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1.
Angew Chem Int Ed Engl ; 63(8): e202314617, 2024 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-38181042

RESUMEN

There is a pressing need, particularly in the field of drug discovery, for general methods that will enable direct coupling of tertiary alkyl fragments to (hetero)aryl halides. Herein a uniquely powerful and simple set of conditions for achieving this transformation with unparalleled generality and chemoselectivity is disclosed. This new protocol is placed in context with other recently reported methods, applied to simplify the routes of known bioactive building blocks molecules, and scaled up in both batch and flow. The role of pyridine additive as well as the mechanism of this reaction are interrogated through Cyclic Voltammetry studies, titration experiments, control reactions with Ni(0) and Ni(II)-complexes, and ligand optimization data. Those studies indicate that the formation of a BINAPNi(0) is minimized and the formation of an active pyridine-stabilized Ni(I) species is sustained during the reaction. Our preliminary mechanistic studies ruled out the involvement of Ni(0) species in this electrochemical cross-coupling, which is mediated by Ni(I) species via a Ni(I)-Ni(II)-Ni(III)-Ni(I) catalytic cycle.

2.
Angew Chem Int Ed Engl ; 61(47): e202204565, 2022 11 21.
Artículo en Inglés | MEDLINE | ID: mdl-36130196

RESUMEN

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl ϵ-N-carboxyacyllysine posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target have been lacking. Here we report the harnessing of aryl fluorosulfate-based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne-tagged affinity-labeling agents recognize and capture overexpressed SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates. It is our hope that these results will serve as inspiration for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.


Asunto(s)
Neoplasias , Sirtuinas , Humanos , Animales , Ratones , Lisina , Células HEK293 , Sirtuinas/química , Neoplasias/genética
3.
Chemistry ; 26(17): 3862-3869, 2020 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-31922630

RESUMEN

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. This work describes the development of peptide activity/affinity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of ϵ-N-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to the effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the ϵ-N-glutaryllysine and ϵ-N-myristoyllysine analogues, were successfully applied for the enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.


Asunto(s)
Lisina/química , Péptidos/química , Secuencia de Aminoácidos , Humanos , Hidrólisis , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo
4.
Int J Cancer ; 144(4): 767-776, 2019 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-30194764

RESUMEN

Ras proteins, most notably KRas, are prevalent oncogenes in human cancer. Plasma membrane localization and thereby signaling of KRas is regulated by the prenyl-binding protein PDEδ. Recently, we have reported the specific anti-proliferative effects of PDEδ inhibition in KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, we investigated the proliferative dependence on the solubilizing activity of PDEδ of human colorectal cancer (CRC) cell lines with or without oncogenic KRas mutations. Our results show that genetic and pharmacologic interference with PDEδ specifically inhibits proliferation and survival of CRC cell lines harboring oncogenic KRas mutations whereas isogenic cell lines in which the KRas oncogene has been removed, or cell lines with oncogenic BRaf mutations or EGFR overexpression are not dependent on PDEδ. Pharmacological PDEδ inhibition is therefore a possible new avenue to target oncogenic KRas bearing CRC.


Asunto(s)
Bencimidazoles/farmacología , Proliferación Celular/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HT29 , Humanos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Interferencia de ARN
5.
Angew Chem Int Ed Engl ; 58(4): 957-966, 2019 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-30024079

RESUMEN

Selective covalent modification of a targeted protein is a powerful tool in chemical biology and drug discovery, with applications ranging from identification and characterization of proteins and their functions to the development of targeted covalent inhibitors. Most covalent ligands contain an affinity motif and an electrophilic warhead that reacts with a nucleophilic residue of the targeted protein. Because the electrophilic warhead is prone to react and modify off-target nucleophiles, its reactivity should be balanced carefully to maximize target selectivity. Arylfluorosulfates have recently emerged as latent electrophiles for selective labeling of context-specific tyrosine and lysine residues in protein pockets. Here, we review the recent but intense introduction of arylfluorosulfates into the arsenal of available warheads for selective covalent modification of proteins. We highlight the untapped potential of this functional group for use in chemical biology and drug discovery.


Asunto(s)
Descubrimiento de Drogas , Proteínas Recombinantes/química , Ésteres del Ácido Sulfúrico/química , Sitios de Unión , Células HeLa , Humanos , Lisina/química , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Tirosina/química
6.
Cell Chem Biol ; 24(5): 589-597.e5, 2017 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-28434875

RESUMEN

Covalent labeling of amino acids in proteins by reactive small molecules, in particular at cysteine SH and lysine NH groups, is a powerful approach to identify and characterize proteins and their functions. However, for the less-reactive carboxylic acids present in Asp and Glu, hardly any methodology is available. Employing the lipoprotein binding chaperone PDE6δ as an example, we demonstrate that incorporation of isoxazolium salts that resemble the structure and reactivity of Woodward's reagent K into protein ligands provides a novel method for selective covalent targeting of binding site carboxylic acids in whole proteomes. Covalent adduct formation occurs via rapid formation of enol esters and the covalent bond is stable even in the presence of strong nucleophiles. This new method promises to open up hitherto unexplored opportunities for chemical biology research.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Glutamatos/química , Coloración y Etiquetado/métodos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Ligandos , Modelos Moleculares , Conformación Proteica
7.
Angew Chem Int Ed Engl ; 56(9): 2423-2428, 2017 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-28106325

RESUMEN

Small-molecule inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6δ impairs KRas spatial organization and signaling in cells. However, despite potent binding in vitro (KD <10 nm), interference with Ras signaling and growth inhibition require 5-20 µm compound concentrations. We demonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6δ by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6δ with up to 7 hydrogen bonds, resulting in picomolar affinity. Their release by Arl2 is greatly decreased, and representative compounds selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6δ interaction may impair the growth of tumors driven by oncogenic KRas.

8.
Biol Chem ; 398(5-6): 535-545, 2017 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-27935847

RESUMEN

The prenyl binding protein PDEδ enhances the diffusion of farnesylated Ras proteins in the cytosol, ultimately affecting their correct localization and signaling. This has turned PDEδ into a promising target to prevent oncogenic KRas signaling. In this review we summarize and describe the structure-guided-development of the three different PDEδ inhibitor chemotypes that have been documented so far. We also compare both their potency for binding to the PDEδ pocket and their in vivo efficiency in suppressing oncogenic KRas signaling, as a result of the inhibition of the PDEδ/KRas interaction.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/antagonistas & inhibidores , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Animales , Bencimidazoles/química , Bencimidazoles/farmacología , Inhibidores Enzimáticos/química , Piridazinas/química , Piridazinas/farmacología
9.
Chemistry ; 23(25): 6083-6093, 2017 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-27809361

RESUMEN

The K-Ras GTPase is a major target in anticancer drug discovery. However, direct interference with signaling by K-Ras has not led to clinically useful drugs yet. Correct localization and signaling by farnesylated K-Ras is regulated by the prenyl binding protein PDEδ. Interfering with binding of PDEδ to K-Ras by means of small molecules provides a novel opportunity to suppress oncogenic signaling. Here we describe the identification and structure-guided development of novel K-Ras-PDEδ inhibitor chemotypes based on pyrrolopyridazinones and pyrazolopyridazinones that bind to the farnesyl binding pocket of PDEδ with low nanomolar affinity. We delineate the structure-property relationship and in vivo pharmacokinetic (PK) and toxicokinetic (Tox) studies for pyrazolopyridazinone-based K-Ras-PDEδ inhibitors. These findings may inspire novel drug discovery efforts aimed at the development of drugs targeting oncogenic Ras.

10.
Sci Rep ; 6: 27285, 2016 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-27271737

RESUMEN

Somatostatin is a 14-residue peptide hormone that regulates the endocrine system by binding to five G-protein-coupled receptors (SSTR1-5). We have designed six new Somatostatin analogs with L-3-(3',5'-difluorophenyl)-alanine (Dfp) as a substitute of Phe and studied the effect of an electron-poor aromatic ring in the network of aromatic interactions present in Somatostatin. Replacement of each of the Phe residues (positions 6, 7 and 11) by Dfp and use of a D-Trp8 yielded peptides whose main conformations could be characterized in aqueous solution by NMR. Receptor binding studies revealed that the analog with Dfp at position 7 displayed a remarkable affinity to SSTR2 and SSTR3. Analogs with Dfp at positions 6 or 11 displayed a π-π interaction with the Phe present at 11 or 6, respectively. Interestingly, these analogs, particularly [D-Trp8,L-Dfp11]-SRIF, showed high selectivity towards SSTR2, with a higher value than that of Octreotide and a similar one to that of native Somatostatin.


Asunto(s)
Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Somatostatina/análogos & derivados , Alanina/química , Secuencia de Aminoácidos , Sitios de Unión , Halogenación , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Péptidos Cíclicos/química , Somatostatina/química , Relación Estructura-Actividad
11.
Nat Commun ; 7: 11360, 2016 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-27094677

RESUMEN

The prenyl-binding protein PDEδ is crucial for the plasma membrane localization of prenylated Ras. Recently, we have reported that the small-molecule Deltarasin binds to the prenyl-binding pocket of PDEδ, and impairs Ras enrichment at the plasma membrane, thereby affecting the proliferation of KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, using structure-based compound design, we have now identified pyrazolopyridazinones as a novel, unrelated chemotype that binds to the prenyl-binding pocket of PDEδ with high affinity, thereby displacing prenylated Ras proteins in cells. Our results show that the new PDEδ inhibitor, named Deltazinone 1, is highly selective, exhibits less unspecific cytotoxicity than the previously reported Deltarasin and demonstrates a high correlation with the phenotypic effect of PDEδ knockdown in a set of human pancreatic cancer cell lines.


Asunto(s)
Antineoplásicos/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Células Epiteliales/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Inhibidores de Fosfodiesterasa/química , Proteínas Proto-Oncogénicas p21(ras)/química , Pirazinas/química , Pirazoles/química , Bibliotecas de Moléculas Pequeñas/química , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/antagonistas & inhibidores , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Expresión Génica , Humanos , Simulación del Acoplamiento Molecular , Conductos Pancreáticos/efectos de los fármacos , Conductos Pancreáticos/metabolismo , Conductos Pancreáticos/patología , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/farmacología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Pirazinas/síntesis química , Pirazinas/farmacología , Pirazoles/síntesis química , Pirazoles/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología
12.
J Am Chem Soc ; 136(34): 12161-5, 2014 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-25127186

RESUMEN

The development of efficient methods for the generation of enantioenriched sulfonamides and sulfones is an important objective for fields such as organic synthesis and medicinal chemistry; however, there have been relatively few reports of direct catalytic asymmetric approaches to controlling the stereochemistry of the sulfur-bearing carbon of such targets. In this report, we describe nickel-catalyzed stereoconvergent Negishi arylations and alkenylations of racemic α-bromosulfonamides and -sulfones that furnish the desired cross-coupling product in very good ee and yield for an array of reaction partners. Mechanistic studies are consistent with the generation of a radical intermediate that has a sufficient lifetime to diffuse out of the solvent cage and to cyclize onto a pendant olefin.


Asunto(s)
Alcanos/química , Alquenos/química , Hidrocarburos Aromáticos/síntesis química , Sulfonamidas/síntesis química , Catálisis , Ciclización , Transporte de Electrón , Hidrocarburos Aromáticos/química , Níquel , Compuestos Organometálicos/química , Estereoisomerismo , Sulfonamidas/química , Sulfonas/síntesis química , Sulfonas/química , Zinc/química , Circonio/química
13.
Bioorg Med Chem Lett ; 24(1): 103-7, 2014 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-24342240

RESUMEN

We described here the first tetradecapeptide somatostatin-analogue where the disulfide bridge has been replaced by a carbon-carbon double bond. This analogue was prepared using microwave assisted ring closing metathesis (RCM) using the 2nd generation Grubbs as catalyst. Under our optimized conditions the cyclization between allylGly 3 and 14 proceeded in moderate yield, excellent cyclic/linear ratio and very high Z-double bond selectivity. NMR studies also demonstrated that the conformational flexibility of this peptide is increased in comparison to that of the natural hormone. Remarkably, this alkene-bridged somatostatin analog is highly selective against somatostatin receptors 1 and 5, suggesting that conformational rigidity is not required for the efficient interaction of somatostatin analogues with these two receptors.


Asunto(s)
Receptores de Somatostatina/antagonistas & inhibidores , Somatostatina/análogos & derivados , Somatostatina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Microondas , Estructura Molecular , Ratas , Receptores de Somatostatina/metabolismo , Somatostatina/síntesis química , Somatostatina/química , Relación Estructura-Actividad
14.
Molecules ; 18(12): 14564-84, 2013 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-24287991

RESUMEN

The non-natural amino acid mesitylalanine (2,4,6-trimethyl-L-phenylalanine; Msa) has an electron-richer and a more conformationally restricted side-chain than that of its natural phenylalanine counterpart. Taking these properties into account, we have synthesized ten somatostatin analogs containing Msa residues in different key positions to modify the intrinsic conformational flexibility of the natural hormone. We have measured the binding affinity of these analogs and correlated it with the main conformations they populate in solution. NMR and computational analysis revealed that analogs containing one Msa residue were conformationally more restricted than somatostatin under similar experimental conditions. Furthermore, we were able to characterize the presence of a hairpin at the pharmacophore region and a non-covalent interaction between aromatic residues 6 and 11. In all cases, the inclusion of a D-Trp in the eighth position further stabilized the main conformation. Some of these peptides bound selectively to one or two somatostatin receptors with similar or even higher affinity than the natural hormone. However, we also found that multiple incorporations of Msa residues increased the life span of the peptides in serum but with a loss of conformational rigidity and binding affinity.


Asunto(s)
Fenilalanina/química , Somatostatina/química , Secuencia de Aminoácidos , Animales , Células CHO , Cricetulus , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Péptidos/síntesis química , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Somatostatina/análogos & derivados , Somatostatina/metabolismo , Relación Estructura-Actividad
16.
Chembiochem ; 12(4): 625-32, 2011 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-21259412

RESUMEN

We prepared the two enantiomers of 3-(3'-quinolyl)-alanine (Qla, 1) in multigram scale by asymmetric hydrogenation. These amino acids, protected as Fmoc derivatives, were then used in the solid-phase synthesis of two new somatostatin 14 (SRIF-14) analogues 8 a and 8 b, tetradecapeptides in which the tryptophan residue (Trp8) is replaced by one of the two enantiomers of 3-(3'-quinolyl)-alanine (Qla8) and therefore lack the N--H bond in residue 8. The selectivity of these new analogues for the somatostatin receptors, SSTR1-5, was measured. Substitution with L-Qla8 yielded peptide 8 a, which was highly selective for SSTR1 and SSTR3, with an affinity similar to that of SRIF-14. Substitution by D-Qla gave the relatively selective analogue 8 b, which showed high affinity for SSTR3 and significant affinity for SSTR1, SSTR2 and SSTR5. The biological results demonstrate that bulky and electronically poor aromatic amino acids at position 8 are compatible with strong activity with SSTR1 and SSTR3. Remarkably, these high affinity levels were achieved with peptides in which the conformational mobility was increased with respect to that of SRIF-14. This observation suggests that conformational rigidity is not required, and might be detrimental to the interaction with receptors SSTR1 and SSTR3. The absence of an indole N proton in Qla8 might also contribute to the increased flexibility observed in these analogues.


Asunto(s)
Alanina/análogos & derivados , Modelos Moleculares , Quinolinas/síntesis química , Receptores de Somatostatina/química , Alanina/síntesis química , Alanina/química , Animales , Bioensayo , Células Cultivadas , Humanos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Quinolinas/química , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Somatostatina/química , Somatostatina/metabolismo , Estereoisomerismo , Especificidad por Sustrato , Triptófano/química
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