Multiparameter Optimization of Trypanocidal Cruzain Inhibitors With In Vivo Activity and Favorable Pharmacokinetics.

Cruzain, the main cysteine protease of Trypanosoma cruzi, plays key roles in all stages of the parasite's life cycle, including nutrition acquisition, differentiation, evasion of the host immune system, and invasion of host cells. Thus, inhibition of this validated target may lead to the development of novel drugs for the treatment of Chagas disease. In this study, a multiparameter optimization (MPO) approach, molecular modeling, and structure-activity relationships (SARs) were employed for the identification of new benzimidazole derivatives as potent competitive inhibitors of cruzain with trypanocidal activity and suitable pharmacokinetics. Extensive pharmacokinetic studies enabled the identification of metabolically stable and permeable compounds with high selectivity indices. CYP3A4 was found to be involved in the main metabolic pathway, and the identification of metabolic soft spots provided insights into molecular optimization. Compound 28, which showed a promising trade-off between pharmacodynamics and pharmacokinetics, caused no acute toxicity and reduced parasite burden both in vitro and in vivo.

A Myristoyl Amide Derivative of Doxycycline Potently Targets Cancer Stem Cells (CSCs) and Prevents Spontaneous Metastasis, Without Retaining Antibiotic Activity.

Here, we describe the chemical synthesis and biological activity of a new Doxycycline derivative, designed specifically to more effectively target cancer stem cells (CSCs). In this analog, a myristic acid (14 carbon) moiety is covalently attached to the free amino group of 9-amino-Doxycycline. First, we determined the IC50 of Doxy-Myr using the 3D-mammosphere assay, to assess its ability to inhibit the anchorage-independent growth of breast CSCs, using MCF7 cells as a model system. Our results indicate that Doxy-Myr is >5-fold more potent than Doxycycline, as it appears to be better retained in cells, within a peri-nuclear membranous compartment. Moreover, Doxy-Myr did not affect the viability of the total MCF7 cancer cell population or normal fibroblasts grown as 2D-monolayers, showing remarkable selectivity for CSCs. Using both gram-negative and gram-positive bacterial strains, we also demonstrated that Doxy-Myr did not show antibiotic activity, against Escherichia coli and Staphylococcus aureus. Interestingly, other complementary Doxycycline amide derivatives, with longer (16 carbon; palmitic acid) or shorter (12 carbon; lauric acid) fatty acid chain lengths, were both less potent than Doxy-Myr for the targeting of CSCs. Finally, using MDA-MB-231 cells, we also demonstrate that Doxy-Myr has no appreciable effect on tumor growth, but potently inhibits tumor cell metastasis in vivo, with little or no toxicity. In summary, by using 9-amino-Doxycycline as a scaffold, here we have designed new chemical entities for their further development as anti-cancer agents. These compounds selectively target CSCs, e.g., Doxy-Myr, while effectively minimizing the risk of driving antibiotic resistance. Taken together, our current studies provide proof-of-principle, that existing FDA-approved drugs can be further modified and optimized, to successfully target the anchorage-independent growth of CSCs and to prevent the process of spontaneous tumor cell metastasis.

Discovery of Potent, Reversible, and Competitive Cruzain Inhibitors with Trypanocidal Activity: A Structure-Based Drug Design Approach.

A virtual screening conducted with nearly 4 000 000 compounds from lead-like and fragment-like subsets enabled the identification of a small-molecule inhibitor (1) of the Trypanosoma cruzi cruzain enzyme, a validated drug target for Chagas disease. Subsequent comprehensive structure-based drug design and structure-activity relationship studies led to the discovery of carbamoyl imidazoles as potent, reversible, and competitive cruzain inhibitors. The most potent carbamoyl imidazole inhibitor (45) exhibited high affinity with a Ki value of 20 nM, presenting both in vitro and in vivo activity against T. cruzi. Furthermore, the most promising compounds reduced parasite burden in vivo and showed no toxicity at a dose of 100 mg/kg. These carbamoyl imidazoles are structurally attractive, nonpeptidic, and easy to prepare and synthetically modify. Finally, these results further advance our understanding of the noncovalent mode of inhibition of this pharmaceutically relevant enzyme, building strong foundations for drug discovery efforts.

Structure-Based and Molecular Modeling Studies for the Discovery of Cyclic Imides as Reversible Cruzain Inhibitors With Potent Anti-Trypanosoma cruzi Activity.

Chagas disease causes ~10,000 deaths each year, mainly in Latin America, where it is endemic. The currently available chemotherapeutic agents are ineffective in the chronic stage of the disease, and the lack of pharmaceutical innovation for Chagas disease highlights the urgent need for the development of new drugs. The enzyme cruzain, the main cysteine protease of Trypanosoma cruzi, has been explored as a validated molecular target for drug discovery. Herein, the design, molecular modeling studies, synthesis, and biological evaluation of cyclic imides as cruzain inhibitors are described. Starting with a micromolar-range cruzain inhibitor (3a, IC50 = 2.2 µM), this molecular optimization strategy resulted in the nanomolar-range inhibitor 10j (IC50 = 0.6 µM), which is highly active against T. cruzi intracellular amastigotes (IC50 = 1.0 µM). Moreover, most compounds were selective toward T. cruzi over human fibroblasts, which were used as host cells, and are less toxic to hepatic cells than the marketed drug benznidazole. This study enabled the discovery of novel chemical diversity and established robust structure-activity relationships to guide the design of optimized cruzain inhibitors as new trypanocidal agents.

Inhibition of Breast Cancer Cell Migration by Cyclotides Isolated from Pombalia calceolaria.

Two new bracelet cyclotides from roots of Pombalia calceolaria with potential anticancer activity have been characterized in this work. The cyclotides Poca A and B (1 and 2) and the previously known CyO4 (3) were de novo sequenced by MALDI-TOF/TOF mass spectrometry (MS). The MS2 spectra were examined and the amino acid sequences were determined. The purified peptides were tested for their cytotoxicity and effects on cell migration of MDA-MB-231, a triple-negative breast cancer cell line. The isolated cyclotides reduced the number of cancer cells by more than 80% at 20 µM, and the concentration-related cytotoxic responses were observed with IC50 values of 1.8, 2.7, and 9.8 µM for Poca A (1), Poca B (2), and CyO4 (3), respectively. Additionally, the inhibition of cell migration (wound-healing assay) exhibited that CyO4 (3) presents an interesting activity profile, in being able to inhibit cell migration (50%) at a subtoxic concentration (2 µM). The distribution of these cyclotides in the roots was analyzed by MALDI imaging, demonstrating that all three compounds are present in the phloem and cortical parenchyma regions.

Recent Advances and Perspectives in Cancer Drug Design.

Cancer is one of the leading causes of death worldwide. With the increase in life expectancy, the number of cancer cases has reached unprecedented levels. In this scenario, the pharmaceutical industry has made significant investments in this therapeutic area. Despite these efforts, cancer drug research remains a remarkably challenging field, and therapeutic innovations have not yet achieved expected clinical results. However, the physiopathology of the disease is now better understood, and the discovery of novel molecular targets has refreshed the expectations of developing improved treatments. Several noteworthy advances have been made, among which the development of targeted therapies is the most significant. Monoclonal antibodies and antibody-small molecule conjugates have emerged as a worthwhile approach to improve drug selectivity and reduce adverse effects, which are the main challenges in cancer drug discovery. This review will examine the current panorama of drug research and development (R&D) with emphasis on some of the major advances brought to clinical trials and to the market in the past five years. Breakthrough discoveries will be highlighted along with the medicinal chemistry strategies used throughout the discovery process. In addition, this review will provide perspectives and updates on the discovery of novel molecular targets as well as drugs with innovative mechanisms of action.

Discovery of a Series of Acridinones as Mechanism-Based Tubulin Assembly Inhibitors with Anticancer Activity.

Microtubules play critical roles in vital cell processes, including cell growth, division, and migration. Microtubule-targeting small molecules are chemotherapeutic agents that are widely used in the treatment of cancer. Many of these compounds are structurally complex natural products (e.g., paclitaxel, vinblastine, and vincristine) with multiple stereogenic centers. Because of the scarcity of their natural sources and the difficulty of their partial or total synthesis, as well as problems related to their bioavailability, toxicity, and resistance, there is an urgent need for novel microtubule binding agents that are effective for treating cancer but do not have these disadvantages. In the present work, our lead discovery effort toward less structurally complex synthetic compounds led to the discovery of a series of acridinones inspired by the structure of podophyllotoxin, a natural product with important microtubule assembly inhibitory activity, as novel mechanism-based tubulin assembly inhibitors with potent anticancer properties and low toxicity. The compounds were evaluated in vitro by wound healing assays employing the metastatic and triple negative breast cancer cell line MDA-MB-231. Four compounds with IC50 values between 0.294 and 1.7 µM were identified. These compounds showed selective cytotoxicity against MDA-MB-231 and DU-145 cancer cell lines and promoted cell cycle arrest in G2/M phase and apoptosis. Consistent with molecular modeling results, the acridinones inhibited tubulin assembly in in vitro polymerization assays with IC50 values between 0.9 and 13 µM. Their binding to the colchicine-binding site of tubulin was confirmed through competitive assays.

Semisynthesis of new aphidicolin derivatives with high activity against Trypanosoma cruzi.

Chagas disease continues to be a difficult disease to eradicate, largely because of the widespread populations it affects as well as the highly toxic effects of current therapies. Thus, the exploration of innovative scaffolds, ideally with distinct mechanisms of action, is urgently needed. The natural product aphidicolin and its effects on cell cycle division have been widely studied; it is a potent inhibitor of parasitic cells. In the present study, we report for the first time the semisynthesis of a series of aphidicolin derivatives, their unique structural features, and demonstration of their activity against Trypanosoma cruzi cells. Two demonstrated high potency and selectivity against parasitic amastigote cells, and thus show promise as new leads for Chagas disease treatment.