RESUMEN
Aldehyde dehydrogenase 1A1 (ALDH1A1) is an isoenzyme that catalyzes the conversion of aldehydes to acids. However, the overexpression of ALDH1A1 in a variety of malignancies is the major cause of resistance to an anti-cancer drug, cyclophosphamide (CP). CP is a prodrug that is initially converted into 4-hydroxycyclophosphamide and its tautomer aldophosphamide, in the liver. These compounds permeate into the cell and are converted as active metabolites, i.e., phosphoramide mustard (PM), through spontaneous beta-elimination. On the other hand, the conversion of CP to PM is diverted at the level of aldophosphamide by converting it into inactive carboxyphosphamide using ALDH1A1, which ultimately leads to high drug inactivation and CP resistance. Hence, in combination with our earlier work on the target of resistance, i.e., ALDH1A1, we hereby report selective ALDH1A1 inhibitors. Herein, we selected a lead molecule from our previous virtual screening and implemented scaffold hopping analysis to identify a novel scaffold that can act as an ALDH1A1 inhibitor. This results in the identification of various novel scaffolds. Among these, on the basis of synthetic feasibility, the benzimidazole scaffold was selected for the design of novel ALDH1A1 inhibitors, followed by machine learning-assisted structure-based virtual screening. Finally, the five best compounds were selected and synthesized. All synthesized compounds were evaluated using in vitro enzymatic assay against ALDH1A1, ALDH2, and ALDH3A1. The results disclosed that three molecules A1, A2, and A3 showed significant selective ALDH1A1 inhibitory potential with an IC50 value of 0.32 µM, 0.55 µM, and 1.63 µM, respectively, and none of the compounds exhibits potency towards the other two ALDH isoforms i.e. ALDH2 and ALDH3A1. Besides, the potent compounds (A1, A2, and A3) have been tested for in vitro cell line assay in combination with mafosfamide (analogue of CP) on two cell lines i.e. A549 and MIA-PaCa-2. All three compounds show significant potency to reverse mafosfamide resistance by inhibiting ALDH1A1 against these cell lines.
RESUMEN
Cyclophosphamide (CP) is one of the most widely used anticancer drugs for various malignancies. However, its long-term use leads to ALDH1A1-mediated inactivation and subsequent resistance which necessitates the development of potential ALDH1A1 inhibitors. Currently, ALDH1A1 inhibitors from different chemical classes have been reported, but these failed to reach the market due to safety and efficacy problems. Developing a new treatment from the ground requires a huge amount of time, effort, and money, therefore it is worthwhile to improve CP efficacy by proposing better adjuvants as ALDH1A1 inhibitors. Herein, the database constituting the FDA-approved drugs with well-established safety and toxicity profiles was screened through already reported machine learning models by our research group. This model is validated for discriminating the ALDH1A1 inhibitors and non-inhibitors. Virtual screening protocol (VS) from this model identified four FDA-approved drugs, raloxifene, bazedoxifene, avanafil, and betrixaban as selective ALDH1A1 inhibitors. The molecular docking, dynamics, and water swap analysis also suggested these drugs to be promising ALDH1A1 inhibitors which were further validated for their CP resistance reversal potential by in-vitro analysis. The in-vitro enzymatic assay results indicated that raloxifene and bazedoxifene selectively inhibited the ALDH1A1 enzyme with IC50 values of 2.35 and 4.41 µM respectively, whereas IC50 values of both the drugs against ALDH2 and ALDH3A1 was >100 µM. Additional in-vitro studies with well-reported ALDH1A1 overexpressing A549 and MIA paCa-2 cell lines suggested that mafosfamide sensitivity was further ameliorated by the combination of both raloxifene and bazedoxifene. Collectively, in-silico and in-vitro studies indicate raloxifene and bazedoxifene act as promising adjuvants with CP that may improve the quality of treatment for cancer patients with minimal toxicities.