , Mioblastos/parasitología , Nematodos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Ratas , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Trypanosoma/efectos de los fármacos , Xenorhabdus/metabolismo
14.
Chemistry ; 22(2): 639-45, 2016 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-26626278

RESUMEN

Many methods have been devised over the decades to trace precursors of specific molecules in cellular environments as, for example, in biosynthesis studies. The advent of click chemistry has facilitated the powerful combination of tracing and at the same time sieving the highly complex metabolome for compounds derived from simple or complex starting materials, especially when the click reaction takes place on a solid support. While the principle of solid-phase click reactions has already been successfully applied for selective protein and peptide enrichment, the successful enrichment of much smaller primary and secondary metabolites, showing great structural diversity and undergoing many different biosynthetic steps, has seen only little development. For bacterial secondary metabolism, a far broader tolerance for "clickable" precursors was observed than in ribosomal proteinogenesis, thus making this method a surprisingly valuable tool for the tracking and discovery of compounds within the cellular biochemical network. The implementation of this method has led to the identification of several new compounds from the bacterial genera Photorhabdus and Xenorhabdus, clearly proving its power.


Asunto(s)
Azidas/síntesis química , Productos Biológicos/síntesis química , Ácidos Grasos/química , Técnicas de Síntesis en Fase Sólida/métodos , Animales , Azidas/química , Productos Biológicos/química , Química Clic , Ácidos Grasos/metabolismo , Peces , Photorhabdus/química , Xenorhabdus/química
15.
Chem Sci ; 7(1): 339-345, 2016 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-29861986

RESUMEN

Despite recent advances, the direct Fmoc-based solid phase synthesis of peptide α-thioesters for the convergent synthesis of proteins via native chemical ligation (NCL) remains a challenge in the field. We herein report a simple and general methodology, enabling access to peptide thioester surrogates. A novel C-terminal N-(2-hydroxybenzyl)cysteine thioesterification device based on an amide-to-thioester rearrangement was developed, and the resulting peptide crypto-thioesters can be directly used in NCL reactions with fast N → S shift kinetics at neutral pH. These fast kinetics arise from our bio-inspired design, via intein-like intramolecular catalysis. Due to a well-positioned phenol moiety, an impressive >50 fold increase in the kinetic rate is observed compared to an O-methylated derivative. Importantly, the synthesis of this new device can be fully automated using inexpensive commercially available materials and does not require any post-synthetic steps prior to NCL. We successfully applied this new method to the synthesis of two long naturally-occurring cysteine-rich peptide sequences.

16.
Angew Chem Int Ed Engl ; 54(43): 12702-5, 2015 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-26465655

RESUMEN

Pyrrolizidine alkaloids (PAs) are widespread plant natural products with potent toxicity and bioactivity. Herein, the identification of bacterial PAs from entomopathogenic bacteria using differential analysis by 2D NMR spectroscopy (DANS) and mass spectrometry is described. Their biosynthesis was elucidated to involve a non-ribosomal peptide synthetase. The occurrence of these biosynthesis gene clusters in Gram-negative and Gram-positive bacteria indicates an important biological function in bacteria.


Asunto(s)
Bacterias/metabolismo , Alcaloides de Pirrolicidina/química , Alcaloides de Pirrolicidina/metabolismo , Bacterias/química , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Péptido Sintasas/metabolismo , Xenorhabdus/química , Xenorhabdus/enzimología , Xenorhabdus/metabolismo
17.
J Med Chem ; 55(20): 8588-602, 2012 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-22984902

RESUMEN

A series of fluorescent benzazepine ligands for the arginine-vasopressin V2 receptor (AVP V2R) was synthesized using "Click" chemistry. Their in vitro pharmacological profile at AVP V2R, V(1a)R, V(1b)R, and oxytocin receptor was measured by binding assay and functional studies. Compound 9p, labeled with Lissamine Rhodamine B using novel solid-phase organic tagging (SPOrT) resin, exhibited a high affinity for V2R (4.0 nM), an excellent selectivity toward V2R and antagonist properties. By changing the nature of the dye, DY647 and Lumi4-Tb probes 44 and 47 still display a high affinity for V2R (5.6 and 5.8 nM, respectively). These antagonists constitute the first high-affinity selective nonpeptidic fluorescent ligands for V2R. They enabled the development of V2R time-resolved FRET-based assay readily amenable to high-throughput screening. Taking advantage of their selectivity, these compounds were also successfully involved in the study of V(1a)R-V2R dimerization on cell surface.


Asunto(s)
Antagonistas de los Receptores de Hormonas Antidiuréticas , Benzazepinas/síntesis química , Colorantes Fluorescentes/síntesis química , Receptores de Vasopresinas/metabolismo , Benzazepinas/química , Benzazepinas/farmacología , Química Clic , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Ensayos Analíticos de Alto Rendimiento , Ligandos , Modelos Moleculares , Multimerización de Proteína , Receptores de Oxitocina/antagonistas & inhibidores , Receptores de Oxitocina/metabolismo , Solubilidad , Relación Estructura-Actividad
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