The S1'-S3' Pocket of the SARS-CoV-2 Main Protease Is Critical for Substrate Selectivity and Can Be Targeted with Covalent Inhibitors.

The main protease (Mpro ) of SARS-CoV-2 is a well-characterized target for antiviral drug discovery. To date, most antiviral drug discovery efforts have focused on the S4-S1' pocket of Mpro ; however, it is still unclear whether the S1'-S3' pocket per se can serve as a new site for drug discovery. In this study, the S1'-S3' pocket of Mpro was found to differentially recognize viral peptidyl substrates. For instance, S3' in Mpro strongly favors Phe or Trp, and S1' favors Ala. The peptidyl inhibitor D-4-77, which possesses an α-bromoacetamide warhead, was discovered to be a promising inhibitor of Mpro , with an IC50 of 0.95 µM and an antiviral EC50 of 0.49 µM. The Mpro /inhibitor co-crystal structure confirmed the binding mode of the inhibitor to the S1'-S3' pocket and revealed a covalent mechanism. In addition, D-4-77 functions as an immune protectant and suppresses SARS-CoV-2 Mpro -induced antagonism of the host NF-κB innate immune response. These findings indicate that the S1'-S3' pocket of SARS-CoV-2 Mpro is druggable, and that inhibiting SARS-CoV-2 Mpro can simultaneously protect human innate immunity and inhibit virion assembly.

N-cystaminylbiguanide MC001 prevents neuron cell death and alleviates motor deficits in the MPTP-model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN), which is highly associated with oxidative stress. Antioxidants are therefore considered as potential therapies in PD treatment. In this study, we examined the neuroprotective effect of a cysteamine-based biguanide N-cystaminylbiguanide (MC001) in the MPTP mouse model of PD. The results showed that MC001 prevented neuron cell death and alleviated motor deficits in the MPTP mouse model of PD. Both in vitro and in vivo data indicated that MC001 may exert its neuroprotective effect by alleviating ROS production, suppressing neuroinflammation, and upregulating BDNF expression. Further mechanistic studies revealed that MC001 promoted GSH synthesis by inducing the expression of Glutamate-cysteine ligase catalytic subunit (Gclc) and enhancing the activity of Glutamate-cysteine ligase (Gcl). Our results suggest that MC001 warrants further investigation as a potential candidate for the treatment of PD.

Biguanide MC001, a Dual Inhibitor of OXPHOS and Glycolysis, Shows Enhanced Antitumor Activity Without Increasing Lactate Production.

Metformin and other biguanides represent a new class of inhibitors of mitochondrial complex I that show promising antitumor effects. However, stronger inhibition of mitochondrial complex I is generally associated with upregulation of glycolysis and higher risk of lactic acidosis. Herein we report a novel biguanide derivative, N-cystaminylbiguanide (MC001), which was found to inhibit mitochondrial complex I with higher potency while inducing lactate production to a similar degree as metformin.Furthermore, MC001 was found to efficiently inhibit a panel of colorectal cancer (CRC) cells in vitro and to suppress tumor growth in a HCT116 xenograft nude mouse model, while not enhancing lactate production relative to metformin, exhibiting a superior safety profile to other potent biguanides such as phenformin. Mechanistically, MC001 efficiently inhibits mitochondrial complex I, activates AMPK, and represses mTOR, leading to cell-cycle arrest and apoptosis. Notably, MC001 inhibits both oxidative phosphorylation (OXPHOS) and glycolysis. We therefore propose that MC001 warrants further investigation in cancer treatment.

Identification of Small Molecule Inhibitors of RNase L by Fragment-Based Drug Discovery.

The pseudokinase-endoribonuclease RNase L plays important roles in antiviral innate immunity and is also implicated in many other cellular activities. The inhibition of RNase L showed therapeutic potential for Aicardi-Goutières syndrome (AGS). Thus, RNase L is a promising drug target. In this study, using an enzyme assay and NMR screening, we discovered 13 inhibitory fragments against RNase L. Cocrystal structures of RNase L separately complexed with two different fragments were determined in which both fragments bound to the ATP-binding pocket of the pseudokinase domain. Myricetin, vitexin, and hyperoside, three natural products sharing similar scaffolds with the fragment AC40357, demonstrated a potent inhibitory activity in vitro. In addition, myricetin has a promising cellular inhibitory activity. A cocrystal structure of RNase L with myricetin provided a structural basis for inhibitor design by allosterically modulating the ribonuclease activity. Our findings demonstrate that fragment screening can lead to the discovery of natural product inhibitors of RNase L.

Improvement on Permeability of Cyclic Peptide/Peptidomimetic: Backbone N-Methylation as A Useful Tool.

Peptides have a three-dimensional configuration that can adopt particular conformations for binding to proteins, which are well suited to interact with larger contact surface areas on target proteins. However, low cell permeability is a major challenge in the development of peptide-related drugs. In recent years, backbone N-methylation has been a useful tool for manipulating the permeability of cyclic peptides/peptidomimetics. Backbone N-methylation permits the adjustment of molecule's conformational space. Several pathways are involved in the drug absorption pathway; the relative importance of each N-methylation to total permeation is likely to differ with intrinsic properties of cyclic peptide/peptidomimetic. Recent studies on the permeability of cyclic peptides/peptidomimetics using the backbone N-methylation strategy and synthetic methodologies will be presented in this review.

GALA peptide improves the potency of nanobody-drug conjugates by lipid-induced helix formation.

A novel nanobody-drug conjugate (NDC) was constructed by incorporating an amphipathic peptide, GALA, which improved the cytotoxicity by one to two orders of magnitude. Mechanistic studies demonstrate that tethering to lipids induces GALA to form a helix, which dramatically enhances endocytosis. Our work provides a general strategy not only for improving the anti-cancer efficacy of protein-drug conjugates but also for increasing the efficiency of other types of endocytosis-dependent cell delivery.

Asymmetric Total Syntheses of Kopsane Alkaloids via a PtCl2 -Catalyzed Intramolecular [3+2] Cycloaddition.

A concise and asymmetric total synthesis of five kopsane alkaloids that share a unique heptacyclic caged ring system was accomplished. The key transformation in the sequence involved a remarkable PtCl2 -catalyzed intramolecular [3+2] cycloaddition, which allowed for the rapid assembly of pentacyclic carbon skeletons bearing 2,3-quaternary functionalized indoline. Expeditious construction of diverse indoline scaffolds with excellent control of diastereoselectivity demonstrated the broad scope and versatility of this key transformation.

Total Synthesis of Hoiamide A Using an Evans-Tishchenko Reaction as a Key Step.

The first total synthesis of neurotoxic cyclodepsipeptide hoiamide A (1) has been accomplished. The synthesis features the use of an Evans-Tishchenko fragment coupling between a five-stereogenic-center-containing ß-hydroxyketone and a chiral aldehyde derived from threonine.

A Cell-Permeant Mimetic of NMN Activates SARM1 to Produce Cyclic ADP-Ribose and Induce Non-apoptotic Cell Death.

SARM1, an NAD-utilizing enzyme, regulates axonal degeneration. We show that CZ-48, a cell-permeant mimetic of NMN, activated SARM1 in vitro and in cellulo to cyclize NAD and produce a Ca2+ messenger, cADPR, with similar efficiency as NMN. Knockout of NMN-adenylyltransferase elevated cellular NMN and activated SARM1 to produce cADPR, confirming NMN was its endogenous activator. Determinants for the activating effects and cell permeability of CZ-48 were identified. CZ-48 activated SARM1 via a conformational change of the auto-inhibitory domain and dimerization of its catalytic domain. SARM1 catalysis was similar to CD38, despite having no sequence similarity. Both catalyzed similar set of reactions, but SARM1 had much higher NAD-cyclizing activity, making it more efficient in elevating cADPR. CZ-48 acted selectively, activating SARM1 but inhibiting CD38. In SARM1-overexpressing cells, CZ-48 elevated cADPR, depleted NAD and ATP, and induced non-apoptotic death. CZ-48 is a specific modulator of SARM1 functions in cells.

Inactivation of endothelial adenosine A2A receptors protects mice from cerebral ischaemia-induced brain injury.

BACKGROUND AND PURPOSE: Inactivation of the gene for adenosine A2A receptors (ADORA2A for humans and Adora2a for rodents) protects against brain injury in experimental stroke. However, the cell-specific pathogenic effects of A2A receptors in thromboembolic stroke and the underlying mechanisms remain undefined. Here, we tested the hypothesis that inhibition of endothelial A2A receptors after thromboembolic stroke improves post-stroke outcomes via down-regulation of inflammation. EXPERIMENTAL APPROACH: Thromboembolic stroke was induced by embolic middle cerebral artery occlusion in mice. Post-stroke outcomes were determined with neurological deficit scoring, infarct volume, inflammatory marker expression, brain leukocyte infiltration, blood-brain barrier (BBB) leakage, and oedema assessment. Anti-inflammatory effects of silencing the gene for A2A receptors or pharmacological antagonism of these receptors were assessed in vitro. KEY RESULTS: Thromboembolic stroke induced Adora2a expression in the brain. Mice globally deficient in Adora2a (Adora2a-/- ) were resistant to stroke injury. Mice specifically deficient in endothelial Adora2a (Adora2aΔVEC ) showed reduced leukocyte infiltration, BBB leakage, and oedema after stroke, along with attenuated downstream proinflammatory markers, both in vivo and in vitro. The A2A receptor antagonist, KW 6002, also reduced brain injury and inflammation after stroke. Inactivation of ADORA2A inhibited endothelial inflammation via suppression of the NLRP3 inflammasome, down-regulating cleaved caspase 1 and IL-1ß expression. CONCLUSIONS AND IMPLICATIONS: Specific inactivation of endothelial A2A receptors mitigated ischaemic brain injury and improved post-stroke outcomes, at least partly, through anti-inflammatory effects via blockade of NLRP3 inflammasome activity. Our findings may open new approaches to vascular protection after ischaemic stroke.

Solution-phase total synthesis of teixobactin.

The first solution-phase total synthesis of the cyclic depsipeptide teixobactin is described. Stereoselective construction of l-allo-enduracididine was established, and the protective groups for the peptide coupling reactions and conditions for the assembly of the fragments were also optimised. The longest linear sequence for the total synthesis was 20 steps from the known l-cis-4-hydroxyproline derivative and gave a 5.6% overall yield. This solution-phase total synthesis could serve as a complement to the current solid-phase synthesis of teixobactin.

Studies toward the Synthesis of Iriomoteolide-2a: Construction of the C(6)-C(28) Fragment.

The synthesis of an appropriately functionalized advanced C(6-28) fragment (3) of the marine macrolide iriomoteolide-2a (1) has been achieved in a highly efficient manner. The C(6)-C(18) fragment of 1 is prepared via a radical cyclization of a vinyl ether intermediate and palladium-promoted hydrostannylation/iodination. Paterson aldol reaction and Peterson olefination are used to construct the C(19)-C(28) fragment. The union of the C(6)-C(18) and C(19)-C(28) fragments is accomplished via a Suzuki-Miyaura coupling reaction.

Regio- and Stereospecific Construction of 3a-(1H-Indol-3-yl)pyrrolidinoindolines and Application to the Formal Syntheses of Gliocladins B and C.

A one-pot regio- and stereospecific strategy for the construction of 3a-(3-indolyl)-hexahydropyrrolo[2,3-b]indoles based on the condensation of an indole and an in situ generated cyclopropylazetoindoline has been developed. This unified strategy works with a variety of substituted indoles to produce 3a-(3-indolyl)hexahydropyrrolo[2,3-b]indole products in high yields. The utility of this transformation was highlighted in the formal total syntheses of gliocladins B and C.

Total syntheses of smenothiazoles A and B.

Concise total syntheses of smenothiazoles A (1) and B (2), two distinguished vinyl chloride containing natural products isolated from the marine sponge S. aurea, have been developed. Silastannation, Stille reaction and a carefully controlled desilylchlorination were employed as key steps to construct unique polyketide acid fragments, and the optimized reaction conditions avoided migration of 2,5-diene to a 2,4-conjugated system. This report unambiguously confirmed the structures of both natural products.

Total Synthesis and Stereochemical Assignment of Nostosin B.

Nostosins A and B were isolated from a hydrophilic extract of Nostoc sp. strain from Iran, which exhibits excellent tryps inhibitory activity. Nostosin A was the most potent natural tripeptide aldehyde as trypsin inhibitor up to now. Both R- and S-2-hydroxy-4-(4-hydroxy-phenyl) butanoic acid (Hhpba) were prepared and incorporated into the total synthesis of nostosin B, respectively. Careful comparison of the NMR spectra and optical rotation data of synthetic nostosin B (1a and 1b) with the natural product led to the unambiguous identification of the R-configuration of the Hhpba fragment, which was further confirmed by co-injection with the authentic sample on HPLC using both reversed phase column and the chiral AD-RH column.

Total Synthesis and Stereochemical Assignment of Actinoranone.

The total synthesis of four actinoranone stereoisomers led to unambiguous assignment of relative and absolute stereochemistry of the natural product. Key features of the convergent, fully stereocontrolled route include the use of a Negishi carbozirconation/iodination, a Friedel-Crafts cyclization, a Felkin-controlled addition reaction, a Mitsunobu reaction, and a late-stage C-H oxidation.

Concise Total Synthesis of Nannocystin†A.

Nannocystin A, a structurally unique 21-membered macrocyclic depsipeptide with low nanomolar inhibitory activity against elongation factor 1A, was synthesized according to a strategy involving the vinylogous Mukaiyama aldol reaction, Sharpless epoxidation, olefin metathesis, the Mitsunobu reaction, and a palladium-catalyzed intramolecular Suzuki coupling of a highly complex cyclization substrate. The overall synthesis is efficient and paves the way for preparation of analogues for drug development efforts.

Discovery, Total Synthesis and Key Structural Elements for the Immunosuppressive Activity of Cocosolide, a Symmetrical Glycosylated Macrolide Dimer from Marine Cyanobacteria.

A new dimeric macrolide xylopyranoside, cocosolide (1), was isolated from the marine cyanobacterium preliminarily identified as Symploca sp. from Guam. The structure was determined by a combination of NMR spectroscopy, HRMS, X-ray diffraction studies and Mosher's analysis of the base hydrolysis product. Its carbon skeleton closely resembles that of clavosolides A-D isolated from the sponge Myriastra clavosa, for which no bioactivity is known. We performed the first total synthesis of cocosolide (1) along with its [α,α]-anomer (26) and macrocyclic core (28), thus leading to the confirmation of the structure of natural 1. The convergent synthesis featured Wadsworth-Emmons cyclopropanation, Sakurai annulation, Yamaguchi macrocyclization/dimerization reaction, α-selective glycosidation and ß-selective glycosidation. Compounds 1 and 26 potently inhibited IL-2 production in both T-cell receptor dependent and independent manners. Full activity requires the presence of the sugar moiety as well as the intact dimeric structure. Cocosolide also suppressed the proliferation of anti-CD3-stimulated T-cells in a dose-dependent manner.

Total Synthesis and Stereochemical Assignment of Callyspongiolide.

Total synthesis of four callyspongiolide stereoisomers led to unambiguous assignment of relative and absolute stereochemistry of the natural product. Key features of the convergent, fully stereocontrolled route include the use of Krische allylation, Kiyooka Aldol reaction, Kociénski-Julia olefination, Still-Gennari olefination, Yamaguchi macrocyclization, and Sonogashira coupling reaction. Biological evaluation of the synthesized compounds against an array of cancer cells revealed that the stereochemistry of the macrolactone core played an important role.

The total synthesis and stereochemical assignment of scytonemin A.

The total synthesis of scytonemin A and its C-9 epimer, as well as elucidation of the absolute stereochemistry of natural scytonemin A is described.