Abstract Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the clinical importance of 12-LOX, the exact mechanisms by which it affects platelet activation are not fully understood, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryo-electron microscopy to determine high-resolution structures (1.7-2.8 Å) of human 12-LOX. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may affect its catalytic activity and membrane association. We also identified different conformations within the 12-LOX dimer, which likely represent different time points in its catalytic cycle. Furthermore, we identified small molecules bound to 12-LOX. The active site of the 12-LOX tetramer was occupied by an endogenous 12-LOX inhibitor, a long-chain acyl coenzyme A. In addition, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase 1 clinical trial for the treatment of heparin-induced thrombocytopenia and received a fast-track designation by the Food and Drug Administration. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, their catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme. 

Cryo-EM structures of human arachidonate 12S-lipoxygenase bound to endogenous and exogenous inhibitors

Abstract Circular RNAs are an unusual class of single-stranded RNAs whose ends are covalently linked via back-splicing. Due to their versatility, the need to express circular RNAs in vivo and in vitro has increased. Efforts have been made to efficiently and precisely synthesize circular RNAs. However, a review on the optimization of the processes of circular RNA design, synthesis, and delivery is lacking. Our review highlights the multifaceted aspects considered when producing optimal circular RNAs and summarizes the available options for each step of exogenous circular RNA design and synthesis, including circularization strategies. Additionally, this review describes several potential applications of circular RNAs. 

pdf/optimal-design-of-synthetic-circular-rnas-q3808vt8js.pdf

Optimal design of synthetic circular RNAs

Human 12-lipoxygenase (12-LOX) is an enzyme involved in platelet activation and is a promising target for antiplatelet therapies. Despite the clinical importance of 12-LOX, the exact mechanisms of how it affects platelet activation are unclear, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryoelectron microscopy to determine the high-resolution structures (1.7 Å - 2.8 Å) of human 12-LOX for the first time. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may impact its catalytic activity and membrane association. We also identified different conformations within a 12-LOX dimer, likely representing different time points in its catalytic cycle. Furthermore, we were able to identify small molecules bound to the 12-LOX structures. The active site of the 12-LOX tetramer is occupied by an endogenous 12-LOX inhibitor, a long-chain acyl-Coenzyme A. Additionally, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase I clinical trial for treating heparin-induced thrombocytopenia and has received fast-track designation by the FDA. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, its catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme. Key Points The first full-length structures of human arachidonate 12S-Lipoxygenase (12-LOX) Reveals mechanisms of oligomeric and conformational states Uncovers natural inhibitor of 12S-Lipoxygenase (12-lox) Reveals a binding site of inhibitor ML355

/pdf/cryo-em-structures-of-human-arachidonate-12s-lipoxygenase-12-23cci9xa.pdf

Cryo-EM structures of human arachidonate 12S-Lipoxygenase (12-LOX) bound to endogenous and exogenous inhibitors

The X-ray crystal structures of soybean lipoxygenase (LOX) and rabbit 15-LOX were reported in the 1990s. Subsequent 3D structures demonstrated a conserved U-like shape of the substrate cavities as reviewed here. The 8-LOX:arachidonic acid (AA) complex showed AA bound to the substrate cavity carboxylate-out with C10 at 3.4 Å from the iron metal center. A recent cryo-electron microscopy (EM) analysis of the 12-LOX:AA complex illustrated AA in the same position as in the 8-LOX:AA complex. The 15- and 12-LOX complexes with isoenzyme-specific inhibitors/substrate mimics confirmed the U-fold. 5-LOX oxidizes AA to leukotriene A4, the first step in biosynthesis of mediators of asthma. The X-ray structure showed that the entrance to the substrate cavity was closed to AA by Phe and Tyr residues of a partly unfolded α2-helix. Recent X-ray analysis revealed that soaking with inhibitors shifted the short α2-helix to a long and continuous, which opened the substrate cavity. The α2-helix also adopted two conformations in 15-LOX. 12-LOX dimers consisted of one closed and one open subunit with an elongated α2-helix. 13C-ENDOR-MD computations of the 9-MnLOX:linoleate complex showed carboxylate-out position with C11 placed 3.4 ± 0.1 Å from the catalytic water. 3D structures have provided a solid ground for future research. 

Thirty years with three-dimensional structures of lipoxygenases

Abstract We developed phosphorylation reagents with a nitrobenzyl hydrophobic tag and used them for 5′-phosphorylation of chemically or transcriptionally synthesized RNA. The capability of hydrophobic tags to synthesize 5′-monophosphorylated RNA was evaluated based on the yield of the desired oligonucleotides, stability of protecting groups during cleavage/deprotection, separation ability in reverse-phase HPLC (RP-HPLC), and deprotection efficiency after RP-HPLC purification. The results showed that a nitrobenzyl derivative with a tert-butyl group at the benzyl position was most suitable for RNA 5′-phosphorylation. Using the developed phosphorylation reagent, we chemically synthesized 5′-phosphorylated RNA and confirmed that it could be purified by RP-HPLC and the following deprotection. In addition, we demonstrated complete chemical synthesis of minimal mRNA by chemical capping of 5′-monophosphorylated RNA. Ribonucleoside 5′-monophosphates with hydrophobic protecting groups have also been developed and used as substrates to transcriptionally synthesize 5′-phosphorylated RNA with &amp;gt;1000 bases. From the mixture of the by-products and the desired RNA, only 5′-monophosphorylated RNA could be effectively isolated by RP-HPLC. Furthermore, monophosphorylated RNA can be converted into circular mRNA via RNA ligase-mediated cyclization. Circular mRNA expression of nanoluciferase in cultured cells and mice. These techniques are important for the production of chemically synthesized mRNA and circular mRNA. 

Development of hydrophobic tag purifying monophosphorylated RNA for chemical synthesis of capped mRNA and enzymatic synthesis of circular mRNA

Classic strategies for circular RNA (circRNA) preparation always introduce large numbers of linear transcripts or extra nucleotides to the circularized product. In this study, we aimed to develop an efficient system for circRNA preparation based on a self-splicing ribozyme derived from an optimized Tetrahymena thermophila group Ⅰ intron. The target RNA sequence was inserted downstream of the ribozyme and a complementary antisense region was added upstream of the ribozyme to assist cyclization. Then, we compared the circularization efficiency of ribozyme or flanking intronic complementary sequence (ICS)-mediated methods through the DNMT1, CDR1as, FOXO3, and HIPK3 genes and found that the efficiency of our system was remarkably higher than that of flanking ICS-mediated method. Consequently, the circularized products mediated by ribozyme are not introduced with additional nucleotides. Meanwhile, the overexpressed circFOXO3 maintained its biological functions in regulating cell proliferation, migration, and apoptosis. Finally, a ribozyme-based circular mRNA expression system was demonstrated with a split green fluorescent protein (GFP) using an optimized Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) sequence, and this system achieved successful translation of circularized mRNA. Therefore, this novel, convenient, and rapid engineering RNA circularization system can be applied for the functional study and large-scale preparation of circular RNA in the future.

/pdf/a-precise-and-efficient-circular-rna-synthesis-system-based-4bm81egh.pdf

A precise and efficient circular RNA synthesis system based on a ribozyme derived from Tetrahymena thermophila.

http://www.jbc.org/article/S0021925822007244/pdf

Helical remodeling augments 5-lipoxygenase activity in the synthesis of proinflammatory mediators

High-resolution structural-omics of human liver enzymes.

Abstract 15-prostaglandin dehydrogenase (15-PGDH) is a negative regulator of tissue stem cells that acts via enzymatic activity of oxidizing and degrading PGE2, and related eicosanoids, that support stem cells during tissue repair. Indeed, inhibiting 15-PGDH markedly accelerates tissue repair in multiple organs. Here we have used cryo-electron microscopy to solve the solution structure of native 15-PGDH and of 15-PGDH individually complexed with two distinct chemical inhibitors. These structures identify key 15-PGDH residues that mediate binding to both classes of inhibitors. Moreover, we identify a dynamic 15-PGDH lid domain that closes around the inhibitors, and that is likely fundamental to the physiologic 15-PGDH enzymatic mechanism. We furthermore identify two key residues, F185 and Y217, that act as hinges to regulate lid closing, and which both inhibitors exploit to capture the lid in the closed conformation, thus explaining their sub-nanomolar binding affinities. These findings provide the basis for further development of 15-PGDH targeted drugs as therapeutics for regenerative medicine. 

/pdf/small-molecule-inhibitors-of-15-pgdh-exploit-a-physiologic-1gs964my.pdf

Small molecule inhibitors of 15-PGDH exploit a physiologic induced-fit closing system

Activation of Glucagon-like peptide-1 receptor (GLP-1R) by peptides has been proven clinically to be a very effective treatment approach for type 2 diabetes (T2D) and obesity. Here we describe a small molecule GLP-1R full agonist RGT-075 identified through Computer Accelerated Rational Design (CARD) (Ma et al., Cell Research 2020) with nM potency in G-protein coupled cAMP signaling. RGT-075 displayed much reduced activity in receptor-mediated β-arrestin recruitment and subsequent internalization. It was shown to be selective against other class B G protein-coupled receptors (GPCRs) and was only active for human and monkey GLP-1R. It demonstrated favorable oral bioavailability and pharmacodynamic profile for once-daily oral dosing in monkeys. In food-induced glucose intolerant and prediabetic cynomolgus monkeys, oral administration of RGT-075 improved glucose tolerance to a level comparable to injectable liraglutide. In food-induced type 2 diabetic monkeys, once-daily oral administration of RGT-075 reduced food intake and fasting plasma glucose levels. Together, these data support the development and advancement of RGT-075 into the clinic as a potential therapeutic for T2D and obesity.
 
 
 F.Liu: Employee; Regor Pharmaceuticals Inc. X.Sun: Employee; AutoDrug Biotech Co. Ltd., Qilu Regor Therapeutics Inc. W.Huang: None. W.Guo: None. J.Lin: None. W.Zhong: None.


753-P: RGT-075, an Orally Efficacious Small Molecule GLP-1R Full Agonist in Cynomolgus Monkey Models

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