Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification.

Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.

Highly Selective Synthesis of Halomon, Plocamenone, and Isoplocamenone.

Over 160 chiral vicinal bromochlorinated natural products have been identified; however, a lack of synthetic methods for the selective incorporation of halogens into organic molecules has hindered their synthesis. Here we disclose the first total synthesis and structural confirmation of isoplocamenone and plocamenone, as well as the first selective and scalable synthesis of the preclinical anticancer natural product halomon. The synthesis of these inter-halogenated compounds has been enabled by our recently developed chemo-, regio-, and enantioselective dihalogenation reaction.

Synthesis and photophysical characterization of stable indium bacteriochlorins.

Bacteriochlorins have wide potential in photochemistry because of their strong absorption of near-infrared light, yet metallobacteriochlorins traditionally have been accessed with difficulty. Established acid-catalysis conditions [BF(3)·OEt(2) in CH(3)CN or TMSOTf/2,6-di-tert-butylpyridine in CH(2)Cl(2)] for the self-condensation of dihydrodipyrrin-acetals (bearing a geminal dimethyl group in the pyrroline ring) afford stable free base bacteriochlorins. Here, InBr(3) in CH(3)CN at room temperature was found to give directly the corresponding indium bacteriochlorin. Application of the new acid catalysis conditions has afforded four indium bacteriochlorins bearing aryl, alkyl/ester, or no substituents at the ß-pyrrolic positions. The indium bacteriochlorins exhibit (i) a long-wavelength absorption band in the 741-782 nm range, which is shifted bathochromically by 22-32 nm versus the analogous free base species, (ii) fluorescence quantum yields (0.011-0.026) and average singlet lifetime (270 ps) diminished by an order of magnitude versus that (0.13-0.25; 4.0 ns) for the free base analogues, and (iii) higher average yield (0.9 versus 0.5) yet shorter average lifetime (30 vs 105 µs) of the lowest triplet excited state compared to the free base compounds. The differences in the excited-state properties of the indium chelates versus free base bacteriochlorins derive primarily from a 30-fold greater rate constant for S(1) → T(1) intersystem crossing, which stems from the heavy-atom effect on spin-orbit coupling. The trends in optical properties of the indium bacteriochlorins versus free base analogues, and the effects of 5-OMe versus 5-H substituents, correlate well with frontier molecular-orbital energies and energy gaps derived from density functional theory calculations. Collectively the synthesis, photophysical properties, and electronic characteristics of the indium bacteriochlorins and free base analogues reported herein should aid in the further design of such chromophores for diverse applications.

Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes.

We compared the ability of short hairpin RNA (shRNA) and CRISPR/Cas9 screens to identify essential genes in the human chronic myelogenous leukemia cell line K562. We found that the precision of the two libraries in detecting essential genes was similar and that combining data from both screens improved performance. Notably, results from the two screens showed little correlation, which can be partially explained by the identification of distinct essential biological processes with each technology.