Applications of CRISPR genome editing technology in drug target identification and validation.

INTRODUCTION: The analysis of pharmaceutical industry data indicates that the major reason for drug candidates failing in late stage clinical development is lack of efficacy, with a high proportion of these due to erroneous hypotheses about target to disease linkage. More than ever, there is a requirement to better understand potential new drug targets and their role in disease biology in order to reduce attrition in drug development. Genome editing technology enables precise modification of individual protein coding genes, as well as noncoding regulatory sequences, enabling the elucidation of functional effects in human disease relevant cellular systems. Areas covered: This article outlines applications of CRISPR genome editing technology in target identification and target validation studies. Expert opinion: Applications of CRISPR technology in target validation studies are in evidence and gaining momentum. Whilst technical challenges remain, we are on the cusp of CRISPR being applied in complex cell systems such as iPS derived differentiated cells and stem cell derived organoids. In the meantime, our experience to date suggests that precise genome editing of putative targets in primary cell systems is possible, offering more human disease relevant systems than conventional cell lines.

Antitubercular drugs for an old target: GSK693 as a promising InhA direct inhibitor.

Despite being one of the first antitubercular agents identified, isoniazid (INH) is still the most prescribed drug for prophylaxis and tuberculosis (TB) treatment and, together with rifampicin, the pillars of current chemotherapy. A high percentage of isoniazid resistance is linked to mutations in the pro-drug activating enzyme KatG, so the discovery of direct inhibitors (DI) of the enoyl-ACP reductase (InhA) has been pursued by many groups leading to the identification of different enzyme inhibitors, active against Mycobacterium tuberculosis (Mtb), but with poor physicochemical properties to be considered as preclinical candidates. Here, we present a series of InhA DI active against multidrug (MDR) and extensively (XDR) drug-resistant clinical isolates as well as in TB murine models when orally dosed that can be a promising foundation for a future treatment.

Over expression of wild type or a catalytically dead mutant of Sirtuin 6 does not influence NFκB responses.

SIRT6 is involved in inflammation, aging and metabolism potentially by modulating the functions of both NFκB and HIF1α. Since it is possible to make small molecule activators and inhibitors of Sirtuins we wished to establish biochemical and cellular assays both to assist in drug discovery efforts and to validate whether SIRT6 represents a valid drug target for these indications. We confirmed in cellular assays that SIRT6 can deacetylate acetylated-histone H3 lysine 9 (H3K9Ac), however this deacetylase activity is unusually low in biochemical assays. In an effort to develop alternative assay formats we observed that SIRT6 overexpression had no influence on TNFα induced nuclear translocation of NFκB, nor did it have an effect on nuclear mobility of RelA/p65. In an effort to identify a gene expression profile that could be used to identify a SIRT6 readout we conducted genome-wide expression studies. We observed that overexpression of SIRT6 had little influence on NFκB-dependent genes, but overexpression of the catalytically inactive mutant affected gene expression in developmental pathways.

Characterisation of 5-HT3C, 5-HT3D and 5-HT3E receptor subunits: evolution, distribution and function.

The 5-HT(3) receptor is a member of the 'Cys-loop' family of ligand-gated ion channels that mediate fast excitatory and inhibitory transmission in the nervous system. Current evidence points towards native 5-HT(3) receptors originating from homomeric assemblies of 5-HT(3A) or heteromeric assembly of 5-HT(3A) and 5-HT(3B). Novel genes encoding 5-HT(3C), 5-HT(3D), and 5-HT(3E) have recently been described but the functional importance of these proteins is unknown. In the present study, in silico analysis (confirmed by partial cloning) indicated that 5-HT(3C), 5-HT(3D), and 5-HT(3E) are not human-specific as previously reported: they are conserved in multiple mammalian species but are absent in rodents. Expression profiles of the novel human genes indicated high levels in the gastrointestinal tract but also in the brain, Dorsal Root Ganglion (DRG) and other tissues. Following the demonstration that these subunits are expressed at the cell membrane, the functional properties of the recombinant human subunits were investigated using patch clamp electrophysiology. 5-HT(3C), 5-HT(3D), and 5-HT(3E) were all non-functional when expressed alone. Co-transfection studies to determine potential novel heteromeric receptor interactions with 5-HT(3A) demonstrated that the expression or function of the receptor was modified by 5-HT(3C) and 5-HT(3E), but not 5-HT(3D). The lack of distinct effects on current rectification, kinetics or pharmacology of 5-HT(3A) receptors does not however provide unequivocal evidence to support a direct contribution of 5-HT(3C) or 5-HT(3E) to the lining of the ion channel pore of novel heteromeric receptors. The functional and pharmacological contributions of these novel subunits to human biology and diseases such as irritable bowel syndrome for which 5-HT(3) receptor antagonists have major clinical usage, therefore remains to be fully determined.

Development of a high-throughput cell-based assay for 11beta-hydroxysteroid dehydrogenase type 1 using BacMam technology.

Cortisol is an important glucocorticoid in humans that regulates many physiological processes. Human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone to cortisol in vivo and has emerged as an appealing therapeutic target for treating metabolic diseases. Here, we report a sensitive and robust high-throughput (HT) cell-based assay for screening 11beta-HSD1 inhibitors. This assay utilizes a HEK293 cell line transduced by a BacMam virus expressing human 11beta-HSD1. The enzyme activity in the cells was measured by quantifying cortisol levels released into the cell culture supernatant via a competitive homogenous time-resolved fluorescence (HTRF) method. We show that 11beta-HSD1 activity in supernatant of BacMam-transduced HEK293 cells increases with 11beta-HSD1 BacMam virus load in a dose-dependent manner, and is comparable to the enzyme activity detected in differentiated mouse adipocytes. In addition, we show that co-expression of hexose-6-phosphate dehydrogenase (H6PDH) is not required for the enzyme to function effectively as an oxo-reductase. This assay has been developed in low-volume 384-well format and it is sensitive, robust, and amenable to HT screening.

Efficient expression of secreted proteases via recombinant BacMam virus.

Baculovirus vectors engineered to contain mammalian cell-active promoter elements have been described as an efficient method for transduction of a broad spectrum of human cell lines at high frequency. In the first large-scale comparative study of secreted protein production using these viral vectors, we have evaluated production of 16 recombinant enzymes--specifically, we exploited these viral vectors, termed 'BacMam' viruses, to drive expression of a panel of proteases selected from all four major mechanistic classes, including secreted, lysosomal, endosomal, and type I transmembrane proteins. To allow a generic purification strategy, coding sequences were truncated to remove transmembrane and/or subcellular retention signals before introduction, in parallel, into a C-terminally Fc-tagged BacMam transfer vector. BacMam viruses were generated and subsequently evaluated for expression of Fc-tagged protein in virus-transduced HEK-F cells. The common Fc-tag enabled single-step affinity purification of secreted recombinant protein from the culture medium. Yields were excellent, with 14 of 16 genes expressed producing 10-30 mg or more purified protein per litre of culture using standardised transduction conditions. At this level, reagent demands for a typical protease high-throughput screen (HTS) could be met from expression cultures as small as 0.1-0.5 L. Our results indicate this expression system offers a highly efficient and scaleable method for production of enzymatically-active secreted proteases and may therefore represent a novel method of protein production for other secreted enzymes with significant advantages over the diverse approaches in current use.