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1.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33536342

RESUMEN

Cooperative ligand binding is an important phenomenon in biological systems where ligand binding influences the binding of another ligand at an alternative site of the protein via an intramolecular network of interactions. The underlying mechanisms behind cooperative binding remain poorly understood, primarily due to the lack of structural data of these ternary complexes. Using time-resolved fluorescence resonance energy transfer (TR-FRET) studies, we show that cooperative ligand binding occurs for RORγt, a nuclear receptor associated with the pathogenesis of autoimmune diseases. To provide the crucial structural insights, we solved 12 crystal structures of RORγt simultaneously bound to various orthosteric and allosteric ligands. The presence of the orthosteric ligand induces a clamping motion of the allosteric pocket via helices 4 to 5. Additional molecular dynamics simulations revealed the unusual mechanism behind this clamping motion, with Ala355 shifting between helix 4 and 5. The orthosteric RORγt agonists regulate the conformation of Ala355, thereby stabilizing the conformation of the allosteric pocket and cooperatively enhancing the affinity of the allosteric inverse agonists.


Asunto(s)
Regulación Alostérica/genética , Descubrimiento de Drogas , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Conformación Proteica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Sitio Alostérico/genética , Sitios de Unión/genética , Fenómenos Biofísicos , Cristalografía por Rayos X , Humanos , Ligandos , Conformación Molecular , Simulación de Dinámica Molecular , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/química , Unión Proteica/genética
2.
Bioorg Med Chem ; 68: 116877, 2022 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-35714534

RESUMEN

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays a central role in metabolic processes. PPARγ full agonists have side effects, arguing for the discovery of PPARγ partial agonists with novel chemotypes. We report the unique binding mode of the known allosteric retinoic acid receptor-related orphan receptor gamma t (RORγt) ligand MRL-871 to PPARγ. MRL-871 binds between PPARγ helices 3, 5, 7 and 11, where it stabilizes the beta-sheet region with a hydrogen bond between its carboxylic acid moiety and PPARγ Ser370. Its unique binding mode differs from that of the benzoyl 2-methyl indoles which are well-studied, structurally similar, PPARγ ligands. MRL-871's high affinity for PPARγ induces only limited coactivator stabilization, highlighting its attractive partial agonistic characteristics. Affinity comparison of MRL-871 and related compounds towards both RORγt and PPARγ indicates the possibility for tuning of selectivity, bringing MRL-871 forward as an interesting starting point for novel PPARγ ligands.


Asunto(s)
Indazoles , PPAR gamma , Indazoles/farmacología , Ligandos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , PPAR gamma/agonistas , Estructura Secundaria de Proteína
3.
J Biol Chem ; 295(27): 9183-9191, 2020 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-32439807

RESUMEN

Nuclear receptors (NRs) are high-interest targets in drug discovery because of their involvement in numerous biological processes and diseases. Classically, NRs are targeted via their hydrophobic, orthosteric pocket. Although successful, this approach comes with challenges, including off-target effects due to lack of selectivity. Allosteric modulation of NR activity constitutes a promising pharmacological strategy. The retinoic acid receptor-related orphan receptor-γt (RORγt) is a constitutively active NR that positively regulates the expression of interleukin-17 in T helper 17 cells. Inhibiting this process is an emerging strategy for managing autoimmune diseases. Recently, an allosteric binding pocket in the C-terminal region of the ligand-binding domain (LBD) of RORγt was discovered that is amenable to small-molecule drug discovery. Compounds binding this pocket induce a reorientation of helix 12, thereby preventing coactivator recruitment. Therefore, inverse agonists binding this site with high affinity are actively being pursued. To elucidate the pocket formation mechanism, verify the uniqueness of this pocket, and substantiate the relevance of targeting this site, here we identified the key characteristics of the RORγt allosteric region. We evaluated the effects of substitutions in the LBD on coactivator, orthosteric, and allosteric ligand binding. We found that two molecular elements unique to RORγt, the length of helix 11' and a Gln-487 residue, are crucial for the formation of the allosteric pocket. The unique combination of elements present in RORγt suggests a high potential for subtype-selective targeting of this NR to more effectively treat patients with autoimmune diseases.


Asunto(s)
Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/ultraestructura , Sitio Alostérico , Sitios de Unión , Agonismo Inverso de Drogas , Humanos , Interleucina-17 , Ligandos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismo , Células Th17/metabolismo
4.
ACS Chem Biol ; 15(12): 3143-3148, 2020 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-33196173

RESUMEN

Protein-protein interaction (PPI) networks are fundamental for cellular processes. Small-molecule PPI enhancers have been shown to be powerful tools to fundamentally study PPIs and as starting points for potential new therapeutics. Yet, systematic approaches for their discovery are not widely available, and the design prerequisites of "molecular glues" are poorly understood. Covalent fragment-based screening can identify chemical starting points for these enhancers at specific sites in PPI interfaces. We recently reported a mass spectrometry-based disulfide-trapping (tethering) approach for a cysteine residue in the hub protein 14-3-3, an important regulator of phosphorylated client proteins. Here, we invert the strategy and report the development of a functional read-out for systematic identification of PPI enhancers based on fluorescence anisotropy (FA-tethering) with the reactive handle now on a client-derived peptide. Using the DNA-binding domain of the nuclear receptor Estrogen Related Receptor gamma (ERRγ), we target a native cysteine positioned at the 14-3-3 PPI interface and identify several fragments that form a disulfide bond to ERRγ and stabilize the complex up to 5-fold. Crystallography indicates that fragments bind in a pocket comprised of 14-3-3 and the ERRγ phosphopeptide. FA-tethering presents a streamlined methodology to discover molecular glues for protein complexes.


Asunto(s)
Polarización de Fluorescencia , Mapas de Interacción de Proteínas , Proteínas/metabolismo , Proteínas 14-3-3/metabolismo , Receptor alfa de Estrógeno/metabolismo , Magnetismo , Receptores de Estrógenos/metabolismo
5.
Mol Cell Endocrinol ; 485: 20-34, 2019 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-30703487

RESUMEN

Nuclear Receptors (NRs) are multi-domain proteins, whose natural regulation occurs via ligands for a classical, orthosteric, binding pocket and via intra- and inter-domain allosteric mechanisms. Allosteric modulation of NRs via synthetic small molecules has recently emerged as an interesting entry to address the need for small molecules targeting NRs in pathology, via novel modes of action and with beneficial profiles. In this review the general concept of allosteric modulation in drug discovery is first discussed, serving as a background and inspiration for NRs. Subsequently, the review focuses on examples of small molecules that allosterically modulate NRs, with a strong focus on structural information and the ligand binding domain. Recently discovered nanomolar potent allosteric site NR modulators are catapulting allosteric targeting of NRs to the center of attention. The obtained insights serve as a basis for recommendations for the next steps to take in allosteric small molecular targeting of NRs.


Asunto(s)
Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico , Sitios de Unión , Diseño de Fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos
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