RESUMEN
General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound 39 engages GCN2 at levels ≥80% with an oral dose of 15 mg/kg BID. We also demonstrate the ability of compound 39 to alleviate MDSC-related T cell suppression and restore T cell proliferation, similar to the effect seen in MDSCs from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI) as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced by treatment with compound 39 demonstrating the complementarity of these two mechanisms.
Asunto(s)
Células Supresoras de Origen Mieloide , eIF-2 Quinasa , Animales , Hemo , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas , Linfocitos T/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
FRET (fluorescence resonance energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of λPR promoter DNA on Escherichia coli RNA polymerase (RNAP) in closed and open complexes (CC and OC, respectively). Here we determine the kinetics and mechanism of DNA bending and wrapping by FRET and of formation of RNAP contacts with -100 and +14 DNA by single-dye protein-induced fluorescence enhancement (PIFE). FRET and PIFE kinetics exhibit two phases: rapidly reversible steps forming a CC ensemble ({CC}) of four intermediates [initial (RPC), early (I1E), mid (I1M), and late (I1L)], followed by conversion of {CC} to OC via I1L. FRET and PIFE are first observed for I1E, not RPc. FRET and PIFE together reveal large-scale bending and wrapping of upstream and downstream DNA as RPC advances to I1E, decreasing the Cy3-Cy5 distance to â¼75 Å and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper ß'-clamp while downstream DNA contacts the top of the ß-pincer in I1E. Converting I1E to I1M (â¼1 s time scale) reduces FRET efficiency with little change in -100 or +14 PIFE, interpreted as clamp opening that moves far-upstream DNA (on ß') away from downstream DNA (on ß) to increase the Cy3-Cy5 distance by â¼14 Å. FRET increases greatly in converting I1M to I1L, indicating bending of downstream duplex DNA into the clamp and clamp closing to reduce the Cy3-Cy5 distance by â¼21 Å. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I1L is converted to the initial unstable OC (I2). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream binding, DNA-bending transcription activators are discussed.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Carbocianinas/química , ADN/química , ADN/genética , ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Cinética , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio-binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.
Asunto(s)
Conformación de Ácido Nucleico , Proteínas de Unión al ARN/genética , Secuencia de Aminoácidos/genética , Humanos , Cinética , Unión Proteica/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/química , Ribosomas/química , Ribosomas/genéticaRESUMEN
Poly-ADP-ribose polymerases (PARPs1-16) play pivotal roles in diverse cellular processes. PARPs that catalyze poly-ADP-ribosylation (PARylation) are the best characterized PARP family members because of the availability of potent and selective inhibitors for these PARPs. There has been comparatively little success in developing selective small-molecule inhibitors of PARPs that catalyze mono-ADP-ribosylation (MARylation), limiting our understanding of the cellular role of MARylation. Here we describe the structure-guided design of inhibitors of PARPs that catalyze MARylation. The most selective analog, ITK7, potently inhibits the MARylation activity of PARP11, a nuclear envelope-localized PARP. ITK7 is greater than 200-fold selective over other PARP family members. Using live-cell imaging, we show that ITK7 causes PARP11 to dissociate from the nuclear envelope. These results suggest that the cellular localization of PARP11 is regulated by its catalytic activity.
Asunto(s)
Biocatálisis/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Quinazolinonas/farmacología , Células HeLa , Humanos , Estructura Molecular , Inhibidores de Poli(ADP-Ribosa) Polimerasas/síntesis química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Transporte de Proteínas/efectos de los fármacos , Quinazolinonas/síntesis química , Quinazolinonas/químicaRESUMEN
Initial recognition of promoter DNA by RNA polymerase (RNAP) is proposed to trigger a series of conformational changes beginning with bending and wrapping of the 40-50 bp of DNA immediately upstream of the -35 region. Kinetic studies demonstrated that the presence of upstream DNA facilitates bending and entry of the downstream duplex (to +20) into the active site cleft to form an advanced closed complex (CC), prior to melting of â¼13 bp (-11 to +2), including the transcription start site (+1). Atomic force microscopy and footprinting revealed that the stable open complex (OC) is also highly wrapped (-60 to +20). To test the proposed bent-wrapped model of duplex DNA in an advanced RNAP-λP(R) CC and compare wrapping in the CC and OC, we use fluorescence resonance energy transfer (FRET) between cyanine dyes at far-upstream (-100) and downstream (+14) positions of promoter DNA. Similarly large intrinsic FRET efficiencies are observed for the CC (0.30 ± 0.07) and the OC (0.32 ± 0.11) for both probe orientations. Fluorescence enhancements at +14 are observed in the single-dye-labeled CC and OC. These results demonstrate that upstream DNA is extensively wrapped and the start site region is bent into the cleft in the advanced CC, reducing the distance between positions -100 and +14 on promoter DNA from >300 to <100 Å. The proximity of upstream DNA to the downstream cleft in the advanced CC is consistent with the proposed mechanism for facilitation of OC formation by upstream DNA.
Asunto(s)
ADN Viral/química , ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Modelos Moleculares , Regiones Promotoras Genéticas , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriófago lambda/metabolismo , Dominio Catalítico , ADN Viral/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Cinética , Conformación Molecular , Estabilidad Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Desplegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología Estructural de Proteína , Thermus thermophilus/enzimologíaRESUMEN
Understanding the functional and structural implication of a protein encoded in novel genes using function association or fold recognition approaches remains to be a challenging task in the current era of genomes, metagenomes and personal genomes. In an attempt to enhance potential-based fold-recognition methods in recognizing remote homology between proteins, we propose a new approach "Higher Order Residue Interaction Based ALgorithm for Fold REcognition (HORIBALFRE)". Higher order residue interactions refer to a class of interactions in protein structures mediated by C(α) or C(ß) atoms within a pre-defined distance cut-off. Higher order residue interactions (pairwise, triplet and quadruplet interactions) play a vital role in attaining the stable conformation of a protein structure. In HORIBALFRE, we incorporated the potential contributions from two body (pairwise) interactions, three body (triplet interactions) and four-body (quadruple interaction) interactions, to implement a new fold recognition algorithm. Core of HORIBALFRE algorithm includes the potentials generated from a library of protein structure derived from manually curated CAMPASS database of structure based sequence alignment. We used Fischer's dataset, with 68 templates and 56 target sequences, derived from SCOP database and performed one-against-all sequence alignment using TCoffee. Various potentials were derived using custom scripts and these potentials were incorporated in the HORIBALFRE algorithm. In this manuscript, we report outline of a novel fold recognition algorithm and initial results. Our results show that inclusion of quadruplet class of higher order residue interaction improves fold recognition.
RESUMEN
BACKGROUND: Folding of a protein into its three dimensional structure is influenced by both local and global interactions within a protein. Higher order residue interactions, like pairwise, triplet and quadruplet ones, play a vital role in attaining the stable conformation of the protein structure. It is generally agreed that higher order interactions make significant contribution to the potential energy landscape of folded proteins and therefore it is important to identify them to estimate their contributions to overall stability of a protein structure. RESULTS: We developed HORI [Higher order residue interactions in proteins], a web server for the calculation of global and local higher order interactions in protein structures. The basic algorithm of HORI is designed based on the classical concept of four-body nearest-neighbour propensities of amino-acid residues. It has been proved that higher order residue interactions up to the level of quadruple interactions plays a major role in the three-dimensional structure of proteins and is an important feature that can be used in protein structure analysis. CONCLUSION: HORI server will be a useful resource for the structural bioinformatics community to perform analysis on protein structures based on higher order residue interactions. HORI server is a highly interactive web server designed in three modules that enables the user to analyse higher order residue interactions in protein structures. HORI server is available from the URL: http://caps.ncbs.res.in/hori.