Small-molecule drug target identification is an essential and often rate-limiting step in phenotypic drug discovery and remains a major challenge. Here, we report a novel platform for target identification of activators of signaling pathways by leveraging the power of a clustered regularly interspaced short palindromic repeats (CRISPR) knockout library. This platform links the expression of a suicide gene to the small-molecule-activated signaling pathway to create a selection system. With this system, loss-of-function screening using a CRISPR single-guide (sg) RNA library positively enriches cells in which the target has been knocked out. The identities of the drug targets and other essential genes required for the activity of small molecules of interest are then uncovered by sequencing. We tested this platform on BDW568, a newly discovered type-I interferon signaling activator, and identified stimulator of interferon genes (STING) as its target and carboxylesterase 1 (CES1) to be a key metabolizing enzyme required to activate BDW568 for target engagement. The platform we present here can be a general method applicable for target identification for a wide range of small molecules that activate different signaling pathways.
Title: Mice with an autism-associated R451C mutation in neuroligin-3 show a cautious but accurate response style in touchscreen attention tasks Burrows EL, May C, Hill T, Churliov L, Johnson KA, Hannan AJ Ref: Genes Brain Behav, :e12757, 2021 : PubMed
One of the earliest identifiable features of Autism Spectrum Disorder (ASD) is altered attention. Mice expressing the ASD-associated R451C mutation in synaptic adhesion protein neuroligin-3 (NL3) exhibit impaired reciprocal social interactions and repetitive and restrictive behaviors. The role of this mutation in attentional abnormalities has not been established. We assessed attention in male NL3(R451C) mice using two well-established tasks in touchscreen chambers. In the 5-choice serial reaction task (5CSRT), rodents were trained to attend to light stimuli that appear in any one of 5 locations. While no differences between NL3(R451C) and WT mice were seen in accuracy or omissions, slower response times and quicker reward collection latencies were seen across all training and probe trials. In the rodent continuous-performance test (rCPT), animals were required to discriminate, and identify a visual target pattern over multiple distractor stimuli. NL3(R451C) mice displayed enhanced ability to attend to stimuli when task-load was low during training and baseline but lost this advantage when difficulty was increased by altering task parameters in probe trials. NL3(R451C) mice made less responses to the distractor stimuli, exhibiting lower false alarm rates during all training stages and in probe trials. Slower response times and quicker reward latencies were consistently seen in NL3(R451C) mice in the rCPT. Slower response times are a major cognitive phenotype reported in ASD patients and are indicative of slower processing speed. Enhanced attention has been shown in a subset of ASD patients and we have demonstrated this phenotype also exists in the NL3(R451C) mouse model.
Title: Functional and structural studies of a novel cold-adapted esterase from an Arctic intertidal metagenomic library Fu J, Leiros HK, de Pascale D, Johnson KA, Blencke HM, Landfald B Ref: Applied Microbiology & Biotechnology, 97:3965, 2013 : PubMed
A novel cold-adapted lipolytic enzyme gene, est97, was identified from a high Arctic intertidal zone sediment metagenomic library. The deduced amino acid sequence of Est97 showed low similarity with other lipolytic enzymes, the maximum being 30 % identity with a putative lipase from Vibrio caribbenthicus. Common features of lipolytic enzymes, such as the GXSXG sequence motif, were detected. The gene product was over-expressed in Escherichia coli and purified. The recombinant Est97 (rEst97) hydrolysed various rho-nitrophenyl esters with the best substrate being rho-nitrophenyl hexanoate (K m and k cat of 39 muM and 25.8 s(-1), respectively). This esterase activity of rEst97 was optimal at 35 degrees C and pH 7.5 and the enzyme was unstable at temperatures above 25 degrees C. The apparent melting temperature, as determined by differential scanning calorimetry was 39 degrees C, substantiating Est97 as a cold-adapted esterase. The crystal structure of rEst97 was determined by the single wavelength anomalous dispersion method to 1.6 A resolution. The protein was found to have a typical alpha/beta-hydrolase fold with Ser144-His226-Asp197 as the catalytic triad. A suggested, relatively short lid domain of rEst97 is composed of residues 80-114, which form an alpha-helix and a disordered loop. The cold adaptation features seem primarily related to a high number of methionine and glycine residues and flexible loops in the high-resolution structures.
Abstract Objective: A naturalistic, prospective study of the influence of genetic variation on dose prescribed, clinical response, and side effects related to stimulant medication in 77 children with attention-deficit/hyperactivity disorder (ADHD) was undertaken. The influence of genetic variation of the CES1 gene coding for carboxylesterase 1A1 (CES1A1), the major enzyme responsible for the first-pass, stereoselective metabolism of methylphenidate, was investigated. Methods: Parent- and teacher-rated behavioral questionnaires were collected at baseline when the children were medication naive, and again at 6 weeks while they were on medication. Medication dose, prescribed at the discretion of the treating clinician, and side effects, were recorded at week 6. Blood and saliva samples were collected for genotyping. Single nucleotide polymorphisms (SNPs) were selected in the coding, non-coding and the 3' flanking region of the CES1 gene. Genetic association between CES1 variants and ADHD was investigated in an expanded sample of 265 Irish ADHD families. Analyses were conducted using analysis of covariance (ANCOVA) and logistic regression models. Results: None of the CES1 gene variants were associated with the dose of methylphenidate provided or the clinical response recorded at the 6 week time point. An association between two CES1 SNP markers and the occurrence of sadness as a side effect of short-acting methylphenidate was found. The two associated CES1 markers were in linkage disequilibrium and were significantly associated with ADHD in a larger sample of ADHD trios. The associated CES1 markers were also in linkage disequilibrium with two SNP markers of the noradrenaline transporter gene (SLC6A2). Conclusions: This study found an association between two CES1 SNP markers and the occurrence of sadness as a side effect of short-acting methylphenidate. These markers were in linkage disequilibrium together and with two SNP markers of the noradrenaline transporter gene.
Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M4 allosteric potentiators. VU10010, the lead compound, potentiates the M4 response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M4 with VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M4 knockout mice. Selective regulation of excitatory transmission by M4 suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure.
