ABSTRACT
Stimulator of Interferon Genes (STING) plays an important role in innate immunity by inducing type I interferon production upon infection with intracellular pathogens. STING activation can promote increased T-cell activation and inflammation in the tumor microenvironment, resulting in antitumor immunity. Natural and synthetic cyclic dinucleotides (CDNs) are known to activate STING, and several synthetic CDN molecules are being investigated in the clinic using an intratumoral administration route. Here, we describe the identification of STING agonist 15a, a cyclic dinucleotide structurally diversified from natural ligands with optimized properties for systemic intravenous (iv) administration. Our studies have shown that STING activation by 15a leads to an acute innate immune response as measured by cytokine secretion and adaptive immune response via activation of CD8+ cytotoxic T-cells, which ultimately provides robust antitumor efficacy.
Subject(s)
Membrane Proteins/agonists , Nucleotides, Cyclic/chemistry , Pyrimidines/chemistry , Administration, Intravenous , Animals , Binding Sites , Cell Line, Tumor , Half-Life , Humans , Immunotherapy , Membrane Proteins/metabolism , Mice , Molecular Docking Simulation , Neoplasms/pathology , Neoplasms/therapy , Nucleotides, Cyclic/metabolism , Nucleotides, Cyclic/therapeutic use , Phosphates/chemistry , Rats , Structure-Activity Relationship , Transplantation, HeterologousABSTRACT
Acyl derivatives of 4-(aminomethyl)-N-hydroxybenzamide are potent sub-type selective HDAC6 inhibitors. Constrained heterocyclic analogs based on 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine show further enhanced HDAC6 selectivity and inhibitory activity in cells. Homology models suggest that the heterocyclic spacer can more effectively access the wider catalytic channel of HDAC6 compared to other HDAC sub-types.
Subject(s)
Histone Deacetylase Inhibitors/metabolism , Hydroxamic Acids/pharmacology , Pyrazines/metabolism , Protein IsoformsABSTRACT
A screen for HDAC6 inhibitors identified acyl derivatives of 4-(aminomethyl)-N-hydroxybenzamide as potent leads with unexpected selectivity over the other subtypes. We designed and synthesized constrained heterocyclic analogues such as tetrahydroisoquinolines that show further enhanced HDAC6 selectivity and inhibitory activity in cellular assays. Selectivity may be attributed to the benzylic spacer more effectively accessing the wider channel of HDAC6 compared to other HDAC subtypes as well as hydrophobic capping groups interacting with the protein surface near the rim of the active site.
Subject(s)
Benzamides/chemical synthesis , Benzamides/pharmacology , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacology , Benzamides/chemistry , Histone Deacetylase 6 , Histone Deacetylase Inhibitors/chemical synthesis , Humans , Substrate SpecificityABSTRACT
We identified N,C-capped dipeptides that are selective for the Mycobacterium tuberculosis proteasome over human constitutive and immunoproteasomes. Differences in the S3 and S1 binding pockets appeared to account for the species selectivity. The inhibitors can penetrate mycobacteria and kill nonreplicating M. tuberculosis under nitrosative stress.
Subject(s)
Dipeptides/pharmacology , Mycobacterium tuberculosis/drug effects , Proteasome Endopeptidase Complex/metabolism , Binding Sites/drug effects , Dipeptides/chemistry , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Mycobacterium tuberculosis/chemistry , Structure-Activity RelationshipABSTRACT
Starting from a tripeptide screening hit, a series of dipeptide inhibitors of the proteasome with Thr as the P3 residue has been optimized with the aid of crystal structures in complex with the ß-5/6 active site of y20S. Derivative 25, (ß5 IC(50)=7.4 nM) inhibits only the chymotryptic activity of the proteasome, shows cellular activity against targets in the UPS, and inhibits proliferation.
Subject(s)
Chymotrypsin/antagonists & inhibitors , Dipeptides/chemistry , Proteasome Endopeptidase Complex/metabolism , Threonine/chemistry , Humans , Models, MolecularABSTRACT
The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome based [corrected] on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the beta5 (chymotrypsin-like) site over the beta1 (caspase-like) and beta2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S beta5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NFkappaB (nuclear factor kappaB) in response to TNF-alpha (tumour necrosis factor-alpha) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the beta5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the beta5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.
Subject(s)
Oligopeptides/pharmacology , Protease Inhibitors/pharmacology , Proteasome Inhibitors , Amino Acid Sequence , Binding Sites , Boronic Acids/pharmacology , Bortezomib , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , HCT116 Cells , HT29 Cells , Humans , Kinetics , Luciferases/genetics , Luciferases/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , NF-kappa B/genetics , NF-kappa B/metabolism , Oligopeptides/chemistry , Oligopeptides/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Structure, Tertiary , Protein Subunits/antagonists & inhibitors , Protein Subunits/genetics , Protein Subunits/metabolism , Pyrazines/pharmacology , RNA Interference , Sequence Homology, Amino Acid , Ubiquitin/genetics , Ubiquitin/metabolismABSTRACT
The design, synthesis, and structure-activity relationship development of naphthalene-derived human CCR8 antagonists is described. In vitro binding assay results of these investigations are reported, critical interactions of the antagonists with CCR8 are defined, and preliminary physicochemical and pharmacokinetic data for the naphthalene scaffold are presented.
Subject(s)
Naphthalenes/chemical synthesis , Receptors, Chemokine/antagonists & inhibitors , Sulfonamides/chemical synthesis , Administration, Oral , Animals , Biological Availability , Biological Transport , Calcium/metabolism , Cell Line , Cricetinae , Cricetulus , Drug Design , Humans , Naphthalenes/pharmacokinetics , Naphthalenes/pharmacology , Rats , Receptors, CCR8 , Solubility , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacologyABSTRACT
N-Substituted cis-4a-(3-hydroxyphenyl)-8a-methyloctahydroisoquinolines (6a-g) were designed and synthesized as conformationally constrained analogues of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine (4) class of opioid receptor pure antagonists. The methyloctahydroisoquinolines 6a-g can exist in conformations where the 3-hydroxyphenyl substituent is either axial or equatorial, similar to the (3-hydroxyphenyl)piperidines 4. The 3-hydroxyphenyl equatorial conformation is responsible for the antagonist activity observed in the (3-hydroxyphenyl)piperidine antagonists. Single-crystal X-ray analysis of 6a shows that the 3-hydroxyphenyl equatorial conformation is favored in the solid state. Molecular modeling studies also suggest that the equatorial conformation has lower potential energy relative to that of the axial conformation. Evaluation of 6a-g in the [(35)S]GTP-gamma-S in vitro functional assay showed that they were opioid receptor pure antagonists. N-[4a-(3-Hydroxyphenyl)-8a-methyl-2-(3-phenylpropyl)octahydroisoquinoline-6-yl]-3-(piperidin-1-yl)propionamide (6d) with a K(e) of 0.27 nM at the kappa opioid receptor with 154- and 46-fold selectivity relative to those of the micro and delta receptors, respectively, possessed the best combination of kappa potency and selectivity.
Subject(s)
Isoquinolines/pharmacology , Narcotic Antagonists , Animals , Benzeneacetamides/pharmacology , CHO Cells , Cricetinae , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/antagonists & inhibitors , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Isoquinolines/chemical synthesis , Models, Molecular , Molecular Conformation , Pyrrolidines/pharmacology , Receptors, Opioid/metabolism , Receptors, Opioid, delta/antagonists & inhibitors , Receptors, Opioid, kappa/antagonists & inhibitors , Receptors, Opioid, kappa/metabolism , Receptors, Opioid, mu/antagonists & inhibitorsABSTRACT
A library of compounds biased toward opioid receptor antagonist activity was prepared by incorporating N-phenylpropyl-4beta-methyl-5-(3-hydroxyphenyl)morphans as the core scaffold using simultaneous solution phase synthetic methodology. From this library, N-phenylpropyl-4beta-methyl-5-(3-hydroxyphenyl)-7alpha-[3-(1-piperidinyl)propanamido]morphan [(-)-3b] was identified as the first potent and selective kappa opioid receptor antagonist from the 5-phenylmorphan class of opioids.