Structure-guided optimization of 3-hydroxybenzoisoxazole derivatives as inhibitors of Aldo-keto reductase 1C3 (AKR1C3) to target prostate cancer.

AKR1C3 is an enzyme that is overexpressed in several types of radiotherapy- and chemotherapy-resistant cancers. Despite AKR1C3 is a validated target for drug development, no inhibitor has been approved for clinical use. In this manuscript, we describe our study of a new series of potent AKR1C3-targeting 3-hydroxybenzoisoxazole based inhibitors that display high selectivity over the AKR1C2 isoform and low micromolar activity in inhibiting 22Rv1 prostate cancer cell proliferation. In silico studies suggested proper substituents to increase compound potency and provided with a mechanistic explanation that could clarify their different activity, later confirmed by X-ray crystallography. Both the in-silico studies and the crystallographic data highlight the importance of 90° rotation around the single bond of the biphenyl group, in ensuring that the inhibitor can adopt the optimal binding mode within the active pocket. The p-biphenyls that bear the meta-methoxy, and the ortho- and meta-trifluoromethyl substituents (in compounds 6a, 6e and 6f respectively) proved to be the best contributors to cellular potency as they provided the best IC50 values in series (2.3, 2.0 and 2.4 µM respectively) and showed no toxicity towards human MRC-5 cells. Co-treatment with scalar dilutions of either compound 6 or 6e and the clinically used drug abiraterone led to a significant reduction in cell proliferation, and thus confirmed that treatment with both CYP171A1-and AKR1C3-targeting compounds possess the potential to intervene in key steps in the steroidogenic pathway. Taken together, the novel compounds display desirable biochemical potency and cellular target inhibition as well as good in-vitro ADME properties, which highlight their potential for further preclinical studies.

Investigation into the Use of Encorafenib to Develop Potential PROTACs Directed against BRAFV600E Protein.

BRAF is a serine/threonine kinase frequently mutated in human cancers. BRAFV600E mutated protein is targeted through the use of kinase inhibitors which are approved for the treatment of melanoma; however, their long-term efficacy is hampered by resistance mechanisms. The PROTAC-induced degradation of BRAFV600E has been proposed as an alternative strategy to avoid the onset of resistance. In this study, we designed a series of compounds where the BRAF kinase inhibitor encorafenib was conjugated to pomalidomide through different linkers. The synthesized compounds maintained their ability to inhibit the kinase activity of mutated BRAF with IC50 values in the 40-88 nM range. Selected compounds inhibited BRAFV600E signaling and cellular proliferation of A375 and Colo205 tumor cell lines. Compounds 10 and 11, the most active of the series, were not able to induce degradation of mutated BRAF. Docking and molecular dynamic studies, conducted in comparison with the efficient BRAF degrader P5B, suggest that a different orientation of the linker bearing the pomalidomide substructure, together with a decreased mobility of the solvent-exposed part of the conjugates, could explain this behavior.

New aldo-keto reductase 1C3 (AKR1C3) inhibitors based on the hydroxytriazole scaffold.

The aldo-keto reductase 1C3 (AKR1C3) enzyme is considered an attractive target in Castration Resistant Prostate Cancer (CRPC) because of its role in the biosynthesis of androgens. Flufenamic acid, a non-selective AKR1C3 inhibitor, has previously been subjected to bioisosteric modulation to give rise to a series of compounds with the hydroxytriazole core. In this work, the hit compound of the previous series has been modulated further, and new, more potent, and selective derivatives have been obtained. The poor solubility of the most active compound (cpd 5) has been improved by substituting the triazole core with an isoxazole heteronucleous, with similar enzymatic activity being retained. Potent AKR1C3 inhibition is translated into antiproliferative effects against the 22RV1 CRPC cellular model, and the in-silico design, synthesis and biological activity of new compounds are described herein. Compounds have also been assayed in combination with two approved antitumor drugs, abiraterone and enzalutamide.

Inhibition of the drug efflux activity of Ptch1 as a promising strategy to overcome chemotherapy resistance in cancer cells.

The development of inhibitors of key biological mechanisms involved in multidrug resistance (MDR) burden meets an important medical need but still represents a challenging task. Major MDR targets in both bacterial and cancer cells are multidrug efflux systems. Several aspects should be considered in the attempt to design efficient inhibitors of these systems such as toxicity, stability, permeability as a few examples. In order to successfully design promising new compounds, a full understanding of the efflux mechanism is required, from both biological and structural points of view. It is nowadays well established that the success rate in classical drug design and biological evaluation improves when combined with in silico methodologies. In this review, we focus on the biological evaluation and molecular mechanistic insights of inhibitors of the drug efflux activity of the Hedgehog receptor Patched1 (Ptch1). Ptch1 is known to be over-expressed in many types of cancers, but its activity and role in the resistance to chemotherapy of cancer cells have been highlighted only recently. Remarkably, due to its peculiar efflux mechanism, inhibition of Ptch1 was shown to be particularly relevant for improving the efficacy of chemotherapy without concomitant toxicity for healthy cells or potential side effects. To date, three compounds have been identified as efficient Ptch1 inhibitors, namely astemizole, methiothepin and panicein A hydroquinone. Due to the chemical and structural differences of these molecules, the hit-to-lead drug design is not straightforward. This review describes how the merging of in vitro, in vivo and in silico studies provides molecular details that could contribute to the rational design of new Ptch1 inhibitors.

Molecular insights into the Patched1 drug efflux inhibitory activity of panicein A hydroquinone: a computational study.

Human Hedgehog receptor Patched1 (PTCH1) is able to efflux chemotherapeutics of different chemical structure out of cancer cells thus contributing to multidrug resistance phenomena in tumor treatment. A screening of natural compounds purified from marine sponges led to the identification of the first PTCH1 efflux inhibitor, panicein A hydroquinone (PAH), demonstrated to increase doxorubicin toxicity in vitro and vemurafenib toxicity in vitro and in vivo. In this work we combined different computational techniques to gain molecular insights of the inhibitory activity of PAH and some of its active and inactive analogues. We first performed a thorough characterization and druggability analysis of the main putative substrate binding pockets known from available cryo-electron microscopy structures. Further, dynamical descriptors of the active and inactive PAH analogues were extracted from microsecond-long all-atom molecular dynamics simulations in water solution. Finally, a blind ensemble docking methodology coupled with the conformational analysis of compounds enabled rationalization of the interaction between PTCH1 and PAH and derivatives in terms of their intrinsic physico-chemical properties. Our results suggest that the Neck pocket is the preferential binding site for PAH analogues on PTCH1, and that compounds assuming an open cylindric-like shape in solution are most likely to be good binders for PTCH1.

Novel peptide-conjugated nanomedicines for brain targeting: In vivo evidence.

Central nervous system (CNS) compartments remain one of the most difficult districts for drug delivery. This is due to the presence of the blood-brain barrier (BBB) that hampers 90% of drug passage, dramatically requiring non-invasive treatment strategies. Here, for the first time, the use of opioid-derived deltorphin-derivative peptides to drive biodegradable and biocompatible polymeric (i.e. poly-lactide-co-glycolide, PLGA) nanomedicines delivery across the BBB was described. Opioid-derived peptides were covalently conjugated to furnish activated polymers which were further used for fluorescently tagged nanoformulations. Beyond reporting production, formulation methodology and full physico-chemical characterization, in vivo tests generated clear proof of BBB crossing and CNS targeting by engineered nanomedicines opening the research to further applications of drug delivery and targeting in CNS disease models.

Iron Metabolism at the Interface between Host and Pathogen: From Nutritional Immunity to Antibacterial Development.

Nutritional immunity is a form of innate immunity widespread in both vertebrates and invertebrates. The term refers to a rich repertoire of mechanisms set up by the host to inhibit bacterial proliferation by sequestering trace minerals (mainly iron, but also zinc and manganese). This strategy, selected by evolution, represents an effective front-line defense against pathogens and has thus inspired the exploitation of iron restriction in the development of innovative antimicrobials or enhancers of antimicrobial therapy. This review focuses on the mechanisms of nutritional immunity, the strategies adopted by opportunistic human pathogen Staphylococcus aureus to circumvent it, and the impact of deletion mutants on the fitness, infectivity, and persistence inside the host. This information finally converges in an overview of the current development of inhibitors targeting the different stages of iron uptake, an as-yet unexploited target in the field of antistaphylococcal drug discovery.