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Highlights d A robust and flexible platform for genetic screens in hPSCs d Essential gene profiles for hPSCs on two substrates and core hPSC fitness gene sets d Previously unidentified regulators of hPSC fitness (DRICH1 and others) d Confirmation of the TP53 pathway, PAWR, PMAIP1 as negative proliferation regulators

The de novo synthesis of fatty acids has emerged as a therapeutic target for various diseases including cancer. Since cancer cells are intrinsically buffered to combat metabolic stress, it is important to understand how cells may adapt to loss of de novo fatty acid biosynthesis. Here we use pooled genome-wide CRISPR screens to systematically map genetic interactions (GIs) in human HAP1 cells carrying a loss-of-function mutation in FASN , whose product catalyzes the formation of long-chain fatty acids. FASN mutant cells show a strong dependence on lipid uptake that is reflected in negative GIs with genes involved in the LDL receptor pathway, vesicle trafficking, and protein glycosylation. Further support for these functional relationships is derived from additional GI screens in query cell lines deficient for other genes involved in lipid metabolism, including LDLR , SREBF1 , SREBF2 , ACACA . Our GI profiles also identify a potential role for the previously uncharacterized gene LUR1 / C12orf49 in exogenous lipid uptake regulation through modulation of SREBF2 signalling in response to lipid starvation. Overall, our data highlight the genetic determinants underlying the cellular adaptation associated with loss of de novo fatty acid synthesis and demonstrate the power of systematic GI mapping for uncovering metabolic buffering mechanisms in human cells.

Spiropyrazolopyridone 1 was identified, as a novel dengue virus (DENV) inhibitor, from a DENV serotype 2 (DENV-2) high-throughput phenotypic screen. As a general trend within this chemical class, chiral resolution of the racemate revealed that R enantiomer was significantly more potent than the S. Cell-based lead optimization of the spiropyrazolopyridones focusing on improving the physicochemical properties is described. As a result, an optimal compound 14a, with balanced in vitro potency and pharmacokinetic profile, achieved about 1.9 log viremia reduction at 3 × 50 mg/kg (bid) or 3 × 100 mg/kg (QD) oral doses in the dengue in vivo mouse efficacy model. KEYWORDS: Dengue virus (DENV), spiropyrazolopyridones, structure−activity relationship, lead optimization D engue fever is a febrile disease caused by dengue virus (DENV), which is transmitted by Aedes aegypti, a mosquito that feeds on humans.1 DENV threatens up to 2.5 billion people in more than 100 endemic countries. According to a World Health Organization (WHO) report, 2 there are 50− 100 million infections annually (and yet this number could be far underestimated), 3 with approximately 500,000 cases of dengue hemorrhagic fever and 22,000 deaths globally. 4,5 Despite the clear unmet medical need, currently there is no clinically approved vaccine or antiviral therapy available for treatment of dengue.6−10 Therefore, it is urgent to develop safe and effective therapeutics.High-throughput phenotypic screening is a powerful tool to identify compounds that are active against DENV by inhibiting either host pathways and/or viral proteins, which are essential for viral replication.11−16 As a result of DENV-2 screening on Novartis compound library, a new chemical class spiropyrazolopyridone 1 was identified to be an interesting starting point for further optimization (Figure 1). As compound 1 was a racemate, two enantiomers were subsequently separated by chiral high-performance purification chromatography (HPLC). The structure of R enantiomer 1a was unambiguously determined by X-ray crystallography (Figure 2). Interestingly, when tested in the DENV-2 in vitro assay, these two individual enantiomers displayed remarkably different potency where R enantiomer 1a was greater than 200-fold more potent than the S enantiomer 1b (Figure 1). Very recently, genetic analysis demonstrated that mutations in dengue viral NS4B conferred the resistance of compound 1a, suggesting that this class of compounds inhibited DENV replication by targeting NS4B protein. 11,17−19 In this report, lead optimization efforts to explore the structure−activity relationships (SARs) and improve the physicochemical properties are described.Following the identification of compound 1, which was subsequently found to be poorly soluble in aqueous media, we initiated a lead optimization effort in an attempt to improve the solubility while maintaining the potency. The general synthetic route to the spiropyrazolopyridone derivatives is outlined in Scheme 1. The synthesis, featuring a three component...

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.

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