Identification of small-molecule ligands that bind to MiR-21 as potential therapeutics for endometriosis by screening ZINC database and in-vitro assays

ABSTRACT The SCO7222 protein and ActR are two of ∼150 TetR-like transcription factors encoded in the Streptomyces coelicolor genome. Using bioluminescence as a readout, we have developed Escherichia coli-based biosensors that accurately report the regulatory activity of these proteins and used it to investigate their interactions with DNA and small-molecule ligands. We found that the SCO7222 protein and ActR repress the expression of their putative target genes, SCO7223 and actII-ORF2 (actA), respectively, by interacting with operator sequence in the promoters. The operators recognized by the two proteins are related such that O 7223 (an operator for SCO7223) could be bound by both the SCO7222 protein and ActR with similar affinities. In contrast, Oact (an operator for actII-ORF2) was bound tightly by ActR and more weakly by the SCO7222 protein. We demonstrated ligand specificity of these proteins by showing that while TetR (but not ActR or the SCO7222 protein) interacts with tetracyclines, ActR (but not TetR or the SCO7222 protein) interacts with actinorhodin and related molecules. Through operator-targeted mutagenesis, we found that at least two nucleotide changes in O 7223 were required to disrupt its interaction with SCO7222 protein, while ActR was more sensitive to changes on Oact . Most importantly, we found that the interaction of each protein with wild-type and mutant operator sequences in vivo and in vitro correlated perfectly. Our data suggest that E. coli-based biosensors of this type should be broadly applicable to TetR-like transcription factors.

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Mechanistic differences between in vitro assays for hydrazone-based small molecule inhibitors of anthrax lethal factor

Bioorganic Chemistry ◽

10.1016/j.bioorg.2006.07.004 ◽

2007 ◽

Vol 35 (1) ◽

pp. 50-58 ◽

Cited By ~ 11

Author(s):

M. Leslie Hanna ◽

Theodore M. Tarasow ◽

Julie Perkins

Keyword(s):

Small Molecule ◽

Small Molecule Inhibitors ◽

Lethal Factor ◽

In Vitro Assays ◽

Anthrax Lethal Factor

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Small-Molecule Inhibitors of the Rce1p CaaX Protease

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057107307226 ◽

2007 ◽

Vol 12 (7) ◽

pp. 983-993 ◽

Cited By ~ 43

Author(s):

Surya P. Manandhar ◽

Emily R. Hildebrandt ◽

Walter K. Schmidt

Keyword(s):

Small Molecule ◽

Small Molecule Inhibitors ◽

Proteolytic Processing ◽

In Vivo Studies ◽

In Vitro Assays ◽

Selective Agents ◽

Wild Type Yeast ◽

Micromolar Range

The Rce1p protease is required for the maturation of the Ras GTPase and certain other isoprenylated proteins and is considered a chemotherapeutic target. To identify new small-molecule inhibitors of Rce1p, the authors screened the National Cancer Institute Diversity Set compound library using in vitro assays to monitor the proteolytic processing of peptides derived from Ras and the yeast a-factor mating pheromone. Of 46 inhibitors initially identified with a Ras-based assay, only 9 were effective in the pheromone-based assay. The IC50 values of these 9 compounds were in the low micromolar range for both yeast (6-35 µM) and human Rce1p (0.4-46 µM). Four compounds were somewhat Rce1p selective in that they partially inhibited the Ste24p protease and did not inhibit Ste14p isoprenylcysteine carboxyl methyltransferase, 2 enzymes also involved in the maturation of isoprenylated proteins. The remaining 5 compounds inhibited all 3 enzymes. The 2 most Rce1p-selective agents were ineffective trypsin inhibitors, further supporting the specificity of these agents for Rce1p. The 5 least specific compounds formed colloidal aggregates, a proposed common feature of promiscuous inhibitors. Interestingly, the most specific Rce1p inhibitor also formed a colloidal aggregate. In vivo studies revealed that treatment of wild-type yeast with 1 compound induced a Ras2p delocalization phenotype that mimics observed effects in rce1 ste24 null yeast. The 9 compounds identified in this study represent new tools for understanding the enzymology of postisoprenylation-modifying enzymes and provide new insight for the future development of Rce1p inhibitors. ( Journal of Biomolecular Screening 2007:983-993)

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Identification of Small-Molecule Inhibitors of FGF23 Signaling via In Silico Hot Spot Prediction and Molecular Docking to α-Klotho

10.26434/chemrxiv.13083224.v2 ◽

2020 ◽

Author(s):

Shih-Hsien Liu ◽

Zhousheng Xiao ◽

Sambit K. Mishra ◽

Julie C. Mitchell ◽

Jeremy C. Smith ◽

...

Keyword(s):

Small Molecule ◽

In Silico ◽

Ternary Complex ◽

Hot Spot ◽

Fibroblast Growth Factor 23 ◽

Regular Function ◽

Fgf Receptor ◽

Zinc Database ◽

Extracellular Signal

Fibroblast growth factor 23 (FGF23) is a therapeutic target for treating hereditary and acquired hypophosphatemic disorders, such as X-linked hypophosphatemic (XLH) rickets and tumor-induced osteomalacia (TIO), respectively. FGF23-induced hypophosphatemia is mediated by signaling through a ternary complex formed by FGF23, FGF receptor (FGFR), and α-Klotho. Currently, disorders of excess FGF23 are treated with an FGF23-blocking antibody, Burosumab. Small-molecule drugs that disrupt protein:protein interactions necessary for the ternary complex formation offer an alternative to disrupt FGF23 signaling. In this study, the FGF23:α-Klotho interface was targeted to identify small-molecule protein:protein interaction inhibitors. We computationally identified “hot spots” in the FGF23:α-Klotho interface of the ternary complex and performed in silico docking of ~5.5 million compounds from the ZINC database to the interface region of α-Klotho from the ternary crystal structure. Following docking, 23 and 18 compounds were chosen based on the lowest binding free energies to α-Klotho and the largest number of contacts with Tyr433, a predicted hot spot, respectively. 5 compounds available were assessed experimentally by their FGF23-mediated extracellular signal-regulated kinase (ERK) activities in vitro, and two of these reduce activities significantly. Both these compounds have a favorable predicted binding affinity, but not a large number of contacts with the hot spot residues. ZINC12409120 was found experimentally to reduce FGF23-mediated ERK activities by 70% and have a half maximal inhibitory concentration (IC50) of 5.0 ± 0.23 uM. ZINC12409120 exhibits contacts with residues on KL1 and KL2 domains and on the linker between the two domains of α-Klotho in in silico binding poses, thereby possibly disrupting the regular function of α-Klotho and impeding FGF23 binding. ZINC12409120 is a candidate for lead optimization.

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Structure-Activity Studies of Novel di-substituted [1,2,5]oxadiazolo[3,4-b]pyrazine Analogs Targeting the A-loop Regulatory Site of p38 MAP Kinase

Current Medicinal Chemistry ◽

10.2174/0929867328666210712165659 ◽

2021 ◽

Vol 28 ◽

Author(s):

Esther Carrasco ◽

Patricia Gomez-Gutierrez ◽

Pedro M. Campos ◽

Angel Messeguer ◽

Juan Jesus Perez ◽

...

Keyword(s):

Molecular Modeling ◽

Map Kinase ◽

Small Molecule ◽

Human Monocyte ◽

P38 Map Kinase ◽

Molecular Structures ◽

In Vitro Assays ◽

Regulatory Site ◽

Screening Study

Introduction: In the quest for novel allosteric inhibitors of the p38 MAP kinase, we recently described the A-loop regulatory site, identified through molecular modeling studies together with the disclosure of a small molecule hit with a moderate inhibitory profile. Starting from this structure, we subsequently identified two additional hits with simpler molecular structures from an in silico screening study, using a substructure search in the SciFinder database. After corroboration of their inhibitory profile, analysis of their structures permitted to conclude about the suitability of the [1,2,5]oxadiazolo[3,4-b]pyrazine (furazano[3,4-b]pyrazine) scaffold for the development of potent A-loop regulatory site p38 MAP kinase inhibitors. Accordingly, we report the synthesis and pharmacological evaluation of a series of di-substituted analogs with a potent inhibitory profile of p38 MAP kinase, as shown by in vitro assays of their capability to inhibit IL-1β secretion in human monocyte-derived macrophages. Background: In the quest for novel allosteric inhibitors of the p38 MAP kinase, we recently described the A-loop regulatory site, identified through molecular modeling studies together with the disclosure of a small molecule hit with a moderate inhibitory profile. Objective: To find small molecule potent inhibitors of the p38 MAP kinase A-loop regulatory site. Methods: Starting from this structure, we subsequently identified two additional hits with simpler molecular structures from an in silico screening study, using a substructure search in the SciFinder database. After corroboration of their inhibitory profile, we carried out a hit-to-lead optimization process guided by molecular modeling using a [1,2,5]oxadiazolo[3,4-b]pyrazine (furazano[3,4-b]pyrazine) scaffold. Results: We report the synthesis and pharmacological evaluation of a series of di-substituted analogs with a potent inhibitory profile of p38 MAP kinase, as shown by in vitro assays of their capability to inhibit IL-1β secretion in human monocyte-derived macrophages. Conclusions: We describe in the present work a series of [1,2,5]oxadiazolo[3,4-b]pyrazine (furazano[3,4-b]pyrazine), potent inhibitors of IL-1β secretion in human monocyte-derived macrophages allosteric modulators of the p38 MAP kinase A-loop regulatory site.

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Identification of Small-Molecule Inhibitors of FGF23 Signaling via In Silico Hot Spot Prediction and Molecular Docking to α-Klotho

10.26434/chemrxiv.13083224 ◽

2020 ◽

Author(s):

Shih-Hsien Liu ◽

Zhousheng Xiao ◽

Sambit K. Mishra ◽

Julie C. Mitchell ◽

Jeremy C. Smith ◽

...

Keyword(s):

Small Molecule ◽

In Silico ◽

Ternary Complex ◽

Hot Spot ◽

Fibroblast Growth Factor 23 ◽

Regular Function ◽

Fgf Receptor ◽

Zinc Database ◽

Extracellular Signal

Fibroblast growth factor 23 (FGF23) is a therapeutic target for treating hereditary and acquired hypophosphatemic disorders, such as X-linked hypophosphatemic (XLH) rickets and tumor-induced osteomalacia (TIO), respectively. FGF23-induced hypophosphatemia is mediated by signaling through a ternary complex formed by FGF23, FGF receptor (FGFR), and α-Klotho. Currently, disorders of excess FGF23 are treated with an FGF23-blocking antibody, Burosumab. Small-molecule drugs that disrupt protein:protein interactions necessary for the ternary complex formation offer an alternative to disrupt FGF23 signaling. In this study, the FGF23:α-Klotho interface was targeted to identify small-molecule protein:protein interaction inhibitors. We computationally identified “hot spots” in the FGF23:α-Klotho interface of the ternary complex and performed in silico docking of ~5.5 million compounds from the ZINC database to the interface region of α-Klotho from the ternary crystal structure. Following docking, 23 and 18 compounds were chosen based on the lowest binding free energies to α-Klotho and the largest number of contacts with Tyr433, a predicted hot spot, respectively. 5 compounds available were assessed experimentally by their FGF23-mediated extracellular signal-regulated kinase (ERK) activities in vitro, and two of these reduce activities significantly. Both these compounds have a favorable predicted binding affinity, but not a large number of contacts with the hot spot residues. ZINC12409120 was found experimentally to reduce FGF23-mediated ERK activities by 70% and have a half maximal inhibitory concentration (IC50) of 5.0 ± 0.23 uM. ZINC12409120 exhibits contacts with residues on KL1 and KL2 domains and on the linker between the two domains of α-Klotho in in silico binding poses, thereby possibly disrupting the regular function of α-Klotho and impeding FGF23 binding. ZINC12409120 is a candidate for lead optimization.

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In Vitro Characterization of FTX6058, a Novel Small Molecule Fetal Hemoglobin Inducer for Sickle Cell Disease

Blood ◽

10.1182/blood-2020-141145 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-12

Author(s):

Billy Stuart ◽

Peter Rahl ◽

Kingsley Appiah ◽

Ivan Efremov ◽

Lorin Thompson ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Small Molecule ◽

Fetal Hemoglobin ◽

In Vitro Assays ◽

Cell Disease ◽

Publicly Traded ◽

Α Subunit ◽

The Past

Red blood cell disorders like Sickle Cell Disease (SCD) and β-thalassemias are caused by mutations within the gene for the hemoglobin β (HBβ) subunit. A fetal ortholog of HBβ, hemoglobin γ (HBγ) can prevent or reduce disease-related pathophysiology in these disorders by forming nonpathogenic complexes with the required hemoglobin α subunit. Globin expression is developmentally regulated, with a reduction in production of the fetal ortholog (γ) occurring shortly after birth and a concomitant increase in the levels of the adult ortholog (β). It has been postulated that maintaining expression of the anti-sickling γ ortholog may be of therapeutic benefit in children and adults with SCD. Indeed, individuals with the SCD mutation who also have genetic variants that maintain HBγ expression and the resulting fetal hemoglobin (HbF) tetramer at clinically meaningful levels do not present with SCD-related symptoms. Parallel target identification efforts using CRISPR and the Fulcrum proprietary, annotated chemical probe screening set in HUDEP2 cells identified a protein complex as a key regulator of HbF expression. Structure-guided medicinal chemistry optimization led to the design of FTX-6058, a novel, potent and selective small molecule. FTX-6058 treatment of differentiated primary CD34+ cells from multiple healthy donors demonstrated target engagement and potent upregulation of HBG1/2 mRNA and HbF protein. Across multiple healthy and SCD donors, FTX-6058 treatment resulted in a clinically desirable globin profile (e.g., up to approximately 30% HbF) accompanied by pancellular HbF expression, resembling the phenotype of SCD mutation carriers with hereditary persistence of fetal hemoglobin. FTX-6058 demonstrated a superior pharmacological profile relative to hydroxyurea and other small molecule compounds whose putative mechanism of action is to induce HbF. Preclinical studies using a variety of in vitro assays have demonstrated the potential of FTX-6058 as a clinical development candidate for potential treatment of hemoglobinopathies, such as SCD and ꞵ-thalassemia, via upregulation of HbF. IND enabling studies for FTX-6058 have been completed. Keywords: hemoglobin, fetal hemoglobin, HbF, HBG1/2, sickle cell disease, gene regulation Disclosures Stuart: Fulcrum Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rahl:Fulcrum Therapeutics: Ended employment in the past 24 months. Appiah:Fulcrum Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Efremov:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Thompson:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Wallace:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Moxham:Fulcrum Therapeutics: Current Employment, Current equity holder in publicly-traded company.

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Application of small molecule FPR1 antagonists in the treatment of cancers

Scientific Reports ◽

10.1038/s41598-020-74350-z ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Djevdet S. Ahmet ◽

Haneen A. Basheer ◽

Anwar Salem ◽

Di Lu ◽

Amin Aghamohammadi ◽

...

Keyword(s):

Small Molecule ◽

Formyl Peptide Receptor ◽

In Vitro Assays ◽

In Vivo Model ◽

Formyl Peptide ◽

Infection And Inflammation ◽

2D And 3D ◽

Receptor Family

Abstract The formylpeptide receptor-1 (FPR1) is a member of the chemotactic GPCR-7TM formyl peptide receptor family, whose principle function is in trafficking of various leukocytes into sites of bacterial infection and inflammation. More recently, FPR1 has been shown to be expressed in different types of cancer and in this context, plays a significant role in their expansion, resistance and recurrence. ICT12035 is a selective and potent (30 nM in calcium mobilisation assay) small molecule FPR1 antagonist. Here, we demonstrate the efficacy of ICT12035, in a number of 2D and 3D proliferation and invasion in vitro assays and an in vivo model. Our results demonstrate that targeting FPR1 by a selective small molecule antagonist, such as ICT12035, can provide a new avenue for the treatment of cancers.

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Progress in the Discovery of Small-Molecule Inhibitors of Bromodomain–Histone Interactions

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111421372 ◽

2011 ◽

Vol 16 (10) ◽

pp. 1170-1185 ◽

Cited By ~ 39

Author(s):

Chun-wa Chung ◽

Jason Witherington

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Transcriptional Activation ◽

Label Free ◽

Protein Protein Interactions ◽

Ligand Selectivity ◽

Small Molecule Ligands ◽

Associated Proteins ◽

In Vitro Systems

Bromodomains are structurally conserved protein modules present in a large number of chromatin-associated proteins and in many nuclear histone acetyltransferases. The bromodomain functions as an acetyl-lysine binding domain and has been shown to be pivotal in regulating protein–protein interactions in chromatin-mediated cellular gene transcription, cell proliferation, and viral transcriptional activation. Structural analyses of these modules in complex with acetyl-lysine peptide ligands provide insights into the molecular basis for recognition and ligand selectivity within this epigenetic reader family. However, there are significant challenges in configuring assays to identify inhibitors of these proteins. This review focuses on the progress made in developing methods to identify peptidic and small-molecule ligands using biophysical label-free and biochemical approaches. The advantage of each technique and the results reported are summarized, highlighting the potential applicably to other reader domains and the caveats in translation from simple in vitro systems to a biological context.

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Identification of Small-Molecule Inhibitors of FGF23 Signaling via In Silico Hot Spot Prediction and Molecular Docking to α-Klotho

10.26434/chemrxiv.13083224.v1 ◽

2020 ◽

Author(s):

Shih-Hsien Liu ◽

Zhousheng Xiao ◽

Sambit K. Mishra ◽

Julie C. Mitchell ◽

Jeremy C. Smith ◽

...

Keyword(s):

Small Molecule ◽

In Silico ◽

Ternary Complex ◽

Hot Spot ◽

Fibroblast Growth Factor 23 ◽

Regular Function ◽

Fgf Receptor ◽

Zinc Database ◽

Extracellular Signal

Fibroblast growth factor 23 (FGF23) is a therapeutic target for treating hereditary and acquired hypophosphatemic disorders, such as X-linked hypophosphatemic (XLH) rickets and tumor-induced osteomalacia (TIO), respectively. FGF23-induced hypophosphatemia is mediated by signaling through a ternary complex formed by FGF23, FGF receptor (FGFR), and alpha-Klotho. Currently, disorders of excess FGF23 are treated with an FGF23-blocking antibody, Burosumab. Small-molecule drugs that disrupt protein:protein interactions necessary for the ternary complex formation offer an alternative to disrupt FGF23 signaling. In this study, the FGF23:alpha-Klotho interface was targeted to identify small-molecule protein:protein interaction inhibitors. We computationally identified “hot spots” in the FGF23:alpha-Klotho interface of the ternary complex and performed in silico docking of ~5.5 million compounds from the ZINC database to the interface region of alpha-Klotho from the ternary crystal structure. Following docking, 23 and 18 compounds were chosen based on the lowest binding free energies to alpha-Klotho and the largest number of contacts with Tyr433, a predicted hot spot, respectively. 5 compounds available were assessed experimentally by their FGF23-mediated extracellular signal-regulated kinase (ERK) activities in vitro, and two of these reduce activities significantly. Both these compounds have a favorable predicted binding affinity, but not a large number of contacts with the hot spot residues. ZINC12409120 was found experimentally to reduce FGF23-mediated ERK activities by 70% and have a half maximal inhibitory concentration (IC50) of 5.0 ± 0.23 uM. ZINC12409120 exhibits contacts with residues on KL1 and KL2 domains and on the linker between the two domains of alpha-Klotho in in silico binding poses, thereby possibly disrupting the regular function of alpha-Klotho and impeding FGF23 binding. ZINC12409120 is a candidate for lead optimization.

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