C-2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV-2 through Interaction with the Viral Spike (S) Protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, by infecting cells via the interaction of its spike protein (S) with the primary cell receptor angiotensin-converting enzyme (ACE2). To search for inhibitors of this key step in viral infection, we screened an in-house library of multivalent tryptophan derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell entry. Thermofluor and microscale thermophoresis studies revealed their binding to S and to its isolated receptor binding domain (RBD), interfering with the interaction with ACE2. High-resolution cryoelectron microscopy structure of S, free or bound to 2, shed light on cell entry inhibition mechanisms by these compounds. Overall, this work identifies and characterizes a new class of SARS-CoV-2 entry inhibitors with clear potential for preventing and/or fighting COVID-19.

Hybridization Approach to Identify Salicylanilides as Inhibitors of Tubulin Polymerization and Signal Transducers and Activators of Transcription 3 (STAT3).

The superimposition of the X-ray complexes of cyclohexanediones (i.e., TUB015), described by our research group, and nocodazole, within the colchicine binding site of tubulin provided an almost perfect overlap of both ligands. This structural information led us to propose hybrids of TUB015 and nocodazole using a salicylanilide core structure. Interestingly, salicylanilides, such as niclosamide, are well-established signal transducers and activators of transcription (STAT3) inhibitors with anticancer properties. Thus, different compounds with this new scaffold have been synthesized with the aim to identify compounds inhibiting tubulin polymerization and/or STAT3 signaling. As a result, we have identified new salicylanilides (6 and 16) that showed significant antiproliferative activity against a panel of cancer cells. Both compounds were able to reduce the levels of p-STAT3Tyr705 without affecting the total expression of STAT3. While compound 6 inhibited tubulin polymerization and arrested the cell cycle of DU145 cells at G2/M, similar to TUB015, compound 16 showed a more potent effect on inhibiting STAT3 phosphorylation and arrested the cell cycle at G1/G0, similar to niclosamide. In both cases, no toxicity towards PBMC cells was detected. Thus, the salicylanilides described here represent a new class of antiproliferative agents affecting tubulin polymerization and/or STAT3 phosphorylation.

Inhibition of XPO-1 Mediated Nuclear Export through the Michael-Acceptor Character of Chalcones.

The nuclear export receptor exportin-1 (XPO1, CRM1) mediates the nuclear export of proteins that contain a leucine-rich nuclear export signal (NES) towards the cytoplasm. XPO1 is considered a relevant target in different human diseases, particularly in hematological malignancies, tumor resistance, inflammation, neurodegeneration and viral infections. Thus, its pharmacological inhibition is of significant therapeutic interest. The best inhibitors described so far (leptomycin B and SINE compounds) interact with XPO1 through a covalent interaction with Cys528 located in the NES-binding cleft of XPO1. Based on the well-established feature of chalcone derivatives to react with thiol groups via hetero-Michael addition reactions, we have synthesized two series of chalcones. Their capacity to react with thiol groups was tested by incubation with GSH to afford the hetero-Michael adducts that evolved backwards to the initial chalcone through a retro-Michael reaction, supporting that the covalent interaction with thiols could be reversible. The chalcone derivatives were evaluated in antiproliferative assays against a panel of cancer cell lines and as XPO1 inhibitors, and a good correlation was observed with the results obtained in both assays. Moreover, no inhibition of the cargo export was observed when the two prototype chalcones 9 and 10 were tested against a XPO1-mutated Jurkat cell line (XPO1C528S), highlighting the importance of the Cys at the NES-binding cleft for inhibition. Finally, their interaction at the molecular level at the NES-binding cleft was studied by applying the computational tool CovDock.

Diphenyl ether derivatives occupy the expanded binding site of cyclohexanedione compounds at the colchicine site in tubulin by movement of the αT5 loop.

Microtubule targeting agents represent a very active arena in the development of anticancer agents. In particular, compounds binding at the colchicine site in tubulin are being deeply studied, and the structural information recently available on this binding site allows structure-directed design of new ligands. Structural comparison of our recently reported high resolution X-Ray structure of the cyclohexanedione derivative TUB075 bound to tubulin and the tubulin-DAMA-colchicine complex has revealed a conformational change in the αT5 loop. By a grid-based computational analysis of the tubulin-DAMA-colchicine binding site, we have identified a new favourable binding area in the colchicine-site that was unexplored by our lead TUB075. Thus, based on a structure-guided design, new cyclohexanedione derivatives have been synthesized and tested for tubulin binding and in cellular assays. As a result, we have identified diphenyl ether derivatives with IC50 values around 10-40 nM against three different tumor cell lines and affinity constants for tubulin similar to that of colchicine around 107 M-1. As expected, they halted the cell cycle progression at G2/M phase at concentrations as low as 0.08 µM.

Chalcones and Chalcone-mimetic Derivatives as Notch Inhibitors in a Model of T-cell Acute Lymphoblastic Leukemia.

Based on hit-likeness and chemical diversity, a number of chalcones and chalcone-mimetic compounds were selected as putative Notch inhibitors. The evaluation of the antiproliferative effect combined with the inhibition of Notch1 expression in KOPTK1 cell line identified compound 18, featuring a tetrahydronaphthalene-based scaffold, as a new promising Notch-blocking agent.

Identification of a novel 2-oxindole fluorinated derivative as in vivo antitumor agent for prostate cancer acting via AMPK activation.

The key metabolic sensor adenosine monophosphate-dependent kinase (AMPK) has emerged as a promising therapeutic target for cancer prevention and treatment. Besides its role in energy homeostasis, AMPK blocks cell cycle, regulates autophagy and suppresses the anabolic processes required for rapid cell growth. AMPK is especially relevant in prostate cancer in which activation of lipogenic pathways correlate with tumor progression and aggressiveness. This study reports the discovery of a new series of 2-oxindole derivatives whose AMPK modulatory ability, as well as the antitumoral profile in prostate cancer cells, was evaluated. One of the assayed compounds, compound 8c, notably activated AMPK in cultured PC-3, DU145 and LNCaP prostate cancer cells. Likewise, compound 8c caused PC-3, DU145 and LNCaP cells viability inhibition. Selective knocking down of α1 or α2 isoforms as well as in vitro assays using human recombinant α1ß1γ1 or α2ß1γ1 AMPK isoforms revealed that compound 8c exhibit preference for AMPKα1. Consistent with efficacy at the cellular level, compound 8c was potent in suppressing the growth of PC-3 xenograft tumors. In conclusion, our results show that a new 2-oxindole fluorinated derivative exerts potent in vivo antitumor actions against prostate cancer cells, indicating a promising clinical therapeutic strategy for the treatment of androgen-independent prostate cancer.