Prospective performance evaluation of selected common virtual screening tools. Case study: Cyclooxygenase (COX) 1 and 2

Computational methods can be applied in drug development for the identification of novel lead candidates, but also for the prediction of pharmacokinetic properties and potential adverse effects, thereby aiding to prioritize and identify the most promising compounds. In principle, several techniques are available for this purpose, however, which one is the most suitable for a specific research objective still requires further investigation. Within this study, the performance of several programs, representing common virtual screening methods, was compared in a prospective manner. First, we selected top-ranked virtual screening hits from the three methods pharmacophore modeling, shape-based modeling, and docking. For comparison, these hits were then additionally predicted by external pharmacophore- and 2D similarity-based bioactivity profiling tools. Subsequently, the biological activities of the selected hits were assessed in vitro, which allowed for evaluating and comparing the prospective performance of the applied tools. Although all methods performed well, considerable differences were observed concerning hit rates, true positive and true negative hits, and hitlist composition. Our results suggest that a rational selection of the applied method represents a powerful strategy to maximize the success of a research project, tightly linked to its aims. We employed cyclooxygenase as application example, however, the focus of this study lied on highlighting the differences in the virtual screening tool performances and not in the identification of novel COX-inhibitors.


Comprehensive discussion of the novel COX-inhibitors
Several of the novel COX-inhibitors identified in this study belong to the class of SERMs, and have been previously described in the context of hormone-sensitive cancer. Bifluranol (1) is a synthetic estrogen that has anti-prostatic effects in a comparable extent to diethylstilbestrol (DES) in mice and rats. In contrast, it exhibits an approximately 8-fold lower estrogenicity compared to DES. It might have beneficial effects compared to DES, because its effects were fully reversible, and unlike DES, it did not impair spermatogenesis or fertility [1].
Research on dienestrol diacetate (5) was mainly performed during the seventies and focused on the beneficial outcome as feeding additives in meat and egg production. Several studies investigated the effects on gain of body weight [2] and the increase in productivity of chicken in respect to laying eggs [3], although the results appeared to be disillusioning [4].
COX-inhibition was often linked to gastrointestinal and other side effects like abdominal pain [5,6], e.g. chlorotrianisene. However, modulation of multiple targets can also have beneficial effects, especially in complex diseases like cancer [7]. COX-2 emerges as new cancer target [8,9] and overexpression of the inducible isoform was observed in a variety of different tumors, including stomach, esophagus, liver, pancreas, neck lung, breast, prostate, and bladder [10]. COX-1, the constitutive isoform, has long been considered to have minor influence in disease progression; however, increased levels have also been detected in ovarian [11] and cervical [12] cancer.
Inhibition of COX has been proved beneficial in reducing the risk of multiple neoplastic diseases including lung, prostate, breast, and colon cancer [13], and positive effects of co-administration of S6 COX-inhibitors to conventional chemotherapeutics have been reported [8]. In addition, there is a cross-regulation between the estrogen and the COX pathway and ongoing efforts are made to investigate the underlying mechanisms [14] and the role of COX in hormone-sensitive cancer [15,16].
Having in mind the beneficial effects of synthetic estrogens in hormone-sensitive cancer and the emerging role of COX in neoplastic diseases, a compound like dienestrol diacetate (5), that is both an estrogen receptor modulator and an unselective COX-inhibitor with an IC50 value comparable to ibuprofen, might have substantial advantages and should be investigated further.
Cyqualon (3) was identified in a screen for synthetic curcumin analogues with improved bioavailability [17]. Despite the remarkable effects of curcumin in vitro, the in vivo effects are moderate due its pharmacokinetic properties [18]. Continuous efforts are made to further investigate the biological activity of cyqualon (3), since multiple targets and effects were discovered. These include inhibition of p38 mitogen activated protein kinase, c-Jun N-terminal kinase, extracellular-signal regulated receptor1/2, nuclear factor ƙb, activator protein-1 transcription factor [19], p300 histone acetyltransferase [20], α-glucosidase [21], the transcriptional regulation of target gene expression in a prostate cancer cell line [22], and antiinflammatory [23], antibacterial [24], antiproliferative and proapoptotic [25] properties. The COXinhibition reported in this study could explain the underlying mechanism for the anitiinflammatory effects of cyqualon (3).
Paxamate (4) and p-kresalol (2) are not very well investigated compounds, and since their IC50 values are relatively high compared to the established COX-inhibitors, the use of these compounds may be limited.