The discovery of quinoline-3-carboxamides as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors.

With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC50 = 220,000 nM, LE = 0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC50 = 3,100 nM, LE = 0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC50 = 9.9 nM, LE = 0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD2 production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.

A high-throughput MALDI-TOF MS biochemical screen for small molecule inhibitors of the antigen aminopeptidase ERAP1.

MALDI-TOF MS is a powerful analytical technique that provides a fast and label-free readout for in vitro assays in the high-throughput screening (HTS) environment. Here, we describe the development of a novel, HTS compatible, MALDI-TOF MS-based drug discovery assay for the endoplasmic reticulum aminopeptidase 1 (ERAP1), an important target in immuno-oncology and auto-immune diseases. A MALDI-TOF MS assay was developed beginning with an already established ERAP1 RapidFire MS (RF MS) assay, where the peptide YTAFTIPSI is trimmed into the product TAFTIPSI. We noted low ionisation efficiency of these peptides in MALDI-TOF MS and hence incorporated arginine residues into the peptide sequences to improve ionisation. The optimal assay conditions were established with these new basic assay peptides on the MALDI-TOF MS platform and validated with known ERAP1 inhibitors. Assay stability, reproducibility and robustness was demonstrated on the MALDI-TOF MS platform. From a set of 699 confirmed ERAP1 binders, identified in a prior affinity selection mass spectrometry (ASMS) screen, active compounds were determined at single concentration and in a dose-response format with the new MALDI-TOF MS setup. Furthermore, to allow for platform performance comparison, the same compound set was tested on the established RF MS setup, as the new basic peptides showed fragmentation in ESI-MS. The two platforms showed a comparable performance, but the MALDI-TOF MS platform had several advantages, such as shorter sample cycle times, reduced reagent consumption, and a lower tight-binding limit.

Improving the efficiency and effectiveness of an industrial SARS-CoV-2 diagnostic facility.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

Enabling lead discovery for histone lysine demethylases by high-throughput RapidFire mass spectrometry.

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.

Lead discovery for microsomal prostaglandin E synthase using a combination of high-throughput fluorescent-based assays and RapidFire mass spectrometry.

Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH(2)) and the challenge of detection of the product (PGE(2)). A coupled fluorescent assay is described for mPGES-1 where PGH(2) is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE(2) is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE(2) and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target.