RESUMEN
Nsp1 is one of the first proteins expressed from the SARS-CoV-2 genome and is a major virulence factor for COVID-19. A rapid multiplexed assay for detecting the action of Nsp1 was developed in cultured lung cells. The assay is based on the acute cytopathic effects induced by Nsp1. Virtual screening was used to stratify compounds that interact with two functional Nsp1 sites: the RNA-binding groove and C-terminal helix-loop-helix region. Experimental screening focused on compounds that could be readily repurposed to treat COVID-19. Multiple synergistic combinations of compounds that significantly inhibited Nsp1 action were identified. Among the most promising combinations are Ponatinib, Rilpivirine, and Montelukast, which together, reversed the toxic effects of Nsp1 to the same extent as null mutations in the Nsp1 gene.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Línea Celular , Humanos , SARS-CoV-2 , Proteínas no Estructurales Virales/metabolismo , Factores de VirulenciaRESUMEN
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a nonenveloped, linear, single-stranded DNA virus belonging to the family Parvoviridae and is a World Organisation for Animal Health (OIE)-notifiable crustacean pathogen. During screening of Penaeus vannamei shrimp from 3 commercial shrimp facilities in the United States for a panel of OIE-listed (n = 7) and nonlisted (n = 2) crustacean diseases, shrimp from these facilities tested positive for IHHNV. Nucleotide sequences of PCR amplicons showed 99%-100% similarity to IHHNV isolates from Latin America and Asia. The whole genome of the isolates also showed high similarity to type 2 infectious forms of IHHNV. Phylogenetic analysis using capsid gene and whole-genome sequences demonstrated that the isolates clustered with an IHHNV isolate from Ecuador. The detection of an OIE-listed crustacean pathogen in the United States highlights the need for biosecurity protocols in hatcheries and grow-out ponds to mitigate losses.
Asunto(s)
Densovirinae , Penaeidae , Animales , Densovirinae/genética , Genoma , Penaeidae/genética , Filogenia , Reacción en Cadena de la Polimerasa , Estados Unidos/epidemiologíaRESUMEN
Toll-like receptors (TLRs) play key role in innate immune response to Damage Associated Molecular Patterns (DAMPs) and Pathogen Associated Molecular Patterns (PAMPs). DAMP/PAMP-mediated activation of TLRs triggers NFκB signaling resulting in pro-inflammatory cytokine release. Using TLR2-Pam2CSK4 agonist co-crystal structure information, we designed and synthesized a novel series of Toll-like Receptor 2 (TLR2) lipid antagonists and identified compounds 14, 15 and 17 with sub-micromolar potency. TLR2 antagonists that we identified are stable forâ¯>â¯1.0â¯h in both gastric juice and PBS buffer and could be used as research tools.
Asunto(s)
Lípidos/química , Oligopéptidos/química , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 9/agonistas , Cristalización , Citocinas/metabolismo , Descubrimiento de Drogas , Humanos , FN-kappa B/metabolismo , Unión Proteica , Transducción de Señal , Relación Estructura-Actividad , Receptor Toll-Like 2/química , Receptor Toll-Like 9/químicaRESUMEN
Riproximin (Rpx) is a type II ribosome-inactivating protein with specific anti-proliferative activity. It was purified from Ximenia americana by affinity chromatography using a resin coupled with lactosyl residues. The same technique facilitated isolation of proteins with lectin-like properties from human Suit2-007 and rat ASML pancreatic cancer cells, which were termed lactosyl-sepharose binding proteins (LSBPs). The role of these proteins in cancer progression was investigated at mRNA level using chip array data of Suit2-007 and ASML cells re-isolated from nude rats. These data compared significant mRNA expression changes when relating primary (pancreas) and metastatic (liver) sites following orthotopic and intraportal implantation of Pancreatic Ductal Adenocarcinoma (PDAC) cells, respectively. The affinity of Rpx to 13 simple sugar structures was modeled by docking experiments, the ranking of which was principally confirmed by NMR-spectroscopy. In addition, Rpx and LSBPs were evaluated for anti-proliferative activity and their cellular uptake was assessed by fluorescence microscopy. From 13 monosaccharides evaluated, open-chain rhamnose, ß-d-galactose, and α-l-galactopyranose showed the highest affinities for site 1 of Rpx's B-chain. NMR evaluation yielded a similar ranking, as galactose was among the best binders. Both, Rpx and LSBPs reduced cell proliferation in vitro, but their anti-proliferative effects were decreased by 15-20% in the presence of galactose. The program "Ingenuity Pathway Analysis" identified 2,415 genes showing significantly modulated mRNA expression following exposure of Suit2-007 cells to Rpx in vitro. These genes were then matched to those 1,639 genes, which were significantly modulated in the rat model when comparing primary and metastatic growth of Suit2-007 cells. In this overlap analysis, LSBP genes were considered separately. The potential suitability of Rpx for treating metastatic Suit2-007 PDAC cells was reflected by those genes, which were modulated by Rpx in a way opposite to that observed in cancer progression. Remarkably, these were 14% of all genes modulated during cancer progression, but 71% of the respective LSBP gene subgroup. Based on these findings, we predict that Rpx has the potential to treat PDAC metastasis by modulating genes involved in metastatic progression, especially by targeting LSBPs.
RESUMEN
Methane-producing archaea are among a select group of microorganisms that utilize tetrahydromethanopterin (H4MPT) as a one-carbon carrier instead of tetrahydrofolate. In H4MPT biosynthesis, ß-ribofuranosylaminobenzene 5'-phosphate (RFAP) synthase catalyzes the production of RFAP, CO2, and pyrophosphate from p-aminobenzoic acid (pABA) and phosphoribosyl-pyrophosphate (PRPP). In this work, to gain insight into amino acid residues required for substrate binding, RFAP synthase from Methanothermobacter thermautotrophicus was produced in Escherichia coli, and site-directed mutagenesis was used to alter arginine 26 (R26) and aspartic acid 19 (D19), located in a conserved sequence of amino acids resembling the pABA binding site of dihydropteroate synthase. Replacement of R26 with lysine increased the KM for pABA by an order of magnitude relative to wild-type enzyme without substantially altering the KM for PRPP. Although replacement of D19 with alanine produced inactive enzyme, asparagine substitution allowed retention of some activity, and the K M for pABA increased about threefold relative to wild-type enzyme. A molecular model developed by threading RFAP synthase onto the crystal structure of homoserine kinase places R26 in the proposed active site. In the static model, D19 is located close to the active site, yet appears too far away to influence ligand binding directly. This may be indicative of the protein conformational change predicted previously in the Bi-Ter kinetic mechanism and/or formation of the active site at the interface of two subunits. Due to the vital role of RFAP synthase in H4MPT biosynthesis, insights into the mode of substrate binding and mechanism could be beneficial for developing RFAP synthase inhibitors designed to reduce the production of methane as a greenhouse gas.
RESUMEN
Psilocybin and its direct precursor baeocystin are indole alkaloids of psychotropic Psilocybe mushrooms. The pharmaceutical interest in psilocybin as a treatment option against depression and anxiety is currently being investigated in advanced clinical trials. Here, we report a biocatalytic route to synthesize 6-methylated psilocybin and baeocystin from 4-hydroxy-6-methyl-l-tryptophan, which was decarboxylated and phosphorylated by the Psilocybe cubensis biosynthesis enzymes PsiD and PsiK. N-Methylation was catalyzed by PsiM. We further present an in silico structural model of PsiM that revealed a well-conserved SAM-binding core along with peripheral nonconserved elements that likely govern substrate preferences.
Asunto(s)
Alcaloides/síntesis química , Indoles/síntesis química , Metiltransferasas/química , Organofosfatos/síntesis química , Psilocibina/análogos & derivados , Psilocibina/síntesis química , Proteínas Bacterianas/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Metilación , Metiltransferasas/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Psilocybe/enzimología , S-Adenosilmetionina/metabolismo , Salmonella enterica/enzimología , Triptófano Sintasa/químicaRESUMEN
Allosteric modulation of receptors provides mechanistic safety while effectively achieving biologic endpoints otherwise difficult or impossible to obtain by other means. The theoretical case has been made for the development of a positive allosteric modulator (PAM) of the type 1 cholecystokinin receptor (CCK1R) having minimal intrinsic agonist activity to enhance meal-induced satiety for the treatment of obesity, while reducing the risk of side effects and/or toxicity. Unfortunately, such a drug does not currently exist. In this work, we have identified a PAM agonist of the CCK1R, SR146131, and determined its putative binding mode and receptor activation mechanism by combining molecular modeling, chimeric CCK1R/CCK2R constructs, and site-directed mutagenesis. We probed the structure-activity relationship of analogs of SR146131 for impact on agonism versus cooperativity of the analogs. This identified structural features that might be responsible for binding affinity and potency while retaining PAM activity. SR146131 and several of its analogs were docked into the receptor structure, which had the natural endogenous peptide agonist, cholecystokinin, already in the bound state (by docking), providing a refined structural model of the intact CCK1R holoreceptor. Both SR146131 and its analogs exhibited unique probe-dependent cooperativity with orthosteric peptide agonists and were simultaneously accommodated in this model, consistent with the derived structure-activity relationships. This provides improved understanding of the molecular basis for CCK1R-directed drug development.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Colecistoquinina/metabolismo , Receptores de Colecistoquinina/agonistas , Receptores de Colecistoquinina/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Células CHO , Línea Celular , Cricetulus , Indoles/farmacología , Mutagénesis Sitio-Dirigida/métodos , Péptidos/metabolismo , Relación Estructura-Actividad , Tiazoles/farmacologíaRESUMEN
Afamin, a human plasma glycoprotein and putative transporter of hydrophobic molecules, has been shown to act as extracellular chaperone for poorly soluble, acylated Wnt proteins, forming a stable, soluble complex with functioning Wnt proteins. The 2.1-Å crystal structure of glycosylated human afamin reveals an almost exclusively hydrophobic binding cleft capable of harboring large hydrophobic moieties. Lipid analysis confirms the presence of lipids, and density in the primary binding pocket of afamin was modeled as palmitoleic acid, presenting the native O-acylation on serine 209 in human Wnt3a. The modeled complex between the experimental afamin structure and a Wnt3a homology model based on the XWnt8-Fz8-CRD fragment complex crystal structure is compelling, with favorable interactions comparable with the crystal structure complex. Afamin readily accommodates the conserved palmitoylated serine 209 of Wnt3a, providing a structural basis how afamin solubilizes hydrophobic and poorly soluble Wnt proteins.
Asunto(s)
Proteínas Portadoras/química , Glicoproteínas/química , Albúmina Sérica Humana/química , Proteína Wnt3A/metabolismo , Acetilación , Sitios de Unión , Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Humanos , Lipoilación , Simulación del Acoplamiento Molecular , Unión Proteica , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Transporte de Proteínas , Albúmina Sérica Humana/metabolismo , Proteína Wnt3A/químicaRESUMEN
Amino-terminal regions of secretin-family peptides contain key determinants for biological activity and binding specificity, although the nature of interactions with receptors is unclear. A helix N-capping motif within this region has been postulated to directly contribute to agonist activity while also stabilizing formation of a helix extending toward the peptide carboxyl terminus and docking within the receptor amino terminus. We used cysteine trapping to systematically explore spatial approximations between cysteines replacing each residue in this motif of secretin (sec), Phe(6), Thr(7), and Leu(10), and cysteines incorporated into the extracellular face of the receptor. Each peptide was a full agonist for cAMP, but had a lower binding affinity than natural hormone. These bound to COS cells expressing 61 receptor constructs incorporating cysteines in every position along each extracellular loop (ECL) and adjacent parts of transmembrane (TM) segments. Patterns of covalent labeling were distinct for each probe, with Cys(6)-sec labeling multiple residues in the carboxyl-terminal half of ECL2 and throughout ECL3, Cys(7)-sec predominantly labeling only single residues in the carboxyl-terminal end of ECL2 and the amino-terminal end of ECL3, and Cys(10)-sec not efficiently labeling any of these residues. These spatial constraints were used to refine our model of secretin bound to its receptor, now bringing ECL3 above the amino terminus of the ligand and revealing possible charge-charge interactions between this part of secretin and receptor residues in TM5, TM6, ECL2, and ECL3, which can orient and stabilize the peptide-receptor complex. This was validated by testing predicted approximations by mutagenesis and residue-residue complementation studies.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Receptores de la Hormona Gastrointestinal/química , Secretina/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Cisteína/genética , Cisteína/metabolismo , Humanos , Datos de Secuencia Molecular , Unión Proteica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Secretina/genética , Secretina/metabolismoRESUMEN
The type 1 cholecystokinin receptor (CCK1R) has multiple physiologic roles relating to nutrient homeostasis, including mediation of postcibal satiety. This effect has been central in efforts to develop agonists of this receptor as part of a program to manage and/or prevent obesity. While a number of small molecule CCK1R agonists have been developed, none have yet been approved for clinical use, based on inadequate efficacy, side effects, or the potential for toxicity. Understanding the molecular details of docking and mechanism of action of these ligands can be helpful in the rational refinement and enhancement of small molecule drug candidates. In the current work, we have defined the mechanism of binding and activity of two triazolobenzodiazepinones, CE-326597 and PF-04756956, which are reported to be full agonist ligands. To achieve this, we utilized receptor binding with a series of allosteric and orthosteric radioligands at structurally related CCK1R and CCK2R, as well as chimeric CCK1R/CCK2R constructs exchanging residues in the allosteric pocket, and assessment of biological activity. These triazolobenzodiazepinones docked within the intramembranous small molecule allosteric ligand pocket, with higher affinity binding to CCK2R than CCK1R, yet with biological activity exclusive to or greatly enhanced at CCK1R. These ligands exhibited cooperativity with benzodiazepine binding across the CCK1R homodimeric complex, resulting in their ability to inhibit only a fraction of the saturable binding of a benzodiazepine radioligand, unlike other small molecule antagonists and agonists of this receptor. This may contribute to the understanding of the unique short duration and reversible gallbladder contraction observed in vivo upon administration of these drugs.
Asunto(s)
Benzodiazepinas/química , Benzodiazepinonas/química , Receptor de Colecistoquinina A/agonistas , Triazoles/química , Sitio Alostérico , Secuencia de Aminoácidos , Animales , Benzodiazepinas/farmacología , Benzodiazepinonas/farmacología , Células CHO , Cricetulus , Humanos , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutación , Multimerización de Proteína , Ensayo de Unión Radioligante , Ratas , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina A/metabolismo , Receptor de Colecistoquinina B/genética , Receptor de Colecistoquinina B/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad , Triazoles/farmacologíaRESUMEN
Cyclophilins are peptidyl cis-trans prolyl isomerases (PPIases), whose activity is typically inhibited by cyclosporine A (CsA), a potent immunosuppressor. Cyclophilins are also chaperones. Emerging evidence supports that cyclophilins present nonoverlapping PPIase and chaperone activities. The proteostasis of the disease-relevant substrates, signal transducer and activator of transcription 3 and 5 (STAT3/STAT5), heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), and M-opsin, is regulated by nonoverlapping chaperone and PPIase activities of the cyclophilin domain (CY) of Ranbp2, a multifunctional and modular scaffold that controls nucleocytoplasmic shuttling and proteostasis of selective substrates. Although highly homologous, CY and the archetypal cyclophilin A (CyPA) present distinct catalytic and CsA-binding activities owing to unique structural features between these cylophilins. We explored structural idiosyncrasies between CY and CyPA to screen in silico nearly 9 million small molecules (SM) against the CY PPIase pocket and identify SMs with selective bioactivity toward STAT3, hnRNPA2B1, or M-opsin proteostasis. We found three classes of SMs that enhance the cytokine-stimulated transcriptional activity of STAT3 without changing latent and activated STAT3 levels, down-regulate hnRNPA2B1 or M-opsin proteostasis, or a combination of these. Further, a SM that suppresses hnRNPA2B1 proteostasis also inhibits strongly and selectively the PPIase activity of CY. This study unravels chemical probes for multimodal regulation of CY of Ranbp2 and its substrates, and this regulation likely results in the allosterism stemming from the interconversion of conformational substates of cyclophilins. The results also demonstrate the feasibility of CY in drug discovery against disease-relevant substrates controlled by Ranbp2, and they open new opportunities for therapeutic interventions.
Asunto(s)
Ciclofilina A/química , Ciclofilina A/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Opsinas/metabolismo , Factor de Transcripción STAT3/metabolismo , Secuencia de Aminoácidos , Ciclofilina A/genética , Células HeLa , Humanos , Ligandos , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Estructura Molecular , Proteínas de Complejo Poro Nuclear/genética , Homología de Secuencia de AminoácidoRESUMEN
Understanding the molecular basis of ligand binding to receptors provides insights useful for rational drug design. This work describes development of a new antagonist radioligand of the type 1 cholecystokinin receptor (CCK1R), (2-fluorophenyl)-2,3-dihydro-3-[(3-isoquinolinylcarbonyl)amino]-6-methoxy-2-oxo-l-H-indole-3-propanoate (T-0632), and exploration of the molecular basis of its binding. This radioligand bound specifically with high affinity within an allosteric pocket of CCK1R. T-0632 fully inhibited binding and action of CCK at this receptor, while exhibiting no saturable binding to the closely related type 2 cholecystokinin receptor (CCK2R). Chimeric CCK1R/CCK2R constructs were used to explore the molecular basis of T-0632 binding. Exchanging exonic regions revealed the functional importance of CCK1R exon 3, extending from the bottom of transmembrane segment (TM) 3 to the top of TM5, including portions of the intramembranous pocket as well as the second extracellular loop region (ECL2). However, CCK1R mutants in which each residue facing the pocket was changed to that present in CCK2R had no negative impact on T-0632 binding. Extending the chimeric approach to ECL2 established the importance of its C-terminal region, and site-directed mutagenesis of each nonconserved residue in this region revealed the importance of Ser(208) at the top of TM5. A molecular model of T-0632-occupied CCK1R was consistent with these experimental determinants, also identifying Met(121) in TM3 and Arg(336) in TM6 as important. Although these residues are conserved in CCK2R, mutating them had a distinct impact on the two closely related receptors, suggesting differential orientation. This establishes the molecular basis of binding of a highly selective nonpeptidyl allosteric antagonist of CCK1R, illustrating differences in docking that extend beyond determinants attributable to distinct residues lining the intramembranous pocket in the two receptor subtypes.
Asunto(s)
Aminoácidos/metabolismo , Indoles/química , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina A/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Células CHO , Células COS , Chlorocebus aethiops , Cricetulus , Humanos , Indoles/farmacología , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Ensayo de Unión Radioligante , Receptor de Colecistoquinina A/antagonistas & inhibidores , Receptor de Colecistoquinina B/genética , Receptor de Colecistoquinina B/metabolismoRESUMEN
The immunophilins, cyclophilins, catalyze peptidyl cis-trans prolyl-isomerization (PPIase), a rate-limiting step in protein folding and a conformational switch in protein function. Cyclophilins are also chaperones. Noncatalytic mutations affecting the only cyclophilins with known but distinct physiological substrates, the Drosophila NinaA and its mammalian homolog, cyclophilin-B, impair opsin biogenesis and cause osteogenesis imperfecta, respectively. However, the physiological roles and substrates of most cyclophilins remain unknown. It is also unclear if PPIase and chaperone activities reflect distinct cyclophilin properties. To elucidate the physiological idiosyncrasy stemming from potential cyclophilin functions, we generated mice lacking endogenous Ran-binding protein-2 (Ranbp2) and expressing bacterial artificial chromosomes of Ranbp2 with impaired C-terminal chaperone and with (Tg-Ranbp2(WT-HA)) or without PPIase activities (Tg-Ranbp2(R2944A-HA)). The transgenic lines exhibit unique effects in proteostasis. Either line presents selective deficits in M-opsin biogenesis with its accumulation and aggregation in cone photoreceptors but without proteostatic impairment of two novel Ranbp2 cyclophilin partners, the cytokine-responsive effectors, STAT3/STAT5. Stress-induced STAT3 activation is also unaffected in Tg-Ranbp2(R2944A-HA)::Ranbp2(-/-). Conversely, proteomic analyses found that the multisystem proteinopathy/amyotrophic lateral sclerosis proteins, heterogeneous nuclear ribonucleoproteins A2/B1, are down-regulated post-transcriptionally only in Tg-Ranbp2(R2944A-HA)::Ranbp2(-/-). This is accompanied by the age- and tissue-dependent reductions of diubiquitin and ubiquitylated proteins, increased deubiquitylation activity, and accumulation of the 26 S proteasome subunits S1 and S5b. These manifestations are absent in another line, Tg-Ranbp2(CLDm-HA)::Ranbp2(-/-), harboring SUMO-1 and S1-binding mutations in the Ranbp2 cyclophilin-like domain. These results unveil distinct mechanistic and biological links between PPIase and chaperone activities of Ranbp2 cyclophilin toward proteostasis of selective substrates and with novel therapeutic potential.
Asunto(s)
Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Pliegue de Proteína , Envejecimiento/metabolismo , Animales , Biocatálisis , Regulación hacia Abajo , Potenciales Evocados Visuales , Proteínas Activadoras de GTPasa/metabolismo , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Ratones , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Proteínas de Complejo Poro Nuclear/deficiencia , Opsinas/metabolismo , Especificidad de Órganos , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/metabolismo , Factores de Transcripción STAT/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Relación Estructura-Actividad , Ubiquitina/metabolismoRESUMEN
Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu(7.39) that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a "trigger" for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu(7.39), whereas the role of this residue was less clear for chemically distinct agonists.
Asunto(s)
Benzodiazepinas/farmacología , Receptor de Colecistoquinina A/agonistas , Secuencia de Aminoácidos , Animales , Benzodiazepinas/química , Células CHO , Cricetinae , Cricetulus , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/agonistas , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Curva ROC , Receptor de Colecistoquinina A/química , Receptor de Colecistoquinina A/metabolismo , Receptor de Colecistoquinina B/química , Receptor de Colecistoquinina B/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein-protein and protein-phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBD(n = 1-4)) with close structural homology to the PH domain of Bruton's tyrosine kinase. The RBD2, kinesin-binding domain (KBD) and RBD3 comprise a tripartite domain (R2KR3) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R2KR3 of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure-function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260,000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD2 and RBD3 towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD2 and RBD3 exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R2KR3 stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R2KR3 is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD2 and RBD3, and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.
Asunto(s)
Cinesinas/metabolismo , Mitocondrias/fisiología , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Animales , Humanos , Cinesinas/química , Cinética , Ratones , Chaperonas Moleculares/química , Células 3T3 NIH , Proteínas de Complejo Poro Nuclear/química , Unión Proteica , Estructura Terciaria de Proteína , Regulación hacia ArribaRESUMEN
Mutations affecting the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) interactome cause syndromic retinal dystrophies. RPGRIP1 interacts with the retinitis pigmentosa GTPase regulator (RPGR) through a domain homologous to RCC1 (RHD), a nucleotide exchange factor of Ran GTPase. However, functional relationships between RPGR and RPGRIP1 and their subcellular roles are lacking. We show by molecular modeling and analyses of RPGR disease-mutations that the RPGR-interacting domain (RID) of RPGRIP1 embraces multivalently the shared RHD of RPGR(1-19) and RPGR(ORF15) isoforms and the mutations are non-overlapping with the interface found between RCC1 and Ran GTPase. RPGR disease-mutations grouped into six classes based on their structural locations and differential impairment with RPGRIP1 interaction. RPGRIP1α(1) expression alone causes its profuse self-aggregation, an effect suppressed by co-expression of either RPGR isoform before and after RPGRIP1α(1) self-aggregation ensue. RPGR(1-19) localizes to the endoplasmic reticulum, whereas RPGR(ORF15) presents cytosolic distribution and they determine uniquely the subcellular co-localization of RPGRIP1α(1). Disease mutations in RPGR(1) (-19), RPGR(ORF15), or RID of RPGRIP1α(1), singly or in combination, exert distinct effects on the subcellular targeting, co-localization or tethering of RPGRIP1α(1) with RPGR(1-19) or RPGR(ORF15) in kidney, photoreceptor and hepatocyte cell lines. Additionally, RPGR(ORF15), but not RPGR(1-19), protects the RID of RPGRIP1α(1) from limited proteolysis. These studies define RPGR- and cell-type-dependent targeting pathways with structural and functional plasticity modulating the expression of mutations in RPGR and RPGRIP1. Further, RPGR isoforms distinctively determine the subcellular targeting of RPGRIP1α(1,) with deficits in RPGR(ORF15)-dependent intracellular localization of RPGRIP1α(1) contributing to pathomechanisms shared by etiologically distinct syndromic retinal dystrophies.
RESUMEN
Protective antigen (PA), lethal factor, and edema factor, the protein toxins of Bacillus anthracis , are among its most important virulence factors and play a key role in infection. We performed a virtual ligand screen of a library of 10000 members to identify compounds predicted to bind to PA and prevent its oligomerization. Four of these compounds slowed PA association in a FRET-based oligomerization assay, and two of those protected cells from intoxication at concentrations of 1-10 µM. Exploration of the protective mechanism by Western blot showed decreased SDS-resistant PA oligomer on cells and, surprisingly, decreased amounts of activated PA. In vitro assays showed that one of the inhibitors blocked furin-mediated cleavage of PA, apparently through its binding to the PA substrate. Thus, we have identified inhibitors that can independently block both PA's cleavage by furin and its subsequent oligomerization. Lead optimization on these two backbones may yield compounds with high activity and specificity for the anthrax toxins.
Asunto(s)
Antígenos Bacterianos/química , Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Multimerización de Proteína/efectos de los fármacos , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/toxicidad , Línea Celular , Biología Computacional , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Simulación del Acoplamiento Molecular , Porosidad , Estructura Cuaternaria de Proteína , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
While it is evident that the carboxyl-terminal region of natural peptide ligands bind to the amino-terminal domain of class B GPCRs, how their biologically critical amino-terminal regions dock to the receptor is unclear. We utilize cysteine trapping to systematically explore spatial approximations among residues in the first five positions of secretin and in every position within the receptor extracellular loops (ECLs). Only Cys(2) and Cys(5) secretin analogues exhibited full activity and retained moderate binding affinity (IC(50): 92±4 and 83±1 nM, respectively). When these peptides probed 61 human secretin receptor cysteine-replacement mutants, a broad network of receptor residues could form disulfide bonds consistent with a dynamic ligand-receptor interface. Two distinct patterns of disulfide bond formation were observed: Cys(2) predominantly labeled residues in the amino terminus of ECL2 and ECL3 (relative labeling intensity: Ser(340), 94±7%; Pro(341), 84±9%; Phe(258), 73±5%; Trp(274) 62±8%), and Cys(5) labeled those in the carboxyl terminus of ECL2 and ECL3 (Gln(348), 100%; Ile(347), 73±12%; Glu(342), 59±10%; Phe(351), 58±11%). These constraints were utilized in molecular modeling, providing improved understanding of the structure of the transmembrane bundle and interconnecting loops, the orientation between receptor domains, and the molecular basis of ligand docking. Key spatial approximations between peptide and receptor predicted by this model (H(1)-W(274), D(3)-N(268), G(4)-F(258)) were supported by mutagenesis and residue-residue complementation studies.
Asunto(s)
Cisteína/metabolismo , Mapeo de Interacción de Proteínas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Secretina/metabolismo , Animales , Unión Competitiva , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Cisteína/química , Cisteína/genética , Disulfuros/química , Disulfuros/metabolismo , Humanos , Ligandos , Modelos Moleculares , Mutación , Péptidos/química , Péptidos/metabolismo , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Prolina/química , Prolina/genética , Prolina/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/genética , Secretina/química , Secretina/genética , Triptófano/química , Triptófano/genética , Triptófano/metabolismoRESUMEN
Allosteric binding pockets in peptide-binding G protein-coupled receptors create opportunities for the development of small molecule drugs with substantial benefits over orthosteric ligands. To gain insights into molecular determinants for this pocket within type 1 and 2 cholecystokinin receptors (CCK1R and CCK2R), we prepared a series of receptor constructs in which six distinct residues in TM2, -3, -6, and -7 were reversed. Two novel iodinated CCK1R- and CCK2R-selective 1,4-benzodiazepine antagonists, differing only in stereochemistry at C3, were used. When all six residues within CCK1R were mutated to corresponding CCK2R residues, benzodiazepine selectivity was reversed, yet peptide binding selectivity was unaffected. Detailed analysis, including observations of gain of function, demonstrated that residues 6.51, 6.52, and 7.39 were most important for binding the CCK1R-selective ligand, whereas residues 2.61 and 7.39 were most important for binding CCK2R-selective ligand, although the effect of substitution of residue 2.61 was likely indirect. Ligand-guided homology modeling was applied to wild type receptors and those reversing benzodiazepine binding selectivity. The models had high predictive power in enriching known receptor-selective ligands from related decoys, indicating a high degree of precision in pocket definition. The benzodiazepines docked in similar poses in both receptors, with C3 urea substituents pointing upward, whereas different stereochemistry at C3 directed the C5 phenyl rings and N1 methyl groups into opposite orientations. The geometry of the binding pockets and specific interactions predicted for ligand docking in these models provide a molecular framework for understanding ligand selectivity at these receptor subtypes. Furthermore, the strong predictive power of these models suggests their usefulness in the discovery of lead compounds and in drug development programs.
Asunto(s)
Benzodiazepinas/metabolismo , Receptores de Colecistoquinina/antagonistas & inhibidores , Sitio Alostérico , Secuencia de Aminoácidos , Animales , Benzodiazepinas/antagonistas & inhibidores , Células COS , Chlorocebus aethiops , Ligandos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Despite remarkable advances in human genetics and other genetic model systems, the fruit fly, Drosophila melanogaster, remains a powerful experimental tool to probe with ease the inner workings of a myriad of biological and pathological processes, even when evolutionary forces impart apparent divergences to some of such processes. The understanding of such evolutionary differences provides mechanistic insights into genotype-phenotype correlations underpinning biological processes across metazoans. The pioneering work developed by the William Pak laboratory for the past four decades, and the work of others, epitomize the notion of how the Drosophila system breaks new fertile ground or complements research fields of high scientific and medical relevance. Among the three major genetic complementation groups produced by the Pak's laboratory and impairing distinct facets of photoreceptor neuronal function, the nina group (ninaA, ., ninaJ) selectively affects the biogenesis of G protein-coupled receptors (GPCRs), mediating the photoconversion and transduction of light stimuli. Among the nina genes identified, ninaA arguably assumes heightened significance for several reasons. First, it presents unique physiological selectivity toward the biogenesis of a subset of GPCRs, a standalone biological manifestation yet to be discerned for most mammalian homologues of NinaA. Second, NinaA belongs to a family of proteins, immunophilins, which are the primary targets for immunosuppressive drugs at the therapeutic forefront of a multitude of medical conditions. Third, NinaA closest homologue, cyclophilin B (CyPB/PPIB), is an immunophilin whose loss-of-function was found recently to cause osteogenesis imperfecta in the human. This report highlights advances made by studies on some members of immunophilins, the cyclophilins. Finally, it reexamines critically data and dogmas derived from past and recent genetic, structural, biological, and pathological studies on NinaA and few other cyclophilins that support some of such paradigms to be less than definite and advance our understanding of the roles of cyclophilins in cell function, disease, and therapeutic interventions.