Structure-based identification of dual ligands at the A2AR and PDE10A with anti-proliferative effects in lung cancer cell-lines.

Enhanced/prolonged cAMP signalling has been suggested as a suppressor of cancer proliferation. Interestingly, two key modulators that elevate cAMP, the A2A receptor (A2AR) and phosphodiesterase 10A (PDE10A), are differentially co-expressed in various types of non-small lung cancer (NSCLC) cell-lines. Thus, finding dual-target compounds, which are simultaneously agonists at the A2AR whilst also inhibiting PDE10A, could be a novel anti-proliferative approach. Using ligand- and structure-based modelling combined with MD simulations (which identified Val84 displacement as a novel conformational descriptor of A2AR activation), a series of known PDE10A inhibitors were shown to dock to the orthosteric site of the A2AR. Subsequent in-vitro analysis confirmed that these compounds bind to the A2AR and exhibit dual-activity at both the A2AR and PDE10A. Furthermore, many of the compounds exhibited promising anti-proliferative effects upon NSCLC cell-lines, which directly correlated with the expression of both PDE10A and the A2AR. Thus, we propose a structure-based methodology, which has been validated in in-vitro binding and functional assays, and demonstrated a promising therapeutic value.

Structure-based design of allosteric calpain-1 inhibitors populating a novel bioactivity space.

Dimeric calpains constitute a promising therapeutic target for many diseases such as cardiovascular, neurodegenerative and ischaemic disease. The discovery of selective calpain inhibitors, however, has been extremely challenging. Previously, allosteric inhibitors of calpains, such as PD150606, which included a specific α-mercaptoacrylic acid sub-structure, were reported to bind to the penta-EF hand calcium binding domain, PEF(S) of calpain. Although these are selective to calpains over other cysteine proteases, their mode of action has remained elusive due to their ability to inhibit the active site domain with and without the presence of PEF(S), with similar potency. These findings have led to the question of whether the inhibitory response can be attributed to an allosteric mode of action or alternatively to inhibition at the active site. In order to address this problem, we report a structure-based virtual screening protocol as a novel approach for the discovery of PEF(S) binders that populate a novel chemical space. We have identified compound 1, Vidupiprant, which is shown to bind to the PEF(S) domain by the TNS displacement method, and it exhibited specificity in its allosteric mode of inhibition. Compound 1 inhibited the full-length calpain-1 complex with a higher potency (IC50 = 7.5 µM) than the selective, cell-permeable non-peptide calpain inhibitor, PD150606 (IC50 = 19.3 µM), where the latter also inhibited the active site domain in the absence of PEF(S) (IC50 = 17.8 µM). Hence the method presented here has identified known compounds with a novel allosteric mechanism for the inhibition of calpain-1. We show for the first time that the inhibition of enzyme activity can be attributed to an allosteric mode of action, which may offer improved selectivity and a reduced side-effects profile.

Computer-aided design of multi-target ligands at A1R, A2AR and PDE10A, key proteins in neurodegenerative diseases.

Compounds designed to display polypharmacology may have utility in treating complex diseases, where activity at multiple targets is required to produce a clinical effect. In particular, suitable compounds may be useful in treating neurodegenerative diseases by promoting neuronal survival in a synergistic manner via their multi-target activity at the adenosine A1 and A2A receptors (A1R and A2AR) and phosphodiesterase 10A (PDE10A), which modulate intracellular cAMP levels. Hence, in this work we describe a computational method for the design of synthetically feasible ligands that bind to A1 and A2A receptors and inhibit phosphodiesterase 10A (PDE10A), involving a retrosynthetic approach employing in silico target prediction and docking, which may be generally applicable to multi-target compound design at several target classes. This approach has identified 2-aminopyridine-3-carbonitriles as the first multi-target ligands at A1R, A2AR and PDE10A, by showing agreement between the ligand and structure based predictions at these targets. The series were synthesized via an efficient one-pot scheme and validated pharmacologically as A1R/A2AR-PDE10A ligands, with IC50 values of 2.4-10.0 µM at PDE10A and Ki values of 34-294 nM at A1R and/or A2AR. Furthermore, selectivity profiling of the synthesized 2-amino-pyridin-3-carbonitriles against other subtypes of both protein families showed that the multi-target ligand 8 exhibited a minimum of twofold selectivity over all tested off-targets. In addition, both compounds 8 and 16 exhibited the desired multi-target profile, which could be considered for further functional efficacy assessment, analog modification for the improvement of selectivity towards A1R, A2AR and PDE10A collectively, and evaluation of their potential synergy in modulating cAMP levels.