Small Molecule Mitochondrial Uncouplers and Their Therapeutic Potential

AbstractApproximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5′ splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases.

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The Role of the Cannabinoid CB2 Receptor in Pain Transmission and Therapeutic Potential of Small Molecule CB2 Receptor Agonists

Current Medicinal Chemistry ◽

10.2174/092986707780363023 ◽

2007 ◽

Vol 14 (8) ◽

pp. 917-936 ◽

Cited By ~ 93

Author(s):

G Whiteside ◽

G. Lee ◽

K Valenzano

Keyword(s):

Small Molecule ◽

Therapeutic Potential ◽

Cb2 Receptor ◽

Receptor Agonists ◽

Pain Transmission

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Idalopirdine – a small molecule antagonist of 5-HT6 with therapeutic potential against obesity

Metabolic Brain Disease ◽

10.1007/s11011-015-9736-3 ◽

2015 ◽

Vol 30 (6) ◽

pp. 1487-1494 ◽

Cited By ~ 20

Author(s):

Magdalena Dudek ◽

Monika Marcinkowska ◽

Adam Bucki ◽

Adrian Olczyk ◽

Marcin Kołaczkowski

Keyword(s):

Small Molecule ◽

Therapeutic Potential ◽

Small Molecule Antagonist

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Discovery and Optimization of Selective Inhibitors of Meprin α (Part II)

10.20944/preprints202012.0383.v1 ◽

2020 ◽

Author(s):

Chao Wang ◽

Juan Diez ◽

Hajeung Park ◽

Christoph Becker-Pauly ◽

Gregg B. Fields ◽

...

Keyword(s):

Drug Discovery ◽

Small Molecules ◽

Small Molecule ◽

Hydroxamic Acid ◽

Therapeutic Potential ◽

Preceding Paper ◽

Close Relative ◽

Specific Inhibition ◽

Selective Inhibitors

Meprin α is a zinc metalloproteinase (metzincin) that has been implicated in multiple diseases, including fibrosis and cancers. It has proven difficult to find small molecules that are capable of selectively inhibiting meprin α, or its close relative meprin β, over numerous other metzincins which, if inhibited, would elicit unwanted effects. We recently identified possible molecular starting points for meprin α-specific inhibition through an HTS effort (see part I, preceding paper). In part II we report the optimization of a potent and selective hydroxamic acid meprin α inhibitor probe which may help define the therapeutic potential for small molecule meprin α inhibition and spur further drug discovery efforts in the area of zinc metalloproteinase inhibition.

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Applicability of Small-Molecule Inhibitors in the Study of Peptidyl Arginine Deiminase 2 (PAD2) and PAD4

Frontiers in Immunology ◽

10.3389/fimmu.2021.716250 ◽

2021 ◽

Vol 12 ◽

Author(s):

María Teresa Martín Monreal ◽

Alexandra Stripp Rebak ◽

Laura Massarenti ◽

Santanu Mondal ◽

Ladislav Šenolt ◽

...

Keyword(s):

T Cells ◽

Synovial Fluid ◽

Small Molecule ◽

Therapeutic Potential ◽

Specific Inhibitor ◽

Arginine Deiminase ◽

Self Tolerance ◽

Inhibition Of Pad ◽

Pad Inhibitor ◽

Dying Cells

Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline, is involved in the breakage of self-tolerance in anti-CCP-positive rheumatoid arthritis. This reaction is catalyzed by peptidyl arginine deiminases (PADs), of which PAD2 and PAD4 are thought to play key pathogenic roles. Small-molecule PAD inhibitors such as the pan-PAD inhibitor BB-Cl-amidine, the PAD2-specific inhibitor AFM-30a, and the PAD4-specific inhibitor GSK199 hold therapeutic potential and are useful tools in studies of citrullination. Using an ELISA based on the citrullination of fibrinogen, we found that AFM-30a inhibited the catalytic activity of PADs derived from live PMNs or lysed PBMCs and PMNs and of PADs in cell-free synovial fluid samples from RA patients, while GSK199 had minor effects. In combination, AFM-30a and GSK199 inhibited total intracellular citrullination and citrullination of histone H3 in PBMCs, as determined by Western blotting. They were essentially nontoxic to CD4+ T cells, CD8+ T cells, B cells, NK cells, and monocytes at concentrations ranging from 1 to 20 μM, while BB-Cl-amidine was cytotoxic at concentrations above 1 μM, as assessed by flow cytometric viability staining and by measurement of lactate dehydrogenase released from dying cells. In conclusion, AFM-30a is an efficient inhibitor of PAD2 derived from PBMCs, PMNs, or synovial fluid. AFM-30a and GSK199 can be used in combination for inhibition of PAD activity associated with PBMCs but without the cytotoxic effect of BB-Cl-amidine. This suggests that AFM-30a and GSK199 may have fewer off-target effects than BB-Cl-amidine and therefore hold greater therapeutic potential.

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Abstract 183: Small Molecule Gβ? Inhibition Reduces Pathologic Activation of Cardiac Fibroblasts

Circulation Research ◽

10.1161/res.113.suppl_1.a183 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Joshua G Travers ◽

Fadia A Kamal ◽

Burns C Blaxall

Keyword(s):

Cardiac Hypertrophy ◽

Small Molecule ◽

Adrenergic Receptor ◽

Cellular Proliferation ◽

Therapeutic Potential ◽

Cardiac Fibroblasts ◽

Akt Phosphorylation ◽

Potential Therapeutic Role ◽

Protease Activated Receptor 1 ◽

Chronic Activation

Heart failure (HF) is a devastating disease characterized by cardiac hypertrophy, fibrosis and inflammation. Excess signaling through Gβγ subunits leads to chronic β-adrenergic receptor (β-AR) downregulation, mediated predominantly by GRK2 in complex with PI3Kγ. Our recent work has demonstrated the therapeutic potential of the small molecule Gβγ-GRK2 inhibitor Gallein in limiting HF progression. Chronic activation of cardiac fibroblasts (CF), critical yet underappreciated myocardial cells, is a key contributor to pathologic cardiac remodeling. We hypothesized that Gβγ-GRK2 inhibition may limit pathologic CF activation. CFs were stimulated with Isoproterenol (Iso, β-AR agonist), AngII, or vehicle (V), +/- Gβγ inhibition for 24hr. Gallein treatment attenuated the induction of αSMA expression, a marker of pathologic CF activation, and two inflammatory cytokines, IL-1β and IL-6 in response to these pathologic stimuli (Iso, AngII), as assessed by real time PCR. This data suggest that Gallein treatment may reduce pathologic CF activation. Iso stimulation also enhances the phosphorylation of Akt, a kinase downstream of PI3Kγ known to be involved in cellular proliferation. Gβγ inhibition mitigated this induction, decreasing Akt phosphorylation >60% in response to Iso. This phenomenon was also observed in failing human CFs, in which Gallein decreased Akt phosphorylation >70%. We have recently demonstrated that the protease-activated receptor 1 (PAR1), a GPCR we have implicated in cardiac hypertrophy, is transactivated via chronic β-AR stimulation by induction of MMP-13, a protease we have found to be elevated in HF. Recent data from our lab and others have demonstrated that PAR1 is the most abundantly expressed GPCR in CFs, and that its stimulation in CFs may be pathologic. Interestingly, Gβγ inhibition treatment reduced PAR1 cleavage and activation in response to chronic Iso. In summary, small molecule Gβγ inhibition appears to reduce pathologic CF activation. The reduction in β-AR-mediated PAR1 cleavage reveals an alternative role for Gβγ inhibition in preventing CF activation and proliferation. These data suggest a potential therapeutic role for small molecule Gβγ inhibition in limiting pathologic CF activation and cardiac hypertrophy.

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Abstract 319: Small Molecule Gβγ Inhibition Attenuates Cardiac Fibroblast Inflammatory and Pro-Fibrotic Signaling

Circulation Research ◽

10.1161/res.115.suppl_1.319 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Joshua G Travers ◽

Fadia A Kamal ◽

Michael S Burhans ◽

Burns C Blaxall

Keyword(s):

Myocardial Fibrosis ◽

Inflammatory Cytokines ◽

Small Molecule ◽

Therapeutic Potential ◽

Interstitial Fibrosis ◽

Pressure Overload ◽

Cardiac Fibroblast ◽

Ventricular Compliance ◽

Pro Inflammatory Cytokines

Heart failure (HF) is a devastating disease characterized by chamber remodeling, interstitial fibrosis and reduced ventricular compliance. Prolonged sympathetic overstimulation promotes excess signaling through G-protein Gβγ subunits and ultimately results in pathologic GRK2-mediated β-adrenergic receptor (β-AR) downregulation. We have recently demonstrated the therapeutic potential of the small molecule Gβγ-GRK2 inhibitor Gallein in limiting HF progression. Pathologic activation of the cardiac fibroblast (CF) induces the transition to a myofibroblast phenotype, which plays a fundamental role in myocardial fibrosis and remodeling. We hypothesized that Gβγ-GRK2 inhibition plays an important functional role in the CF to attenuate pathologic CF activation, inflammation and interstitial fibrosis. To explore the effect of Gβγ-GRK2 inhibition on inflammation and pro-fibrotic signaling, mice were subjected to 7 days of transverse aortic constriction, a pressure-overload model of HF. In addition to the attenuation in overall cardiac hypertrophy, animals treated with Gallein displayed reduced expression of pro-inflammatory cytokines, including macrophage inflammatory protein 1 alpha (MIP-1α) and MIP-1β, along with Interleukin-6, as assessed by qPCR. Gallein-treated animals also exhibited diminished pro-fibrotic signaling, as evidenced by a reduction in the expression of TGFβ, a major driver of myocardial fibrosis, and decreased expression of collagen. To recapitulate these findings in vitro, primary adult mouse ventricular fibroblasts were pathologically stimulated using Isoproterenol (ISO, β-AR agonist) or Angiotensin II and treated +/- Gallein for 24 hours. CFs treated with Gallein displayed an analogous reduction in the expression of these pro-inflammatory cytokines and collagen. In summary, these data suggest a potential therapeutic role for small molecule Gβγ-GRK2 inhibition in limiting pathologic myofibroblast activation, inflammation and interstitial fibrosis. We believe this fibroblast-targeted approach will lead to the refinement of existing targets and compounds, and possibly the generation of novel therapeutics for the treatment of HF.

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Cdc-Like Kinases (CLKs): Biology, Chemical Probes, and Therapeutic Potential

International Journal of Molecular Sciences ◽

10.3390/ijms21207549 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7549

Author(s):

Paula Martín Moyano ◽

Václav Němec ◽

Kamil Paruch

Keyword(s):

Clinical Trials ◽

Protein Kinases ◽

Small Molecule ◽

Chemical Biology ◽

Small Molecule Inhibitors ◽

Therapeutic Potential ◽

Chemical Probes ◽

The Family

Protein kinases represent a very pharmacologically attractive class of targets; however, some members of the family still remain rather unexplored. The biology and therapeutic potential of cdc-like kinases (CLKs) have been explored mainly over the last decade and the first CLK inhibitor, compound SM08502, entered clinical trials only recently. This review summarizes the biological roles and therapeutic potential of CLKs and their heretofore published small-molecule inhibitors, with a focus on the compounds’ potential to be utilized as quality chemical biology probes.

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A phenolic small molecule inhibitor of RNase L prevents cell death from ADAR1 deficiency

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2006883117 ◽

2020 ◽

Vol 117 (40) ◽

pp. 24802-24812 ◽

Cited By ~ 1

Author(s):

Salima Daou ◽

Manisha Talukdar ◽

Jinle Tang ◽

Beihua Dong ◽

Shuvojit Banerjee ◽

...

Keyword(s):

Cell Death ◽

Protein Kinase ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Therapeutic Potential ◽

Protein Kinase Inhibitors ◽

Response To Treatment ◽

Rnase L ◽

Oligoadenylate Synthetase ◽

In Cells

The oligoadenylate synthetase (OAS)–RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.

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