ABSTRACT
The RNA-binding protein LIN28 is a regulator of miRNA let-7 biogenesis. Inhibitors of LIN28 are highly sought after given the central role that LIN28 plays in tumorigenesis and development of cancer stem cells as well as LIN28's association with poor clinical prognosis. Although LIN28 inhibitors of different scaffolds have been reported, the potential of most LIN28 inhibiting small molecules was not fully explored since very limited structure-activity relationship (SAR) studies have been performed. We previously identified trisubstituted pyrrolinones as a new class of LIN28 inhibitors disrupting the LIN28-let-7 interaction. Here, we performed extensive SAR by evaluating 95 small molecules and identified new trisubstituted pyrrolinones featuring either an N-biphenyl or N-dibenzofuran substituent, overthrowing the existing conclusion that a salicylic acid moiety is indispensable for activity. Exchange of the negatively charged salicylic acid moiety in LIN28 inhibitors with a heterocyclic substituent is beneficial for membrane permeability, leading to increased activity in a cellular assay, and will potentially reduce toxicity.
ABSTRACT
The ubiquitously expressed glucocorticoid receptor (GR) is a nuclear receptor that controls a broad range of biological processes and is activated by steroidal glucocorticoids such as hydrocortisone or dexamethasone. Glucocorticoids are used to treat a wide variety of conditions, from inflammation to cancer but suffer from a range of side effects that motivate the search for safer GR modulators. GR is also regulated outside the steroid-binding site through protein-protein interactions (PPIs) with 14-3-3 adapter proteins. Manipulation of these PPIs will provide insights into noncanonical GR signaling as well as a new level of control over GR activity. We report the first molecular glues that selectively stabilize the 14-3-3/GR PPI using the related nuclear receptor estrogen receptor α (ERα) as a selectivity target to drive design. These 14-3-3/GR PPI stabilizers can be used to dissect noncanonical GR signaling and enable the development of novel atypical GR modulators.
Subject(s)
Glucocorticoids , Receptors, Glucocorticoid , Glucocorticoids/metabolism , Receptors, Glucocorticoid/metabolism , 14-3-3 Proteins/metabolism , Gene Expression Regulation , Binding Sites , Steroids , DexamethasoneABSTRACT
Thiazolidinedione PPARγ agonists such as rosiglitazone and pioglitazone are effective antidiabetic drugs, but side effects have limited their use. It has been posited that their positive antidiabetic effects are mainly mediated by the inhibition of the CDK5-mediated Ser273 phosphorylation of PPARγ, whereas the side effects are linked to classical PPARγ agonism. Thus compounds that inhibit PPARγ Ser273 phosphorylation but lack classical PPARγ agonism have been sought as safer antidiabetic therapies. Herein we report the discovery by virtual screening of 10, which is a potent PPARγ binder and in vitro inhibitor of the CDK5-mediated phosphorylation of PPARγ Ser273 and displays negligible PPARγ agonism in a reporter gene assay. The pharmacokinetic properties of 10 are compatible with oral dosing, enabling preclinical in vivo testing, and a 7 day treatment demonstrated an improvement in insulin sensitivity in the ob/ob diabetic mouse model.
ABSTRACT
OBJECTIVE: In vivo studies have reported several beneficial metabolic effects of ß-adrenergic receptor agonist administration in skeletal muscle, including increased glucose uptake, fatty acid metabolism, lipolysis and mitochondrial biogenesis. Although these effects have been widely studied in vivo, the in vitro data are limited to mouse and rat cell lines. Therefore, we sought to discover the effects of the ß2-adrenergic receptor agonist terbutaline on metabolism and protein synthesis in human primary skeletal muscle cells. METHODS: Human cultured myotubes were exposed to terbutaline in various concentrations (0.01-30 âµM) for 4 or 96 âh. Thereafter uptake of [14C]deoxy-D-glucose, oxydation of [14C]glucose and [14C]oleic acid were measured. Incorporation of [14C]leucine, gene expression by qPCR and proteomics analyses by mass spectrometry by the STAGE-TIP method were performed after 96 âh exposure to 1 and 10 âµM of terbutaline. RESULTS: The results showed that 4 âh treatment with terbutaline in concentrations up to 1 âµM increased glucose uptake in human myotubes, but also decreased both glucose and oleic acid oxidation along with oleic acid uptake in concentrations of 10-30 âµM. Moreover, administration of terbutaline for 96 âh increased glucose uptake (in terbutaline concentrations up to 1 âµM) and oxidation (1 âµM), as well as oleic acid oxidation (0.1-30 âµM), leucine incorporation into cellular protein (1-10 âµM) and upregulated several pathways related to mitochondrial metabolism (1 âµM). Data are available via ProteomeXchange with identifier PXD024063. CONCLUSION: These results suggest that ß2-adrenergic receptor have direct effects in human skeletal muscle affecting fuel metabolism and net protein synthesis, effects that might be favourable for both type 2 diabetes and muscle wasting disorders.
ABSTRACT
Ketohexokinase (KHK) catalyzes the first step of fructose metabolism. Inhibitors of KHK enzymatic activity are being evaluated in clinical trials for the treatment of non-alcoholic fatty liver disease (NAFLD) and diabetes. Here, we present a luminescence-based protocol to quantify KHK activity. The accuracy of this technique has been validated using knockdown and overexpression of KHK in vivo and in vitro. The specificity of the assay has been verified using 3-O-methyl-D-fructose, a non-metabolizable analog of fructose, heat inactivation of hexokinases, and depletion of potassium. For complete details on the use of this protocol, please refer to Damen et al. (2021).
Subject(s)
Enzyme Assays/methods , Fructokinases/metabolism , Fructose/metabolism , Luminescent Measurements/methods , Animals , Carbohydrate Metabolism , Fructokinases/antagonists & inhibitors , Hepatocytes/enzymology , Hepatocytes/metabolism , Humans , Luminescence , Mice , Non-alcoholic Fatty Liver DiseaseABSTRACT
Protein-protein interactions (PPIs) remain poorly explored targets for the treatment of Alzheimer's disease. The interaction of 14-3-3 proteins with Tau was shown to be linked to Tau pathology. This PPI is therefore seen as a potential target for Alzheimer's disease. When Tau is phosphorylated by PKA (Tau-PKA), several phosphorylation sites are generated, including two known 14-3-3 binding sites, surrounding the phosphorylated serines 214 and 324 of Tau. The crystal structures of 14-3-3 in complex with peptides surrounding these Tau phosphosites show that both these motifs are anchored in the amphipathic binding groove of 14-3-3. However, in the absence of structural data with the full-length Tau protein, the stoichiometry of the complex or the interface and affinity of the partners is still unclear. In this work, we addressed these points, using a broad range of biophysical techniques. The interaction of the long and disordered Tau-PKA protein with 14-3-3σ is restricted to two short sequences, containing phosphorylated serines, which bind in the amphipathic binding groove of 14-3-3σ. Phosphorylation of Tau is fundamental for the formation of this stable complex, and the affinity of the Tau-PKA/14-3-3σ interaction is in the 1-10 micromolar range. Each monomer of the 14-3-3σ dimer binds one of two different phosphorylated peptides of Tau-PKA, suggesting a 14-3-3/Tau-PKA stoichiometry of 2 : 1, confirmed by analytical ultracentrifugation. These results contribute to a better understanding of this PPI and provide useful insights for drug discovery projects aiming at the modulation of this interaction.
Subject(s)
14-3-3 Proteins/metabolism , Alzheimer Disease/metabolism , Protein Multimerization , tau Proteins/metabolism , 14-3-3 Proteins/chemistry , 14-3-3 Proteins/genetics , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Binding Sites , Cyclic AMP-Dependent Protein Kinases/metabolism , Exoribonucleases/chemistry , Exoribonucleases/genetics , Exoribonucleases/metabolism , Humans , Magnetic Resonance Spectroscopy , Mutation , Phosphorylation , Protein Binding , Protein Conformation , Serine/chemistry , Serine/metabolism , Surface Plasmon Resonance , tau Proteins/chemistry , tau Proteins/geneticsABSTRACT
Protein-protein interactions (PPIs) of 14-3-3 proteins are a model system for studying PPI stabilization. The complex natural product Fusicoccinâ A stabilizes many 14-3-3 PPIs but is not amenable for use in SAR studies, motivating the search for more drug-like chemical matter. However, drug-like 14-3-3 PPI stabilizers enabling such studies have remained elusive. An X-ray crystal structure of a PPI in complex with an extremely low potency stabilizer uncovered an unexpected non-protein interacting, ligand-chelated Mg2+ leading to the discovery of metal-ion-dependent 14-3-3 PPI stabilization potency. This originates from a novel chelation-controlled bioactive conformation stabilization effect. Metal chelation has been associated with pan-assay interference compounds (PAINS) and frequent hitter behavior, but chelation can evidently also lead to true potency gains and find use as a medicinal chemistry strategy to guide compound optimization. To demonstrate this, we exploited the effect to design the first potent, selective, and drug-like 14-3-3 PPI stabilizers.
Subject(s)
14-3-3 Proteins/chemistry , Biological Products/chemistry , Chelating Agents/chemistry , Metals/chemistry , Drug Discovery , Glycosides , Humans , Molecular Conformation , Protein BindingABSTRACT
OBJECTIVE: Peroxisome proliferator-activated receptors (PPARs) are key transcription factors that regulate adipose development and function, and the conversion of white into brown-like adipocytes. Here we investigated whether PPARα and PPARγ activation synergize to induce the browning of white fat. METHODS: A selection of PPAR activators was tested for their ability to induce the browning of both mouse and human white adipocytes in vitro, and in vivo in lean and obese mice. RESULTS: All dual PPARα/γ activators tested robustly increased uncoupling protein 1 (Ucp1) expression in both mouse and human adipocytes in vitro, with tesaglitazar leading to the largest Ucp1 induction. Importantly, dual PPARα/γ activator tesaglitazar strongly induced browning of white fat in vivo in both lean and obese male mice at thermoneutrality, greatly exceeding the increase in Ucp1 observed with the selective PPARγ activator rosiglitazone. While selective PPARγ activation was sufficient for the conversion of white into brown-like adipocytes in vitro, dual PPARα/γ activation was superior to selective PPARγ activation at inducing white fat browning in vivo. Mechanistically, the superiority of dual PPARα/γ activators is mediated at least in part via a PPARα-driven increase in fibroblast growth factor 21 (FGF21). Combined treatment with rosiglitazone and FGF21 resulted in a synergistic increase in Ucp1 mRNA levels both in vitro and in vivo. Tesaglitazar-induced browning was associated with increased energy expenditure, enhanced insulin sensitivity, reduced liver steatosis, and an overall improved metabolic profile compared to rosiglitazone and vehicle control groups. CONCLUSIONS: PPARγ and PPARα synergize to induce robust browning of white fat in vivo, via PPARγ activation in adipose, and PPARα-mediated increase in FGF21.
Subject(s)
Adipose Tissue, White/metabolism , PPAR alpha/metabolism , PPAR gamma/metabolism , Adipocytes, Brown/metabolism , Adipocytes, White/metabolism , Adipose Tissue, Brown/metabolism , Animals , Energy Metabolism , Male , Mice , Mice, Inbred C57BL , PPAR alpha/genetics , PPAR gamma/genetics , Thermogenesis/genetics , Transcription Factors/metabolism , Uncoupling Protein 1/metabolismABSTRACT
Macrophages are important regulators of obesity-associated inflammation and PPARα and -γ agonism in macrophages has anti-inflammatory effects. In this study, we tested the efficacy with which liposomal delivery could target the PPARα/γ dual agonist tesaglitazar to macrophages while reducing drug action in common sites of drug toxicity: the liver and kidney, and whether tesaglitazar had anti-inflammatory effects in an in vivo model of obesity-associated dysmetabolism. Methods: Male leptin-deficient (ob/ob) mice were administered tesaglitazar or vehicle for one week in a standard oral formulation or encapsulated in liposomes. Following the end of treatment, circulating metabolic parameters were measured and pro-inflammatory adipose tissue macrophage populations were quantified by flow cytometry. Cellular uptake of liposomes in tissues was assessed using immunofluorescence and a broad panel of cell subset markers by flow cytometry. Finally, PPARα/γ gene target expression levels in the liver, kidney, and sorted macrophages were quantified to determine levels of drug targeting to and drug action in these tissues and cells. Results: Administration of a standard oral formulation of tesaglitazar effectively treated symptoms of obesity-associated dysmetabolism and reduced the number of pro-inflammatory adipose tissue macrophages. Macrophages are the major cell type that took up liposomes with many other immune and stromal cell types taking up liposomes to a lesser extent. Liposome delivery of tesaglitazar did not have effects on inflammatory macrophages nor did it improve metabolic parameters to the extent of a standard oral formulation. Liposomal delivery did, however, attenuate effects on liver weight and liver and kidney expression of PPARα and -γ gene targets compared to oral delivery. Conclusions: These findings reveal for the first time that tesaglitazar has anti-inflammatory effects on adipose tissue macrophage populations in vivo. These data also suggest that while nanoparticle delivery reduced off-target effects, yet the lack of tesaglitazar actions in non-targeted cells such (as hepatocytes and adipocytes) and the uptake of drug-loaded liposomes in many other cell types, albeit to a lesser extent, may have impacted overall therapeutic efficacy. This fulsome analysis of cellular uptake of tesaglitazar-loaded liposomes provides important lessons for future studies of liposome drug delivery.
Subject(s)
Alkanesulfonates/pharmacology , Kidney/drug effects , Liposomes/administration & dosage , Liver/drug effects , Macrophages/drug effects , Obesity/drug therapy , PPAR alpha/agonists , PPAR gamma/agonists , Phenylpropionates/pharmacology , Adipose Tissue/drug effects , Adipose Tissue/metabolism , Animals , Diet, High-Fat , Disease Models, Animal , Drug Delivery Systems , Inflammation/metabolism , Kidney/metabolism , Liposomes/chemistry , Liver/metabolism , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/metabolism , Obesity/pathologyABSTRACT
Targeted nanoparticle delivery is a promising strategy for increasing efficacy and limiting side effects of therapeutics. When designing a targeted liposomal formulation, the in vivo biodistribution of the particles must be characterized to determine the value of the targeting approach. Peroxisome proliferator-activated receptor (PPAR) agonists effectively treat metabolic syndrome by decreasing dyslipidemia and insulin resistance but side effects have limited their use, making them a class of compounds that could benefit from targeted liposomal delivery. The adipose targeting sequence peptide (ATS) could fit this role, as it has been shown to bind to adipose tissue endothelium and induce weight loss when delivered conjugated to a pro-apoptotic peptide. To date, however, a full assessment of ATS in vivo biodistribution has not been reported, leaving important unanswered questions regarding the exact mechanisms whereby ATS targeting enhances therapeutic efficacy. We designed this study to evaluate the biodistribution of ATS-conjugated liposomes loaded with the PPARα/γ dual agonist tesaglitazar in leptin-deficient ob/ob mice. The ATS-liposome biodistribution in adipose tissue and other organs was examined at the cellular and tissue level using microscopy, flow cytometry, and fluorescent molecular tomography. Changes in metabolic parameters and gene expression were measured by target and off-target tissue responses to the treatment. Unexpectedly, ATS targeting did not increase liposomal uptake in adipose relative to other tissues, but did increase uptake in the kidneys. Targeting also did not significantly alter metabolic parameters. Analysis of the liposome cellular distribution in the stromal vascular fraction with flow cytometry revealed high uptake by multiple cell types. Our findings highlight the need for thorough study of in vivo biodistribution when evaluating a targeted therapy.
Subject(s)
Adipose Tissue/cytology , Adipose Tissue/metabolism , Drug Delivery Systems , Adipose Tissue/drug effects , Alkanesulfonates/pharmacology , Animals , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Leukocytes/drug effects , Leukocytes/metabolism , Liposomes , Macrophages/drug effects , Macrophages/metabolism , Male , Mice, Inbred C57BL , Organ Specificity/drug effects , Peptides/pharmacology , Phenylpropionates/pharmacologyABSTRACT
The mechanism-based risk for hyperkalemia has limited the use of mineralocorticoid receptor antagonists (MRAs) like eplerenone in cardio-renal diseases. Here, we describe the structure and property-driven lead generation and optimization, which resulted in identification of MR modulators ( S)-1 and ( S)-33. Both compounds were partial MRAs but still demonstrated equally efficacious organ protection as eplerenone after 4 weeks of treatment in uni-nephrectomized rats on high-salt diet and aldosterone infusion. Importantly, and in sharp contrast to eplerenone, this was achieved without substantial changes to the urine Na+/K+ ratio after acute treatment in rat, which predicts a reduced risk for hyperkalemia. This work led to selection of ( S)-1 (AZD9977) as the clinical candidate for treating MR-mediated cardio-renal diseases, including chronic kidney disease and heart failure. On the basis of our findings, we propose an empirical model for prediction of compounds with low risk of affecting the urinary Na+/K+ ratio in vivo.
Subject(s)
Homeostasis/drug effects , Mineralocorticoid Receptor Antagonists/pharmacology , Oxazines/pharmacology , Potassium/metabolism , Protective Agents/pharmacology , Sodium/metabolism , Animals , Heart/drug effects , Humans , Kidney/drug effects , Male , Mineralocorticoid Receptor Antagonists/chemical synthesis , Mineralocorticoid Receptor Antagonists/metabolism , Molecular Structure , Oxazines/chemical synthesis , Oxazines/metabolism , Potassium/urine , Protective Agents/chemical synthesis , Protective Agents/metabolism , Rats, Sprague-Dawley , Rats, Wistar , Receptors, Mineralocorticoid/metabolism , Renal Insufficiency, Chronic/drug therapy , Sodium/urine , Structure-Activity RelationshipABSTRACT
Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein-protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as "undruggable" targets, the last two decades have seen an increasing number of successful examples of PPI modulators, resulting in growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This perspective focuses on the hub-protein 14-3-3, which has several hundred identified protein interaction partners, and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide mimetics, and natural products.
Subject(s)
14-3-3 Proteins/metabolism , Drug Discovery/methods , 14-3-3 Proteins/antagonists & inhibitors , Animals , Humans , Protein Binding , Protein Stability/drug effectsABSTRACT
BACKGROUND: Composite grafts consisting of adipose tissue and skin have been reported in the literature but have been restricted to areas smaller than 4 to 5 cm. The senior author (D.T.W.C.) has developed a technique of adipose tissue-preserved full-thickness skin grafts for larger areas with success similar to that achieved with conventional full-thickness skin grafts. METHODS: All cases of the senior author involving a full-thickness skin graft were identified and reviewed to identify cases in which adipose tissue-preserved full-thickness skin grafts were used. Indication for skin grafting, anatomical location of recipient and donor sites, size of graft, total number of grafts received by each patient, and percentage take were extracted from patient charts. Graft take was measured between days 5 and 14. RESULTS: A total of 72 adipose tissue-preserved skin grafts on 47 patients were identified from 1994 to 2009, with a median follow-up of 8 years. The size of defect ranged from 0.7 to 210 cm, with a median area of 6 cm (interquartile range, 2.5 to 15 cm). Only six of 72 grafts were found to have less than 100 percent take. The lowest take percentage was 85 percent in a graft with an area of 2.6 cm. There were no graft failures. CONCLUSIONS: This study confirms the proof-of-concept that both larger and distant donor- site adipose tissue-preserved skin grafts are a viable alternative to conventional defatted full-thickness skin grafting. It appears that there is a low complication rate with respect to graft failure or incomplete graft take. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.
Subject(s)
Adipose Tissue/transplantation , Skin Transplantation/methods , Adult , Aged , Aged, 80 and over , Feasibility Studies , Female , Graft Survival , Humans , Male , Middle Aged , Skin Neoplasms/surgery , Wounds and Injuries/surgeryABSTRACT
INTRODUCTION: PPIs are involved in every disease and specific modulation of these PPIs with small molecules would significantly improve our prospects of developing therapeutic agents. Both industry and academia have engaged in the identification and use of PPI inhibitors. However in comparison, the opposite strategy of employing small-molecule stabilizers of PPIs is underrepresented in drug discovery. Areas covered: PPI stabilization has not been exploited in a systematic manner. Rather, this concept validated by a number of therapeutically used natural products like rapamycin and paclitaxel has been shown retrospectively to be the basis of the activity of synthetic molecules originating from drug discovery projects among them lenalidomide and tafamidis. Here, the authors cover the growing number of synthetic small-molecule PPI stabilizers to advocate for a stronger consideration of this as a drug discovery approach. Expert opinion: Both the natural products and the growing number of synthetic molecules show that PPI stabilization is a viable strategy for drug discovery. There is certainly a significant challenge to adapt compound libraries, screening techniques and downstream methodologies to identify, characterize and optimize PPI stabilizers, but the examples of molecules reviewed here in our opinion justify these efforts.
Subject(s)
Drug Design , Drug Discovery/methods , Proteins/metabolism , Biological Products/pharmacology , Humans , Pharmaceutical Preparations/chemical synthesis , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/metabolism , Protein Binding , Protein Stability , Small Molecule LibrariesABSTRACT
AIMS: Molecular imaging of the free fatty acid receptor 1 (FFAR1) would be a valuable tool for drug development by enabling in vivo target engagement studies in human. It has also been suggested as a putative target for beta cell imaging, but the inherent lipophilicity of most FFAR1 binders produces high off-target binding, which has hampered progress in this area. The aim of this study was to generate a suitable lead compound for further PET labeling. METHODS: In order to identify a lead compound for future PET labeling for quantitative imaging of FFAR1 in human, we evaluated tritiated small molecule FFAR1 binding probes ([3H]AZ1, [3H]AZ2 and [3H]TAK-875) for their off-target binding, receptor density and affinity in human pancreatic tissue (islets and exocrine) and rodent insulinoma. RESULTS: [3H]AZ1 showed improved specificity to FFAR1, with decreased off-target binding compared to [3H]AZ2 and [3H]TAK-875, while retaining high affinity in the nanomolar range. FFAR1 density in human islets was approximately 50% higher than in exocrine tissue. CONCLUSIONS: AZ1 is a suitable lead compound for PET labeling for molecular imaging of FFAR1 in humans, due to high affinity and reduced off-target binding.
Subject(s)
Insulin-Secreting Cells/metabolism , Molecular Imaging/methods , Receptors, G-Protein-Coupled/analysis , Receptors, G-Protein-Coupled/metabolism , Animals , Benzofurans/chemistry , Benzofurans/pharmacokinetics , Cell Line, Tumor , Drug Discovery/methods , HEK293 Cells , Humans , Insulin/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Pancreas/metabolism , Protein Binding , Rats , Sulfones/chemistry , Sulfones/pharmacokinetics , Tritium/chemistry , Tritium/pharmacokineticsABSTRACT
PURPOSE: To test the biomechanical properties of the dorsoradial capsulodesis procedure. METHODS: Six cadaveric hands were used. After exposing the trapeziometacarpal (TMC) joint, we placed Kirschner wires in the distal radius and thumb metacarpal. The rotation shear test was then performed to test the joint axial laxity, and angular measurements using Kirschner wires as reference points were documented. The dorsoradial (DR) ligament and capsule were released, followed by the intermetacarpal (IM) ligament; angular measurements were obtained. Finally, the DR capsulodesis procedure was performed, and final measurements were obtained. Comparisons were made among the various stages of ligament integrity to determine the amount of stability provided by DR capsulodesis. RESULTS: All cadavers demonstrated axial laxity with transection of the DR ligament; an increase in stability was obtained after DR capsulodesis. Transection of the capsule and IM ligament caused increased laxity relative to the native joint (median, 24° and 35°, respectively, on rotational testing). After we performed DR capsulodesis, rotational stability improved by a median of 41° compared with DR ligament transection, 49° compared with DR and IM ligament transection, and 18° relative to the native joint. CONCLUSIONS: Dorsoradial capsulodesis restores rotational stability for TMC joint after division of the DR and IM ligaments. The stability achieved was statistically significant compared with both an intact native TMC joint and induced laxity of the TMC joint. CLINICAL RELEVANCE: The DR capsulodesis procedure may improve rotational stability to the TMC joint.
Subject(s)
Carpometacarpal Joints/surgery , Joint Instability/surgery , Thumb/surgery , Trapezium Bone/surgery , Adult , Aged , Biomechanical Phenomena , Bone Wires , Cadaver , Carpometacarpal Joints/physiology , Female , Humans , Joint Capsule/surgery , Joint Instability/physiopathology , Ligaments, Articular/physiology , Ligaments, Articular/surgery , Male , Middle Aged , Thumb/physiology , Trapezium Bone/physiologyABSTRACT
The mineralocorticoid receptor (MR) is a nuclear hormone receptor involved in the regulation of body fluid and electrolyte homeostasis. In this study we explore selectivity triggers for a series of nonsteroidal MR antagonists to improve selectivity over other members of the oxosteroid receptor family. A biaryl sulfonamide compound was identified in a high-throughput screening (HTS) campaign. The compound bound to MR with pKi =6.6, but displayed poor selectivity over the glucocorticoid receptor (GR) and the progesterone receptor (PR). Following X-ray crystallography of MR in complex with the HTS hit, a compound library was designed that explored an induced-fit hypothesis that required movement of the Met852 side chain. An improvement in MR selectivity of 11- to 79-fold over PR and 23- to 234-fold over GR was obtained. Given the U-shaped binding conformation, macrocyclizations were explored, yielding a macrocycle that bound to MR with pKi =7.3. Two protein-ligand X-ray structures were determined, confirming the hypothesized binding mode for the designed compounds.
Subject(s)
Drug Design , Mineralocorticoid Receptor Antagonists/chemistry , Mineralocorticoid Receptor Antagonists/pharmacology , Receptors, Glucocorticoid/chemistry , Receptors, Glucocorticoid/metabolism , Receptors, Mineralocorticoid/metabolism , Dose-Response Relationship, Drug , High-Throughput Screening Assays , Humans , Mineralocorticoid Receptor Antagonists/chemical synthesis , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The peroxisome proliferator-activated receptor gamma (PPARγ) is the target for the thiazolidinedione class of potent insulin-sensitizing drugs, which includes rosiglitazone and pioglitazone. However, their usage has been restricted due to severe side effects. Recent data have shown that specifically inhibiting the cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of PPARγ at Ser273 may lead to novel insulin sensitizers with fewer side effects. Here we describe a novel enzyme-linked immunosorbent assay (ELISA) in the 384-well format, which enables screening for PPARγ ligands that inhibit phosphorylation at Ser273 by Cdk5. The assay is robust with a Z-factor > 0.6, demonstrating its suitability for high-throughput screening. We demonstrate the suitability of this assay for profiling of published PPARγ ligands and identification of novel compounds that prevent the Cdk5-mediated phosphorylation of PPARγ at Ser273 in a 622 compound pilot study. Our assay enables the discovery and development of novel therapeutic agents for use in type-2 diabetes. Furthermore, our results in combination with structural analysis of reported PPARγ ligand binding domain X-ray structures give a molecular rationale for the Cdk5-mediated phosphorylation of PPARγ at Ser273.
Subject(s)
Cyclin-Dependent Kinase 5/antagonists & inhibitors , Cyclin-Dependent Kinase 5/metabolism , High-Throughput Screening Assays/methods , PPAR gamma/metabolism , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay/methods , HEK293 Cells , Humans , Phosphorylation/drug effects , Phosphorylation/physiologyABSTRACT
Background: In a cadaveric model, we evaluated thumb metacarpal subsidence, indicated by a decreased metacarpal-to-scaphoid distance, after 2 surgical procedures used to treat thumb carpometacarpal (CMC) osteoarthritis (OA): partial trapeziectomy with capsular interposition (PTCI), which involves removal of 2 mm of both the distal trapezium and base of the metacarpal; and total trapeziectomy with capsular interposition (TTCI). Methods: Nine matched pairs of cadaveric hands were randomly assigned to undergo either PTCI or TTCI. Preoperatively, physiologic forces were applied across the thumb CMC joint by loading 6 tendons, simulating lateral pinch. Anteroposterior radiographs were obtained, and the metacarpal-to-scaphoid distance on each image was estimated independently by 3 separate readers using customized software. A hand surgeon then performed the PTCI and TTCI procedures, and the measurements under loading were repeated. The results were assessed for interrater reliability. Mean values for metacarpal-to-scaphoid distance before and after the surgical procedures were compared. Results: Preoperatively, the metacarpal-to-scaphoid distance in the PTCI and TTCI groups was not significantly different. Postoperatively, metacarpal subsidence was significantly less in the PTCI group (17% compared with 34% for TTCI; P = .05). Conclusions: Metacarpal subsidence occurred after both PTCI and TTCI, but significantly less subsidence was observed after PTCI; thus, thumb length was better preserved. Previous research has shown an inverse correlation between maintenance of thumb length and overall Disabilities of the Arm, Shoulder, and Hand (DASH) score. A procedure for treating thumb CMC OA that preserves thumb length and minimizes disruption of stabilizing joint tissue may provide enhanced maintenance of thumb stability and improved patient outcomes.