FANCJ^K1249R or FANCJ^K1249Q promotes polη- or Rad54-dependent repair, respectively.

<p>A. FA-J cells expressing acetylation mutants have a distinct response and reliance on repair or tolerance factors for DNA damage survival. The FA-J cell lines were transfected with siRNA against Luc, Polη, or Rad54. The cells were treated with indicated doses of zeocin, UV, or as in B., with MMC and the percent survival was calculated 8 days later. Data represent mean percent ± s.d. of survival from three independent experiments. C. Cells were collected and analyzed for expression with the indicated antibodies.</p

FANCJ acetylation is induced after DNA damage.

<p>A. Endogenous FANCJ is acetylated in response DNA damage. MCF7 and HeLa cells were left untreated (UT) or treated with zeocin (6.25 µg/ml for 1 h), MMC (250 nM for 1 h), UV (30 J/m²), MMS (300 µg/ml for 4 h), HU (1 mM for 24 h), or CPT (1 µM for 1 h). Cell lysates were collected at distinct times post damage (zeocin 24 h, MMC 24 h or as indicated, UV 6 h, CPT 24 h, MMS 4 h, and HU 24 h) and analyzed for expression and/or acetylation following immunoprecipitation with the indicated antibodies. B. Exogenous FANCJ is acetylated on lysine 1249 in response to DNA damage. Myc-tagged FANCJ wild-type or mutant species and CBP were co-transfected into 293T cells and left untreated (UT) or treated with zeocin (12.5 µg/ml for 1 h or C. CPT (1 µM for 1 h). Cells were processed at different time points post DNA damage and analyzed for expression and/or acetylation following immunoprecipitation with the indicated antibodies.</p

FANCJ and its acetylation at 1249 promote an RPA response.

<p>A. Deficiency in FANCJ or its acetylation impairs the CPT-induced RPA focus formation. The FA-J cell lines were seeded onto 6-well plates, incubated overnight, left untreated or treated with CPT 1 h and immunoflourescence was performed with the indicated antibodies. The percent of cells with RPA foci was quantified and graphed. Data represent mean ± s.d. from three independent experiments. B. FANCJ and its acetylation promote RPA phosphorylation at 1 h post-CPT. The complemented FA-J cell lines were either left untreated or treated for 1 h with the indicated dose of CPT and analyzed 1 h post-treatment. Cell lysates were collected, lysed, and analyzed with the indicated antibodies. C. FANCJ acetylation promotes RPA phosphorylation at times greater than 1 h post-CPT. Same as above but collected at the time points indicated.</p

FANCJ and its dynamic regulation by acetylation promote resection-associated events.

<p>A. Deficiency in FANCJ or its acetylation impairs the CPT-induced Rad51 focus formation. The FA-J cell lines were seeded onto 6-well plates, incubated overnight, left untreated or treated with CPT 6 h and immunoflourescence was performed with the indicated antibodies. The percent of cells with γ-H2AX and Rad51 foci was quantified and graphed. Data represent mean percent ± s.d. from three independent experiments. B. Deficiency in FANCJ or its ability to be regulated by acetylation impairs the CPT-induced checkpoint response. FA-J cell lines were either left untreated or treated for 1 h with the 0.1 µM or 1 µM of CPT and analyzed at the indicated time points. Cell lysates were collected, lysed, and analyzed with the indicated antibodies. C. Model depicts function of FANCJ acetylation in the DDR. FANCJ acetylation at lysine 1249 promotes resection and HR whereas de-acetylation promotes translesion synthesis (TLS). Maintenance of the checkpoint response; however requires the dynamic regulation of FANCJ acetylation.</p

FANCJ acetylation mutants are functional.

<p>A. The acetylation mutants are expressed in FA-J cells. FA-J cells were complemented with vector, FANCJ^WT, FANCJ^K1249R, or FANCJ^K1249Q. The FA-J cell lines were collected and analyzed or B. lysates were immunoprecipitated with FANCJ antibodies and immunoblot was performed with the indicated antibodies. C. The acetylation mutants localize in nuclear foci of FA-J cells. The FA-J cell lines were seeded onto 6-well plates and incubated overnight. The cells were treated with 1 mM HU and 24 h later immunoflourescence was performed with the indicated antibodies. D. The FA-J cell lines have similar cell cycle profiles. The FA-J cells lines were collected and analyzed by FACS to determine the percentage of cells with 2N and 4N DNA content. E. Expression of acetylation mutants restores MMC resistance. The FA-J cell lines were seed onto 6 well plates and incubated overnight. The cells were either left untreated or treated with increasing doses of MMC. Cells were counted 8 days later and percent survival was calculated. Data represent mean percent ± s.d. of survival from three independent experiments. F. Expression of acetylation mutants restores G2/M checkpoint exit. The FA-J cell lines were untreated or treated with 0.25 µg/ml melphalan, collected at the indicated times, and analyzed by FACS to determine the percentage of cells in G2/M. Data represent mean percent ± s.d. of survival from three independent experiments.</p

Potent and Selective Agonists of Sphingosine 1‑Phosphate 1 (S1P₁): Discovery and SAR of a Novel Isoxazole Based Series

Sphingosine 1-phosphate (S1P) is the endogenous ligand for the sphingosine 1-phosphate receptors (S1P_1–5) and evokes a variety of cellular responses through their stimulation. The interaction of S1P with the S1P receptors plays a fundamental physiological role in a number of processes including vascular development and stabilization, lymphocyte migration, and proliferation. Agonism of S1P₁, in particular, has been shown to play a significant role in lymphocyte trafficking from the thymus and secondary lymphoid organs, resulting in immunosuppression. This article will detail the discovery and SAR of a potent and selective series of isoxazole based full agonists of S1P₁. Isoxazole <b>6d</b> demonstrated impressive efficacy when administered orally in a rat model of arthritis and in a mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis

Discovery and Structure–Activity Relationship (SAR) of a Series of Ethanolamine-Based Direct-Acting Agonists of Sphingosine-1-phosphate (S1P₁)

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates a multitude of physiological processes such as lymphocyte trafficking, cardiac function, vascular development, and inflammation. Because of the ability of S1P₁ receptor agonists to suppress lymphocyte egress, they have great potential as therapeutic agents in a variety of autoimmune diseases. In this article, the discovery of selective, direct acting S1P₁ agonists utilizing an ethanolamine scaffold containing a terminal carboxylic acid is described. Potent S1P₁ agonists such as compounds <b>18a</b> and <b>19a</b> which have greater than 1000-fold selectivity over S1P₃ are described. These compounds efficiently reduce blood lymphocyte counts in rats through 24 h after single doses of 1 and 0.3 mpk, respectively. Pharmacodynamic properties of both compounds are discussed. Compound <b>19a</b> was further studied in two preclinical models of disease, exhibiting good efficacy in both the rat adjuvant arthritis model (AA) and the mouse experimental autoimmune encephalomyelitis model (EAE)

Identification and Preclinical Pharmacology of BMS-986104: A Differentiated S1P₁ Receptor Modulator in Clinical Trials

Clinical validation of S1P receptor modulation therapy was achieved with the approval of fingolimod (Gilenya, <b>1</b>) as the first oral therapy for relapsing remitting multiple sclerosis. However, <b>1</b> causes a dose-dependent reduction in the heart rate (bradycardia), which occurs within hours after first dose. We disclose the identification of clinical compound BMS-986104 (<b>3d</b>), a novel S1P₁ receptor modulator, which demonstrates ligand-biased signaling and differentiates from <b>1</b> in terms of cardiovascular and pulmonary safety based on preclinical pharmacology while showing equivalent efficacy in a T-cell transfer colitis model

Identification of Tricyclic Agonists of Sphingosine-1-phosphate Receptor 1 (S1P₁) Employing Ligand-Based Drug Design

Fingolimod (<b>1</b>) is the first approved oral therapy for the treatment of relapsing remitting multiple sclerosis. While the phosphorylated metabolite of fingolimod was found to be a nonselective S1P receptor agonist, agonism specifically of S1P₁ is responsible for the peripheral blood lymphopenia believed to be key to its efficacy. Identification of modulators that maintain activity on S1P₁ while sparing activity on other S1P receptors could offer equivalent efficacy with reduced liabilities. We disclose in this paper a ligand-based drug design approach that led to the discovery of a series of potent tricyclic agonists of S1P₁ with selectivity over S1P₃ and were efficacious in a pharmacodynamic model of suppression of circulating lymphocytes. Compound <b>10</b> had the desired pharmacokinetic (PK) and pharmacodynamic (PD) profile and demonstrated maximal efficacy when administered orally in a rat adjuvant arthritis model