Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50 values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.

A library of analogues of the cyanobacterium-derived depsipeptide natural product gallinamide A were designed and prepared using a highly efficient and convergent synthetic route. Analogues were shown to exhibit potent inhibitory activity against the Plasmodium falciparum cysteine proteases falcipain 2 and falcipain 3 and against cultured chloroquine-sensitive (3D7) and chloroquine-resistant (W2) strains of P. falciparum. Three lead compounds were selected for evaluation of in vivo efficacy against Plasmodium berghei infection in mice on the basis of their improved blood, plasma, and microsomal stability profiles compared with the parent natural product. One of the lead analogues cured P. berghei-infected mice in the Peters 4 day-suppressive test when administered 25 mg kg −1 intraperitoneally daily for 4 days. The compound was also capable of clearing parasites in established infections at 50 mg kg −1 intraperitoneally daily for 4 days and exhibited moderate activity when administered as four oral doses of 100 mg kg −1 . ■ INTRODUCTIONMalaria is a mosquito-transmitted disease caused by parasites of the genus Plasmodium. 1 Almost half the global population lives in malaria endemic areas and is at high risk of infection. The most virulent human parasite is Plasmodium falciparum, which leads to a severe infection that is often deadly, especially for children. Each year there are over 200 million new cases of malaria, and in 2017, the disease was responsible for an estimated 435 000 deaths. 2 A major global health concern is that the currently employed drug regimens for malaria are rapidly losing their effectiveness due to the emergence of drugresistant parasites. Particularly concerning is the rapid emergence of resistance against the artemisinin (ART)-based combination therapies that serve as the cornerstone of antimalarial therapy. 2,3 The widespread resistance of Plasmodium against currently available drugs and the lack of an efficacious vaccine underscores the need for the development of novel antimalarials that operate through unique mechanisms of action. Many therapeutics in clinical use are either natural products or natural product derivatives. 4 The use of natural products as privileged biologically active scaffolds from which to develop new anti-infectives has proven to be a highly successful strategy, 5 e.g., in the development and clinical 49 approval of lipopeptide and glycopeptide antibiotic analogues 6 50 and antimalarial artemisinins. 7 51 We have recently explored analogues of gallinamide A (1), a 52 linear depsipeptide natural product isolated independently in 53 2009 from Schizothrix 8 and Symploca 9 species of cyanobacteria 54 f1 as antimalarial leads (Figure 1). 10 The natural product and 55 structural analogues have been shown to possess potent 56 inhibitory activity against P. falciparum growth in vitro. The 57 mechanism of action of these natural product analogues is due 58 to the inhibition of cysteine proteases, namely, falcipain 2 59 (FP2) and falcipain 3 (FP3), 11 which are critical for...

A robust methodology for the stereocontrolled chemical glycosylation of pseudaminic acid has been developed to afford both α-(axial) and β-(equatorial) glycosides reliably with complete stereoselectivity, using a common glycosyl donor (7N-Cbz/5N-azido Pse thioglycoside) simply by changing the reaction conditions. In the CH 2 Cl 2 /MeCN cosolvent, highly β-selective pseudaminylation was observed, while addition of 5.0 equiv DMF in CH 2 Cl 2 gave the α-pseudaminosides.

Gallinamide A, a metabolite of the marine cyanobacterium Schizothrix sp., selectively inhibits cathepsin L-like cysteine proteases. We evaluated the potency of gallinamide A and 23 synthetic analogues against intracellular Trypanosoma cruzi amastigotes and the cysteine protease, cruzain. We determined the co-crystal structures of cruzain with gallinamide A and two synthetic analogues at ∼2 Å. SAR data revealed that the N-terminal end of gallinamide A is loosely bound and weakly contributes in drug–target interactions. At the C-terminus, the intramolecular π–π stacking interactions between the aromatic substituents at P1′ and P1 restrict the bioactive conformation of the inhibitors, thus minimizing the entropic loss associated with target binding. Molecular dynamics simulations showed that in the absence of an aromatic group at P1, the substituent at P1′ interacts with tryptophan-184. The P1–P1′ interactions had no effect on anti-cruzain activity, whereas anti-T. cruzi potency increased by ∼fivefold, likely due to an increase in solubility/permeability of the analogues.