Pharmacologic inhibition of the Menin-MLL interaction blocks progression of MLL leukemia in vivo

Chromosomal translocations affecting mixed lineage leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report the development of highly potent and orally bioavailable small-molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, and show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate the efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification

Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents

Smad3-dependent CCN2 mediates fibronectin expression in human skin dermal fibroblasts.

The potential involvement of connective tissue growth factor (CCN2/CTGF) in extracellular matrix (ECM) production is recognized. However, the role CCN2 in fibronectin (FN) gene expression has remained incompletely understood and even controversial. Here we report that CCN2 is absolutely necessary for FN expression in primary human skin dermal fibroblasts, the major cells responsible for ECM production in skin. Gain- and loss-of-function approaches demonstrate that CCN2 is an essential component of FN expression in both basal and stimulation by TGF-β signaling, the major regulator of FN expression. CCN2 is significantly induced by Smad3, a critical mediator of TGF-β signaling. CCN2 acts as a downstream mediator of TGF-β/Smad signaling and acting synergistically with TGF-β to regulate FN gene expression. Finally, we observed that CCN2 and FN predominantly expressed in the dermis of normal human skin, stromal tissues of skin squamous cell carcinoma (SCC), and simultaneously induced in wounded human skin in vivo. These findings provide evidence that CCN2 is responsible for mediating the stimulatory effects of TGF-β/Smad on FN gene expression, and attenuation of CCN2 expression may benefit to reduce fibrotic ECM microenvironment in disease skin

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