Identification of 2-Aminoacyl-1,3,4-thiadiazoles as Prostaglandin E(2) and Leukotriene Biosynthesis Inhibitors.

pubs.acs.org/acsmedchemlett Letter Identification of 2‑Aminoacyl-1,3,4-thiadiazoles as Prostaglandin E2 and Leukotriene Biosynthesis Inhibitors Marianna Potenza,‡ Assunta Giordano,‡ Maria G. Chini, Anella Saviano, Christian Kretzer, Federica Raucci, Marina Russo, Gianluigi Lauro, Stefania Terracciano, Ines Bruno, Maria Iorizzi, Robert K. Hofstetter, Simona Pace, Francesco Maione, Oliver Werz,* and Giuseppe Bifulco* Cite This: ACS Med. Chem. Lett. 2023, 14, 26−34 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: The application of a multi-step scientific workflow revealed an unprecedented class of PGE2/leukotriene biosynthesis inhibitors with in vivo activity. Specifically, starting from a combinatorial virtual library of ∼4.2 × 105 molecules, a small set of compounds was identified for the synthesis. Among these, four novel 2-aminoacyl-1,3,4-thiadiazole derivatives (3, 6, 7, and 9) displayed marked anti-inflammatory properties in vitro by strongly inhibiting PGE2 biosynthesis, with IC50 values in the nanomolar range. The hit compounds also efficiently interfered with leukotriene biosynthesis in cell-based systems and modulated IL6 and PGE2 biosynthesis in a lipopolysaccharide-stimulated J774A.1 macrophage cell line. The most promising compound 3 showed prominent in vivo anti-inflammatory activity in a mouse model, with efficacy comparable to that of dexamethasone, attenuating zymosan-induced leukocyte migration in mouse peritoneum with considerable modulation of the levels of typical pro-/antiinflammatory cytokines. KEYWORDS: Combinatorial virtual screening, 2-Aminoacyl-1,3,4-thiadiazole, mPGES-1, Leukotriene biosynthesis pathway, Anti-inflammatory activity M icrosomal prostaglandin E2 synthase-1 (mPGES-1),1 a downstream PG synthase, is a membrane-integrated protein able to convert the cyclooxygenase (COX)-derived unstable prostaglandin H2 (PGH2) to the bioactive prostaglandin E2 (PGE2). This enzyme is one of the membraneassociated proteins involved in the metabolism of glutathione and prostanoids (MAPEG), a family of proteins including several key targets, such as the 5-lipoxygenase-activating protein (FLAP), leukotriene C4 synthase (LTC4S), and microsomal glutathione S-transferases, useful for the development of anti-inflammatory and anticancer drugs interfering with prostaglandin and leukotriene biosynthesis.2 Contrary to the classical non-steroidal anti-inflammatory drugs (NSAIDs), namely blockers of cyclooxygenases (COX-1 and COX-2) and coxibs (COX-2 selective inhibitors), the inhibition of mPGES1 does not affect the biosynthesis of the other physiologically important PGs.3,4 Consequently, mPGES-1 inhibitors show a safer profile with respect to fewer gastrointestinal and cardiovascular complications, like thrombosis and vascular inflammation.5,6 Several studies reported the involvement of this synthase in different types of cancer,7−9 liver diseases, like viral hepatitis, and drug-induced injury.10 To date, only two drug candidates are currently in Phase II clinical trials: GRC 27864 is being evaluated for efficacy in patients with osteoarthritic pain; GS-248 is currently being tested in a © 2022 The Authors. Published by American Chemical Society Phase II trial (https://clinicaltrials.gov/ct2/show/ NCT04744207) in Europe with systemic sclerosis patients (https://clinicaltrials.gov/ct2/results?term=mPGES-1). Thus, the development of mPGES-11,10,11 inhibitors represents an urgent issue. Furthermore, in recent years, different series of dual- and/or multi-target inhibitors of eicosanoid biosynthesis targets have been developed. In fact, the use of this type of agents able to block the targets belonging to the three different branches of the arachidonic acid cascade, namely lipoxygenases (LOs), cyclooxygenases (COXs) and cytochrome P 450 monooxygenases (CYP450), may increase the anti-inflammatory effects and reduce the side effects. Indeed, the moderate interference with multiple biological macromolecules may provide advantages in re-adjusting and regulating homeostasis compared to single-target drugs, obtaining the next generation of more efficient and safer anti-inflammatory agents.12 In the continuous effort to identify mPGES-1 inhibitors, computaReceived: July 25, 2022 Accepted: November 29, 2022 Published: December 9, 2022 26 https://doi.org/10.1021/acsmedchemlett.2c00343 ACS Med. Chem. Lett. 2023, 14, 26−34 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett tional tools have always played a central role.13−15 In this context, considering the broad spectrum of biological activities16,17 of the 2-amino-thiadiazole derivatives, such as antifungal18 and antiparasitic activities,19 and also encouraged by the inhibitory activity shown by 2-aminothiazole-based mPGES-1 inhibitors,20,21 we investigated the privileged scaffold 2-aminoacyl-1,3,4-thiadiazole as central core for designing potential mPGES-1-blocking agents. To target mPGES-1 protein, in these past few years, we improved and optimized a multi-step computational workflow integrated with robust in vitro, in vivo, and ex vivo experimental analyses22,23 that allowed us to identify novel dual mPGES-1 and leukotriene biosynthesis inhibitors. Therefore, the generation of a novel library of compounds was the first step in starting our investigation by identifying promising specific chemical platforms for a punctual decoration to be performed according to a selected synthetic approach. Thus, according to the generic scheme reported in Figure 1A, the 2-amino-5-(4- Letter belonging to the 2-amino-thiadiazole-based library, obtaining ∼4.2 × 105 entities. After that, QikProp and LigFilter software was used to filter out only compounds presenting the wellknown “drug-like” properties. To discard “non-drug-like” compounds and possible false positives in high-throughput screening (HTS) assays, QikProp software24 was used for the calculation of the pharmacokinetic properties, physically significant descriptors, and pharmaceutically relevant parameters for prediction of absorption, distribution, metabolism, and excretion (ADME). Accordingly, the functional groups generally responsible for reactivity, toxicity, or decomposition problems in vivo were filtered out before the subsequent molecular docking step, in order to rule out “non-drug-like” molecules (Table S1, Supporting Information). Then, the virtual screening workflow (VSW) on mPGES-1 (PDB code: 4BPM)25 was applied to the final library, containing 1.5 × 105 compounds that passed several filters (vide supra), using Glide software.24 Specifically, the VSW consisted of three subsequent steps, each of them yielding a ranking of compounds according to docking score value: (i) high-throughput virtual screening phase (HTVS); (ii) standard precision phase (SP); and (iii) extra precision phase (XP). The computational analyses of docking results were performed by combining the docking score with a qualitativ (...truncated)