Target-guided synthesis approach to the discovery of novel bivalent inhibitors of Glutathione Transferases

Target-guided synthesis is an approach to drug discovery that uses the biological target as a template to direct synthesis of its own best inhibitors from small molecule fragments. The process bridges the gap between chemical synthesis of drug candidates and their biological binding assay, merging the two operations into a single process whereby the active site or a binding pocket within the structure of the biological target directly controls the assembly of the best inhibitor in situ. Two different approaches to target-guided synthesis, the thermodynamic approach, making use of reversible reactions, and the kinetic approach, which uses an irreversible reaction, have been employed to discover novel, isoform selective inhibitors of the glutathione transferase (GST) enzyme family – possible drug targets in cancer and parasitic disease treatments. The thermodynamic approach described in this thesis uses the aniline-catalysed reversible acyl hydrazone formation reaction to create a dynamic covalent library of bivalent ligands designed to bind the dimeric structure of GST. In the presence of GST one of the bivalent ligands was selectively amplified at the expense of the other library members. This ligand was shown, via biological assays, to be a specific inhibitor for one isoform of GST, the mu isoform mGSTM1-1. A kinetic approach has also been investigated as a way to identify novel bivalent GST inhibitors utilising the Huisgen 1, 3 dipolar cycloaddition reaction. An azide and alkyne fragment library was designed to bind across the dimeric GST structure. The inhibitor structures are therefore bivalent, containing two anchoring fragments known to bind to the GST active site, linked by a triazolopeptide spacer. The triazole provides the click chemistry disconnection, enabling rapid in situ screening of candidate alkyne and azide fragments for inhibitor discovery. Whilst the in situ reaction with GST yielded inconclusive results, a number of the triazole products were found to have low nanomolar inhibitory activity towards GST

The clinical utility of plasma cell-free DNA (cfDNA) in NET-02::Randomised, phase II trial of liposomal irinotecan (nal-IRI)/5-fluorouracil (5-FU)/folinic acid or docetaxel as second-line (2L) therapy in patients (pts) with progressive poorly differentiated extra-pulmonary neuroendocrine carcinoma (PD-EP-NEC).

Background: In NET-02, nal-IRI/5-FU, but not docetaxel, met the primary endpoint of 6-month (mo) progression-free survival (PFS) rate in pts with progressive PD-EP-NEC (1). The prognostic potential of cfDNA in progressive PD-EP-NEC has not been explored in a prospective randomised trial. Methods: NET-02 was a multi-centre, randomised (1:1), phase II trial of IV nal-IRI/5-FU/folinic acid, Q14 days (ARM A), or IV docetaxel, Q21 days (ARM B), as 2L therapy in pts with progressive PD-EP-NEC. Plasma samples were taken at baseline (T0), following 6 weeks of treatment (T1) and at progression (T2). Samples were analysed using a multi-omic next-generation sequencing (NGS) approach (T7-MBD-seq) (measures genome-wide methylation and low pass whole genome copy number alterations (CNA)). The tumour fraction (TF) of cfDNA was determined using ichorCNA and correlated with clinical outcomes using Cox proportional hazards model and Logistic regression. Results: Of 56 pts evaluable for efficacy, there were 54 (96%) T0 samples (48 passed quality control), 31 (55%) at T1 and 20 (36%) at T2. At T0, 36/48 (75%) samples had detectable TF by copy number (using 3% TF cut-off). For the entire cohort, T0 TF was negatively prognostic for overall survival (OS) (Hazard Ratio (HR) 1.17, 95% Confidence interval (CI) 1.00-1.37, P=.044), but not for 6 mo PFS rate (Odds ratio (OR) 1.25, 95% CI 0.91-1.73, P=.17), response rate (RR) (OR 1.11, 95% CI 0.67-1.76, P=.65) or PFS (HR 1.04, 95%CI 0.91-1.20, P=.53). In the entire cohort, median OS split by TF (N=16 each) was 10.2 mo (95% CI 4.1-not available (NA)) for low (TF≤8.6%), 6.9 mo (95% CI 3.4-14.8) for medium (8.6<TF≤24.3%) and 3.7 mo (95% CI 2.8-NA) for high (TF>24.3%). T0 TF was also negatively prognostic for 6 mo PFS rate and OS in ARM A (P=.02, P=.03), but not ARM B (P=.61, P=.48), but was not prognostic for RR or PFS (Table). TF correlated with the presence of liver metastases (Wilcoxon Rank Sum P=.009), but not with ECOG PS (P=.14), sex (P=.10), Ki-67 (P=.39) or age (Spearman rank correlation P=.45). Longitudinal copy number and methylation cfDNA analysis is on-going. Conclusions: These results suggest that it may be possible to stratify prognosis based on amount of baseline TF in patients with progressive PD-EP-NEC, and may also identify patients who would benefit most from the nal-IRI combination. 1. McNamara 2023, EClin Med. Clinical trial information: 03837977.Impact of TF (Continuous Variable) at T0 on: ARM A nal-IRI/5-FU (N=24) ARM B Docetaxel (N=24)6 mo PFS rate OR 1.87 (95% CI 1.15-3.65, P=.02) OR 0.85 (95% CI 0.39-1.49, P=.61)RR OR 1.27 (95% CI 0.60-2.52, P=.46) OR 0.98 (95% CI 0.43-1.91, P = .95)PFS HR 1.23 (95% CI 0.97-1.57, P=.09) HR 0.93 (95% CI 0.77-1.11, P=.42)OS HR 1.37 (95% CI 1.03-1.81, P=.03) HR 1.07 (95% CI 0.88-1.31, P=.48