Screening methods for identifying pharmacological chaperones

2017 ◽  
Vol 13 (4) ◽  
pp. 638-647 ◽  
Author(s):  
Min Hyeon Shin ◽  
Hyun-Suk Lim
Keyword(s):  
Small Molecules ◽  
Misfolded Proteins ◽  
Screening Methods ◽  
Pharmacological Chaperones

This review highlights recent screening methods for identifying pharmacological chaperones, which are small-molecules capable of rescuing misfolded proteins.

scholarly journals Druggable Transient Pockets in Protein Kinases

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 651
Author(s):  
Koji Umezawa ◽  
Isao Kii
Keyword(s):  
Drug Discovery ◽  
Small Molecules ◽  
Protein Kinases ◽  
Binding Sites ◽  
Kinase Inhibitors ◽  
Screening Methods ◽  
Research Attention ◽  
Folding Process ◽  
Chemical Screening ◽  
Inhibitory Mechanisms

Drug discovery using small molecule inhibitors is reaching a stalemate due to low selectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of “cryptic inhibitor-binding sites.” These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors.

Targeting LEDGF Interactions in MLL Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2500-2500
Author(s):  
Tomasz Cierpicki ◽  
Shihan He ◽  
Trupta Purohit ◽  
Marcelo Murai ◽  
Thomas Hartley ◽  
...  
Keyword(s):  
Small Molecules ◽  
Fusion Proteins ◽  
High Throughput Screening ◽  
Hox Genes ◽  
Conflicts Of Interest ◽  
Leukemia Cells ◽  
Screening Methods ◽  
Fusion Partner ◽  
Acute Leukemias ◽  
N Terminus

Abstract Abstract 2500 Chromosomal translocations of MLL (Mixed Lineage Leukemia) gene result in aggressive acute leukemias, affecting both children and adults. Fusion of MLL to one of more than 60 partner genes results in MLL fusion oncoproteins which upregulate expression of Hox genes required for normal blood cell development, ultimately leading to development of acute leukemia. Regardless of the fusion partner, the presence of MLL translocations is associated with early relapse and poor prognosis. Survival rates are particularly low for infants and there is a pressing need for the development of targeted therapies against leukemias with MLL translocations. The oncogenic activity of MLL fusion proteins is dependent on association with LEDGF (lens epithelium-derived growth factor) and menin, both of which interact with the N-terminus of MLL retained in all MLL fusion proteins. LEDGF is a chromatin-associated protein, which interacts conjointly with MLL and menin on the chromatin of the cancer associated genes, and both interactions are required for the MLL-mediated leukemogenesis and misregulation of HOXA9 expression. Therefore, LEDGF functions as an essential oncogenic cofactor in MLL related leukemias, and may represent a valuable molecular target for therapeutic intervention with small molecules. We have performed rigorous biophysical and biochemical studies and revealed that LEDGF is involved in simultaneous interaction with menin and with the N-terminus of MLL. Interestingly, the association of LEDGF with the menin-MLL complex has relatively low affinity which limits the application of conventional screening methods for lead identification. To develop small molecule inhibitors targeting LEDGF interactions we have employed two strategies: Fragment Based Drug Discovery (FBDD) approach and High Throughput Screening (HTS). We have identified several compounds that bind directly to LEDGF. By applying NMR spectroscopy we discovered that these compounds interact with the menin binding site on LEDGF. Then we have assessed the activity of these compounds using a broad range of cell based assays. We found that compounds targeting LEDGF specifically inhibit proliferation of the MLL leukemia cells without affecting the non-MLL leukemia cells. They also induce apoptosis and differentiation of MLL leukemia cells as assessed by increased expression of CD11b differentiation marker and substantial change in morphology of these cells. Furthermore, these compounds reduce transforming properties of MLL fusion proteins and downregulate expression of Hoxa9 and Meis1 genes confirming a highly specific mode of action. Overall, our results demonstrate that targeting of LEDGF by small molecules is feasible and may results in development of potent inhibitors of LEDGF interaction with menin and MLL fusion proteins in leukemias with MLL rearrangements. Such compounds might provide a new therapeutic approach for the treatment of MLL-rearranged leukemias. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expanding Small-Molecule Functional Metagenomics through Parallel Screening of Broad-Host-Range Cosmid Environmental DNA Libraries in Diverse Proteobacteria

2010 ◽  
Vol 76 (5) ◽  
pp. 1633-1641 ◽  
Author(s):  
Jeffrey W. Craig ◽  
Fang-Yuan Chang ◽  
Jeffrey H. Kim ◽  
Steven C. Obiajulu ◽  
Sean F. Brady
Keyword(s):  
Small Molecules ◽  
Host Range ◽  
Small Molecule ◽  
Metagenomic Library ◽  
Environmental Dna ◽  
Culture Broth ◽  
Screening Methods ◽  
Broad Host Range ◽  
Functional Metagenomics ◽  
Dna Libraries

ABSTRACT The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.

scholarly journals A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Michael J. Munson ◽  
Gwen O’Driscoll ◽  
Andreia M. Silva ◽  
Elisa Lázaro-Ibáñez ◽  
Audrey Gallud ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
Mrna Translation ◽  
Fine Tuning ◽  
Endosomal Escape ◽  
Screening Methods ◽  
Cellular Delivery ◽  
Sufficient Detail ◽  
Screening Platform ◽  
High Throughput Imaging

AbstractRNA-based therapies have great potential to treat many undruggable human diseases. However, their efficacy, in particular for mRNA, remains hampered by poor cellular delivery and limited endosomal escape. Development and optimisation of delivery vectors, such as lipid nanoparticles (LNPs), are impeded by limited screening methods to probe the intracellular processing of LNPs in sufficient detail. We have developed a high-throughput imaging-based endosomal escape assay utilising a Galectin-9 reporter and fluorescently labelled mRNA to probe correlations between nanoparticle-mediated uptake, endosomal escape frequency, and mRNA translation. Furthermore, this assay has been integrated within a screening platform for optimisation of lipid nanoparticle formulations. We show that Galectin-9 recruitment is a robust, quantitative reporter of endosomal escape events induced by different mRNA delivery nanoparticles and small molecules. We identify nanoparticles with superior escape properties and demonstrate cell line variances in endosomal escape response, highlighting the need for fine-tuning of delivery formulations for specific applications.

scholarly journals Galactosemia: Towards Pharmacological Chaperones

2021 ◽  
Vol 11 (2) ◽  
pp. 106
Author(s):  
Samantha Banford ◽  
Thomas J. McCorvie ◽  
Angel L. Pey ◽  
David J. Timson
Keyword(s):  
Small Molecules ◽  
Enzyme Function ◽  
Current Therapy ◽  
Cognitive Disability ◽  
Novel Therapies ◽  
Biochemical Basis ◽  
Pharmacological Chaperones ◽  
Exciting Potential ◽  
Fundamental Cause ◽  
Life Threatening

Galactosemia is a rare inherited metabolic disease resulting from mutations in the four genes which encode enzymes involved in the metabolism of galactose. The current therapy, the removal of galactose from the diet, is inadequate. Consequently, many patients suffer lifelong physical and cognitive disability. The phenotype varies from almost asymptomatic to life-threatening disability. The fundamental biochemical cause of the disease is a decrease in enzymatic activity due to failure of the affected protein to fold and/or function correctly. Many novel therapies have been proposed for the treatment of galactosemia. Often, these are designed to treat the symptoms and not the fundamental cause. Pharmacological chaperones (PC) (small molecules which correct the folding of misfolded proteins) represent an exciting potential therapy for galactosemia. In theory, they would restore enzyme function, thus preventing downstream pathological consequences. In practice, no PCs have been identified for potential application in galactosemia. Here, we review the biochemical basis of the disease, identify opportunities for the application of PCs and describe how these might be discovered. We will conclude by considering some of the clinical issues which will affect the future use of PCs in the treatment of galactosemia.

scholarly journals Pharmacological Chaperones: A Therapeutic Approach for Diseases Caused by Destabilizing Missense Mutations

2020 ◽  
Vol 21 (2) ◽  
pp. 489 ◽  
Author(s):  
Ludovica Liguori ◽  
Maria Monticelli ◽  
Mariateresa Allocca ◽  
Bruno Hay Mele ◽  
Jan Lukas ◽  
...  
Keyword(s):  
Small Molecules ◽  
Specific Binding ◽  
Total Activity ◽  
Missense Mutations ◽  
Lysosomal Storage ◽  
Pharmacological Chaperone ◽  
Mutant Proteins ◽  
Pharmacological Chaperones ◽  
Protein Mutants ◽  
Competitive Inhibitors

The term “pharmacological chaperone” was introduced 20 years ago. Since then the approach with this type of drug has been proposed for several diseases, lysosomal storage disorders representing the most popular targets. The hallmark of a pharmacological chaperone is its ability to bind a protein specifically and stabilize it. This property can be beneficial for curing diseases that are associated with protein mutants that are intrinsically active but unstable. The total activity of the affected proteins in the cell is lower than normal because they are cleared by the quality control system. Although most pharmacological chaperones are reversible competitive inhibitors or antagonists of their target proteins, the inhibitory activity is neither required nor desirable. This issue is well documented by specific examples among which those concerning Fabry disease. Direct specific binding is not the only mechanism by which small molecules can rescue mutant proteins in the cell. These drugs and the properly defined pharmacological chaperones can work together with different and possibly synergistic modes of action to revert a disease phenotype caused by an unstable protein.

Gap junctions in the prothoracic glands of the tobacco hornworm, Manduca sexta

1992 ◽  
Vol 50 (1) ◽  
pp. 706-707
Author(s):  
Ji-da Dai ◽  
M. Joseph Costello ◽  
Lawrence I. Gilbert
Keyword(s):  
Gap Junctions ◽  
Small Molecules ◽  
Manduca Sexta ◽  
Freeze Fracture ◽  
Cell Communication ◽  
Cellular Level ◽  
Thin Sections ◽  
Prothoracic Glands ◽  
Polyhydroxylated Steroids ◽  
Stimulation Of

Insect molting and metamorphosis are elicited by a class of polyhydroxylated steroids, ecdysteroids, that originate in the prothoracic glands (PGs). Prothoracicotropic hormone stimulation of steroidogenesis by the PGs at the cellular level involves both calcium and cAMP. Cell-to-cell communication mediated by gap junctions may play a key role in regulating signal transduction by controlling the transmission of small molecules and ions between adjacent cells. This is the first report of gap junctions in the PGs, the evidence obtained by means of SEM, thin sections and freeze-fracture replicas.

Fusion and Fission Processes in Exocytosis: Possible Roles for Synexin and Osmotic Lysis in the Two Events

1980 ◽  
Vol 38 ◽  
pp. 594-597
Author(s):  
H.B. Pollard ◽  
C.E. Creutz ◽  
C.J. Pazoles ◽  
J.H. Scott
Keyword(s):  
Small Molecules ◽  
Chromaffin Cells ◽  
Adrenal Glands ◽  
General Concept ◽  
Extracellular Calcium ◽  
Large Protein ◽  
Granule Membrane ◽  
Osmotic Lysis ◽  
Per Se ◽  
Chemical Evidence

Exocytosis is a general concept describing secretion of enzymes, hormones and transmitters that are otherwise sequestered in intracellular granules. Chemical evidence for this concept was first gathered from studies on chromaffin cells in perfused adrenal glands, in which it was found that granule contents, including both large protein and small molecules such as adrenaline and ATP, were released together while the granule membrane was retained in the cell. A number of exhaustive reviews of this early work have been published and are summarized in Reference 1. The critical experiments demonstrating the importance of extracellular calcium for exocytosis per se were also first performed in this system (2,3), further indicating the substantial service given by chromaffin cells to those interested in secretory phenomena over the years.

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