A Chemical Proteomics Approach to Defining the Mechanism of Artemisinin Action and Resistance in PfK13 Resistant parasites

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Synthesis, structure and antileishmanial evaluation of endoperoxide–pyrazole hybrids

Publication . Amado, Patrícia S. M.; Costa, Inês C. C.; Paixão, José A.; Mendes, Ricardo F.; Cortes, Sofia; Cristiano, Maria L.

Leishmaniases are among the most impacting neglected tropical diseases. In attempts to repurpose antimalarial drugs or candidates, it was found that selected 1,2,4-trioxanes, 1,2,4,5-tetraoxanes, and pyrazole-containing chemotypes demonstrated activity against Leishmania parasites. This study reports the synthesis and structure of trioxolane–pyrazole (OZ1, OZ2) and tetraoxane–pyrazole (T1, T2) hybrids obtained from the reaction of 3(5)-aminopyrazole with endoperoxide-containing building blocks. Interestingly, only the endocyclic amine of 3(5)-aminopyrazole was found to act as nucleophile for amide coupling. However, the fate of the reaction was influenced by prototropic tautomerism of the pyrazole heterocycle, yielding 3- and 5-aminopyrazole containing hybrids which were characterized by different techniques, including X-ray crystallography. The compounds were evaluated for in vitro antileishmanial activity against promastigotes of L. tropica and L. infantum, and for cytotoxicity against THP-1 cells. Selected compounds were also evaluated against intramacrophage amastigote forms of L. infantum. Trioxolane–pyrazole hybrids OZ1 and OZ2 exhibited some activity against Leishmania promastigotes, while tetraoxane–pyrazole hybrids proved inactive, most likely due to solubility issues. Eight salt forms, specifically tosylate, mesylate, and hydrochloride salts, were then prepared to improve the solubility of the corresponding peroxide hybrids and were uniformly tested. Biological evaluations in promastigotes showed that the compound OZ1•HCl was the most active against both strains of Leishmania. Such finding was corroborated by the results obtained in assessments of the L. infantum amastigote susceptibility. It is noteworthy that the salt forms of the endoperoxide–pyrazole hybrids displayed a broader spectrum of action, showing activity in both strains of Leishmania. Our preliminary biological findings encourage further optimization of peroxide–pyrazole hybrids to identify a promising antileishmanial lead.

2022-08-24Journal article

Open access

Molecular and crystal structure, spectroscopy, and photochemistry of a dispiro compound bearing the tetraoxane pharmacophore

Publication . Amado, Patrícia; Lopes, Susy; Brás, Elisa M.; Paixão, José A.; Takano, Ma‐aya; Abe, Manabu; Fausto, Rui; Cristiano, Maria De Lurdes

The molecular structure and photochemistry of dispiro[cyclohexane-1,3′-[1,2,4,5]tetraoxane-6′,2′′-tricyclo[3.3.1.13,7]decan]-4-one (TX), an antiparasitic 1,2,4,5-tetraoxane was investigated using matrix isolation IR and EPR spectroscopies, together with quantum chemical calculations undertaken at the DFT(B3LYP)/6-311++G(3df,3pd) level of theory, with and without Grimme's dispersion correction. Photolysis of the matrix-isolated TX, induced by in situ broadband (λ>235 nm) or narrowband (λ in the range 220–263 nm) irradiation, led to new bands in the infrared spectrum that could be ascribed to two distinct photoproducts, oxepane-2,5-dione, and 4-oxohomoadamantan-5-one. Our studies show that these photoproducts result from initial photoinduced cleavage of an O−O bond, with the formation of an oxygen-centered diradical that regioselectivity rearranges to a more stable (secondary carbon-centered)/(oxygen-centered) diradical, yielding the final products. Formation of the diradical species was confirmed by EPR measurements, upon photolysis of the compound at λ=266 nm, in acetonitrile ice (T=10–80 K). Single-crystal X-ray diffraction (XRD) studies demonstrated that the TX molecule adopts nearly the same conformation in the crystal and matrix-isolation conditions, revealing that the intermolecular interactions in the TX crystal are weak. This result is in keeping with observed similarities between the infrared spectrum of the crystalline material and that of matrix-isolated TX. The detailed structural, vibrational, and photochemical data reported here appear relevant to the practical uses of TX in medicinal chemistry, considering its efficient and broad parasiticidal properties.

2023Journal article

Open access

Recent advances of DprE1 inhibitors against mycobacterium tuberculosis: computational analysis of physicochemical and ADMET properties

Publication . Amado, Patrícia; Woodley, Christopher; Lurdes S. Cristiano, M.; O’Neill, Paul M.

D e cap renylp ho sp ho ryl-beta-D-rib os e 2 '-epimerase (DprE1) is a critical flavoenzyme in Mycobacterium tuberculosis, catalyzing a vital step in the production of lipoarabinomannan and arabinogalactan, both of which are essential for cell wall biosynthesis. Due to its periplasmic localization, DprE1 is a susceptible target, and several compounds with diverse scaffolds have been discovered that inhibit this enzyme, covalently or noncovalently. We evaluated a total of similar to 1519 DprE1 inhibitors disclosed in the literature from 2009 to April 2022 by performing an in-depth analysis of physicochemical descriptors and absorption, distribution, metabolism, excretion, and toxicity (ADMET), to gain new insights into these properties in DprE1 inhibitors. Several molecular properties that should facilitate the design and optimization of future DprE1 inhibitors are described, allowing for the development of improved analogues targeting M. tuberculosis.

2022-11Journal article

Open access

Unravelling the structure of peroxides with antiparasitic activity: the relative impact of a trioxolane or a tetraoxane pharmacophore on the overall molecular structure

Publication . Amado, Patrícia; Jesus, A. J. Lopes; Paixão, José A.; Fausto, Rui; Cristiano, Maria De Lurdes

Plasmodium falciparum artemisinin-resistance boosted the quest for novel plasmodial "fast killers," uncovering antimalarial candidates OZ439 and E209, whose peroxide precursors are 1,2,4-trioxolane (1) and 1,2,4,5-tetraoxane (2), differing solely in the pharmacophore (trioxolane or tetraoxane). Combining X-ray crystallography and vibrational spectroscopy, along with Hirsh-feld surface analysis and calculations (CE-B3LYP/6-31G(d,p)) of pairwise interaction energies of intermolecular contacts existing in the crystal structure, may deepen the understanding of relative reactivity and properties of these endoperoxides classes. In the crystal, the tetraoxane ring in 2 and the trioxolane-adamantyl fragment in 1 are disordered, with molecules 1 and 2 existing as two distinct, stable conformations. Whereas the dominant C-H center dot center dot center dot O H-bonds in 1 connect an adamantyl C-H and O1 or O2 of the trioxolane ring, in 2 they involve the carbonyl oxygen, acting as a double acceptor from phenyl ring C-H groups. C-H center dot center dot center dot O and C-H center dot center dot center dot pi H-bonds define the molecular packing of 2, while C-H center dot center dot center dot H-C van der Waals interactions determine the packing of 1. The dispersive component dominates the interaction energies calculated for the most representative molecular pairs.

2022Journal article

Restricted access

Design, synthesis and in vitro evaluation of a series of endoperoxide hybrids designed to tackle latent tuberculosis

Publication . Amado, Patrícia Sofia Menalha; Cristiano, Maria de Lurdes dos Santos; O’Neill, Paul Michael

Mycobacterium tuberculosis (Mtb) is the world's second leading cause of death from infectious diseases (after COVID-19). The ability of Mtb to enter the nonreplicating persistence (NRP) and then transition to latent TB contributes to Mtb's drug tolerance and treatment failure in chronically infected individuals. The DosRST two-component regulatory system regulates the Mtb physiology to promote NRP, in which peroxides such as the natural antimalarial drug artemisinin and synthetic 1,2,4-trioxolanes have been demonstrated to inhibit this system and re-sensitize Mtb. Hence, we proposed hybridizing two separate anti-TB classes by combining the 1,2,4-trioxane-containing moieties with the indole-2-carboxamide scaffold (MmpL3 inhibitors) and the benzothiazinone scaffold (DprE1 inhibitors), to establish a dual mode of action, by increasing Mtb's sensitivity to the active anti-TB pharmacophore while also targeting the DosRST signalling. These hybrid compounds prepared were evaluated for their in vitro antimycobacterial activity, and the drug metabolism and pharmacokinetics parameters were also assessed. Additionally, we evaluated ~1519 DprE1 inhibitors disclosed in the literature from 2009-2022 by performing an in-depth analysis of physicochemical descriptors and absorption, distribution, metabolism, elimination, and toxicology properties to deepen our understanding of DprE1 inhibitors and build machine learning classification models.

2023-07-11Doctoral thesis

Open access