CCN2 functions primary mediator in Smad3-dependent expression of FN in primary dermal fibroblasts.

<p>Primary dermal fibroblasts (1 × 10⁶) were transfected with the indicated siRNAs (20nM) and vectors (1μg). Total RNA and whole cell extract were prepared 48 hours after transfection. mRNA and protein levels were quantified by real-time RT-PCR and Western analysis, respectively. mRNA levels were normalized to mRNA for 36B4, a ribosomal protein used as an internal control for quantitation. Protein levels were normalized by β-actin (loading control). Insets show representative Western blots. (A, C) CCN2 and FN mRNA levels. (B, D) CCN2, FN, Smad2, and Smad3 protein levels. (E) The ability of CCN2 to regulate FN expression is dependent on intact TGF-β signaling. 32 hours after transfection, cell were treated with TGF-β1 (5 ng/ml) for 16 hours. Data are expressed as mean±SEM, N = 3, *p<0.05 vs control.</p

FN expression is regulated by CCN2 in primary dermal fibroblasts.

<p>Primary dermal fibroblasts (1 × 10⁶) were transfected with non-specific control siRNA or CCN2 siRNAs (20nM) (A, B, C), or control vector (pCDNA3.1, 2μg) or increasing amounts of CCN2 vector (0.5, 1, and 2μg) (D, E). Total RNA and whole cell extract were prepared 48 hours after transfection. (A) CCN2 mRNA levels. (B) FN mRNA levels. (C) CCN and FN Protein levels. (D) FN mRNA levels. (E) CCN and FN protein levels. mRNA levels were quantified by real-time RT-PCR. Protein levels were determined by ProteinSimple capillary electrophoresis immunoassay (C) and Western analysis (E). mRNA levels were normalized to mRNA for 36B4, a ribosomal protein used as an internal control quantitation. Protein levels were normalized by β-actin (loading control). Insets show representative digital images (C) and Western blots (E). Data are expressed as mean±SEM, N = 3–5, *p<0.05.</p

CCN2 and FN are primarily expressed in the dermis of normal human skin, stromal tissues of skin SCC, and wounded human skin.

<p>(A, B) Epidermis and dermis were captured by LCM. Total RNA was extracted from captured tissue, and mRNA levels were quantified by real-time RT-PCR. CCN2 (A) and FN (B) mRNA levels were normalized to the housekeeping gene 36B4, as an internal control for quantification. Data are relative levels to 36B4 (mean±SEM), N = 6, *p<0.05. (C) Double immunostaining for CCN2 and FN in normal human skin. OCT-embedded normal human skin sections (7μm) were co-immunofluorescence stained with CCN2 and FN. Representative of six individuals. Bar = 50μm. (D) Expression of CCN2 and FN in the stromal tissues of SCC was determined by immunohistology. Arrow heads indicate tumor islands. Representative of five SCC. Bar = 100μm. (E) Double immunostaining for CCN2 and FN. Representative of six individuals. Bar = 50μm. (F) Partial thickness wounds were made in forearm skin of healthy adult individuals by CO₂ laser (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173191#sec002" target="_blank">Methods</a> for details). Skin samples were obtained at indicated times, and mRNA levels were quantified by real-time RT-PCR. CCN2 and FN mRNA levels were normalized to the housekeeping gene 36B4, as an internal control for quantification. Mean±SEM, N = 6, *p<0.05 vs control.</p

CCN2 and FN expression are primarily regulated by Smad3 in primary dermal fibroblasts.

<p>Primary dermal fibroblasts (1 × 10⁶) were transfected with the indicated siRNAs (20nM). Total RNA and whole cell extract were prepared 48 hours after transfection. mRNA levels were quantified by real-time RT-PCR. Protein levels were determined by ProteinSimple capillary electrophoresis immunoassay (B, C) and Western blots (E). mRNA levels were normalized to mRNA for 36B4, a ribosomal protein used as an internal control for quantitation. Protein levels were normalized by β-actin (loading control). Insets show representative digital images (B, C) and Western blots (E). (A) Smad2 and Smad3 mRNA levels. (B) CCN2, FN, and Smad3 protein levels. (C) CCN2, FN, and Smad2 protein levels. (D) CCN2 and FN mRNA levels. 32 hours after transfection, cell were treated with TGF-β1 (5 ng/ml) for 16 hours. (E) CCN2, FN, Smad2, and Smad3 protein levels. 32 hours after transfection, cell were treated with TGF-β1 (5 ng/ml) for 16 hours. Data are expressed as mean±SEM, N = 3, *p<0.05 vs control.</p

Rational Design of Orthogonal Multipolar Interactions with Fluorine in Protein–Ligand Complexes

Multipolar interactions involving fluorine and the protein backbone have been frequently observed in protein–ligand complexes. Such fluorine–backbone interactions may substantially contribute to the high affinity of small molecule inhibitors. Here we found that introduction of trifluoromethyl groups into two different sites in the thienopyrimidine class of menin–MLL inhibitors considerably improved their inhibitory activity. In both cases, trifluoromethyl groups are engaged in short interactions with the backbone of menin. In order to understand the effect of fluorine, we synthesized a series of analogues by systematically changing the number of fluorine atoms, and we determined high-resolution crystal structures of the complexes with menin. We found that introduction of fluorine at favorable geometry for interactions with backbone carbonyls may improve the activity of menin–MLL inhibitors as much as 5- to 10-fold. In order to facilitate the design of multipolar fluorine–backbone interactions in protein–ligand complexes, we developed a computational algorithm named FMAP, which calculates fluorophilic sites in proximity to the protein backbone. We demonstrated that FMAP could be used to rationalize improvement in the activity of known protein inhibitors upon introduction of fluorine. Furthermore, FMAP may also represent a valuable tool for designing new fluorine substitutions and support ligand optimization in drug discovery projects. Analysis of the menin–MLL inhibitor complexes revealed that the backbone in secondary structures is particularly accessible to the interactions with fluorine. Considering that secondary structure elements are frequently exposed at protein interfaces, we postulate that multipolar fluorine–backbone interactions may represent a particularly attractive approach to improve inhibitors of protein–protein interactions

Rational Design of Orthogonal Multipolar Interactions with Fluorine in Protein–Ligand Complexes

Multipolar interactions involving fluorine and the protein backbone have been frequently observed in protein–ligand complexes. Such fluorine–backbone interactions may substantially contribute to the high affinity of small molecule inhibitors. Here we found that introduction of trifluoromethyl groups into two different sites in the thienopyrimidine class of menin–MLL inhibitors considerably improved their inhibitory activity. In both cases, trifluoromethyl groups are engaged in short interactions with the backbone of menin. In order to understand the effect of fluorine, we synthesized a series of analogues by systematically changing the number of fluorine atoms, and we determined high-resolution crystal structures of the complexes with menin. We found that introduction of fluorine at favorable geometry for interactions with backbone carbonyls may improve the activity of menin–MLL inhibitors as much as 5- to 10-fold. In order to facilitate the design of multipolar fluorine–backbone interactions in protein–ligand complexes, we developed a computational algorithm named FMAP, which calculates fluorophilic sites in proximity to the protein backbone. We demonstrated that FMAP could be used to rationalize improvement in the activity of known protein inhibitors upon introduction of fluorine. Furthermore, FMAP may also represent a valuable tool for designing new fluorine substitutions and support ligand optimization in drug discovery projects. Analysis of the menin–MLL inhibitor complexes revealed that the backbone in secondary structures is particularly accessible to the interactions with fluorine. Considering that secondary structure elements are frequently exposed at protein interfaces, we postulate that multipolar fluorine–backbone interactions may represent a particularly attractive approach to improve inhibitors of protein–protein interactions

High-Affinity Small-Molecule Inhibitors of the Menin-Mixed Lineage Leukemia (MLL) Interaction Closely Mimic a Natural Protein–Protein Interaction

The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ∼288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in <b>MIV</b>-<b>6<i>R</i></b>, which inhibits the menin–MLL interaction with IC₅₀ = 56 nM. Treatment with <b>MIV</b>-<b>6</b> demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules

Property Focused Structure-Based Optimization of Small Molecule Inhibitors of the Protein–Protein Interaction between Menin and Mixed Lineage Leukemia (MLL)

Development of potent small molecule inhibitors of protein–protein interactions with optimized druglike properties represents a challenging task in lead optimization process. Here, we report synthesis and structure-based optimization of new thienopyrimidine class of compounds, which block the protein–protein interaction between menin and MLL fusion proteins that plays an important role in acute leukemias with <i>MLL</i> translocations. We performed simultaneous optimization of both activity and druglike properties through systematic exploration of substituents introduced to the indole ring of lead compound <b>1</b> (MI-136) to identify compounds suitable for in vivo studies in mice. This work resulted in the identification of compound <b>27</b> (MI-538), which showed significantly increased activity, selectivity, polarity, and pharmacokinetic profile over <b>1</b> and demonstrated a pronounced effect in a mouse model of MLL leukemia. This study, which reports detailed structure–activity and structure–property relationships for the menin–MLL inhibitors, demonstrates challenges in optimizing inhibitors of protein–protein interactions for potential therapeutic applications

Complexity of Blocking Bivalent Protein–Protein Interactions: Development of a Highly Potent Inhibitor of the Menin–Mixed-Lineage Leukemia Interaction

The protein–protein interaction between menin and mixed-lineage leukemia 1 (MLL1) plays an important role in development of acute leukemia with translocations of the <i>MLL1</i> gene and in solid tumors. Here, we report the development of a new generation of menin–MLL1 inhibitors identified by structure-based optimization of the thieno­pyrimidine class of compounds. This work resulted in compound <b>28</b> (<b>MI-1481</b>), which showed very potent inhibition of the menin–MLL1 interaction (IC₅₀ = 3.6 nM), representing the most potent reversible menin–MLL1 inhibitor reported to date. The crystal structure of the menin-<b>28</b> complex revealed a hydrogen bond with Glu366 and hydrophobic interactions, which contributed to strong inhibitory activity of <b>28</b>. Compound <b>28</b> also demonstrates pronounced activity in MLL leukemia cells and <i>in vivo</i> in MLL leukemia models. Thus, <b>28</b> is a valuable menin–MLL1 inhibitor that can be used for potential therapeutic applications and in further studies regarding the role of menin in cancer

Complexity of Blocking Bivalent Protein–Protein Interactions: Development of a Highly Potent Inhibitor of the Menin–Mixed-Lineage Leukemia Interaction

The protein–protein interaction between menin and mixed-lineage leukemia 1 (MLL1) plays an important role in development of acute leukemia with translocations of the <i>MLL1</i> gene and in solid tumors. Here, we report the development of a new generation of menin–MLL1 inhibitors identified by structure-based optimization of the thieno­pyrimidine class of compounds. This work resulted in compound <b>28</b> (<b>MI-1481</b>), which showed very potent inhibition of the menin–MLL1 interaction (IC₅₀ = 3.6 nM), representing the most potent reversible menin–MLL1 inhibitor reported to date. The crystal structure of the menin-<b>28</b> complex revealed a hydrogen bond with Glu366 and hydrophobic interactions, which contributed to strong inhibitory activity of <b>28</b>. Compound <b>28</b> also demonstrates pronounced activity in MLL leukemia cells and <i>in vivo</i> in MLL leukemia models. Thus, <b>28</b> is a valuable menin–MLL1 inhibitor that can be used for potential therapeutic applications and in further studies regarding the role of menin in cancer