Structural insight into an anti-BRIL Fab as a G-protein-coupled receptor crystallization chaperone.

Structure determination of G-protein-coupled receptors (GPCRs) is key for the successful development of efficient drugs targeting GPCRs. BRIL is a thermostabilized apocytochrome b562 (with M7W/H102I/R106L mutations) from Escherichia coli and is often used as a GPCR fusion protein for expression and crystallization. SRP2070Fab, an anti-BRIL antibody Fab fragment, has been reported to facilitate and enhance the crystallization of BRIL-fused GPCRs as a crystallization chaperone. This study was conducted to characterize the high-resolution crystal structure of the BRIL-SRP2070Fab complex. The structure of the BRIL-SRP2070Fab complex was determined at 2.1 Šresolution. This high-resolution structure elucidates the binding interaction between BRIL and SRP2070Fab. When binding to BRIL, SRP2070Fab recognizes conformational epitopes, not linear epitopes, on the surface of BRIL helices III and IV, thereby binding perpendicularly to the helices, which indicates stable binding. Additionally, the packing contacts of the BRIL-SRP2070Fab co-crystal are largely due to SRP2070Fab rather than BRIL. The accumulation of SRP2070Fab molecules by stacking is remarkable and is consistent with the finding that stacking of SRP2070Fab is predominant in known crystal structures of BRIL-fused GPCRs complexed with SRP2070Fab. These findings clarified the mechanism of SRP2070Fab as a crystallization chaperone. Moreover, these data will be useful in the structure-based drug design of membrane-protein drug targets.

In Vitro Pharmacological Profile of KW-6356, a Novel Adenosine A2A Receptor Antagonist/Inverse Agonist.

KW-6356 is a novel adenosine A2A (A2A) receptor antagonist/inverse agonist, and its efficacy as monotherapy in Parkinson's disease (PD) patients has been reported. Istradefylline is a first-generation A2A receptor antagonist approved for use as adjunct treatment to levodopa/decarboxylase inhibitor in adult PD patients experiencing "OFF" episodes. In this study, we investigated the in vitro pharmacological profile of KW-6356 as an A2A receptor antagonist/inverse agonist and the mode of antagonism and compared them with istradefylline. In addition, we determined cocrystal structures of A2A receptor in complex with KW-6356 and istradefylline to explore the structural basis of the antagonistic properties of KW-6356. Pharmacological studies have shown that KW-6356 is a potent and selective ligand for the A2A receptor (the -log of inhibition constant = 9.93 ± 0.01 for human receptor) with a very low dissociation rate from the receptor (the dissociation kinetic rate constant = 0.016 ± 0.006 minute-1 for human receptor). In particular, in vitro functional studies indicated that KW-6356 exhibits insurmountable antagonism and inverse agonism, whereas istradefylline exhibits surmountable antagonism. Crystallography of KW-6356- and istradefylline-bound A2A receptor have indicated that interactions with His2506.52 and Trp2466.48 are essential for the inverse agonism, whereas the interactions at both deep inside the orthosteric pocket and the pocket lid stabilizing the extracellular loop conformation may contribute to the insurmountable antagonism of KW-6356. These profiles may reflect important differences in vivo and help predict better clinical performance. SIGNIFICANCE STATEMENT: KW-6356 is a potent and selective adenosine A2A receptor antagonist/inverse agonist and exhibits insurmountable antagonism, whereas istradefylline, a first-generation adenosine A2A receptor antagonist, exhibits surmountable antagonism. Structural studies of adenosine A2A receptor in complex with KW-6356 and istradefylline explain the characteristic differences in the pharmacological properties of KW-6356 and istradefylline.

The dual pocket binding novel tankyrase inhibitor K-476 enhances the efficacy of immune checkpoint inhibitor by attracting CD8+ T cells to tumors.

The Wnt/ß-catenin pathway, which is associated with disease progression, is activated in many cancers. Tankyrase (TNKS) has received attention as a target molecule for Wnt/ß-catenin pathway inhibition. We identified K-476, a novel TNKS inhibitor, a dual pocket binder that binds to both the nicotinamide and ADP-ribose pockets. In a human colon cancer cell line, K-476 specifically and potently inhibited TNKS and led to stabilization of the Axin protein, resulting in Wnt/ß-catenin pathway suppression. Aberrant Wnt/ß-catenin pathway activation was recently reported as a possible mechanism of ineffectiveness in immune checkpoint inhibitor (ICI) treatment. Because the Wnt/ß-catenin pathway activation causes dendritic cell inactivation and suppresses chemokine production, resulting in a paucity of CD8+ T cells in tumor tissue, which is an important effector of ICIs. Thus, TNKS inhibitors may enhance the efficacy of ICIs. To examine whether K-476 enhances the antitumor effect of anti-PD-L1 antibodies, K-476 was administered orally with an anti-PD-L1 antibody to melanoma-bearing C57BL/6J mice. Although K-476 was ineffective as a monotherapy, it significantly enhanced the antitumor effect in combination with anti-PD-L1 antibody. In mice, intra-tumor infiltration of CD8+ T cells was increased by combination treatment. K-476 upregulated the chemokine expression (e.g., Ccl3 and Ccl4), which attracted CD8+ T cells. This was considered to contribute to the increased CD8+ T cells in the tumor microenvironment. Furthermore, while the potential gastrointestinal toxicity of TNKS inhibitors has been reported, it was not observed at effective doses. Thus, K-476 could be an attractive therapeutic option to enhance the efficacy of ICIs.

siRNA potency enhancement via chemical modifications of nucleotide bases at the 5'-end of the siRNA guide strand.

Small interfering RNAs (siRNAs) can be utilized not only as functional biological research tools but also as therapeutic agents. For the clinical use of siRNA as drugs, various chemical modifications have been used to improve the activity of siRNA drugs, and further chemical modifications are expected to improve the utility of siRNA therapeutics. As the 5' nucleobase of the guide strand affects the interaction between an siRNA and AGO2 and target cleavage activity, structural optimization of this specific position may be a useful strategy for improving siRNA activity. Here, using the in silico model of the complex between human AGO2 MID domain and nucleoside monophosphates, we screened and synthesized an original adenine-derived analog, 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), that fits better than the natural nucleotide bases into the MID domain of AGO2. Introduction of the 6-mCEPh-purine analog at the 5'-end of the siRNA guide strand significantly enhanced target knockdown activity in both cultured cell lines and in vivo animal models. Our findings can help expand strategies for rationally optimizing siRNA activity via chemical modifications of nucleotide bases.

Structure-guided screening strategy combining surface plasmon resonance with nuclear magnetic resonance for identification of small-molecule Argonaute 2 inhibitors.

Argonaute (AGO) proteins are the key component of the RNA interference machinery that suppresses gene expression by forming an RNA-induced silencing complex (RISC) with microRNAs (miRNAs). Each miRNA is involved in various cellular processes, such as development, differentiation, tumorigenesis, and viral infection. Thus, molecules that regulate miRNA function are expected to have therapeutic potential. In addition, the biogenesis of miRNA is a multistep process involving various proteins, although the complete pathway remains to be elucidated. Therefore, identification of molecules that can specifically modulate each step will help understand the mechanism of gene suppression. To date, several AGO2 inhibitors have been identified. However, these molecules were identified through a single screening method, and no studies have specifically evaluated a combinatorial strategy. Here, we demonstrated a combinatorial screening (SCR) approach comprising an in silico molecular docking study, surface plasmon resonance (SPR) analysis, and nuclear magnetic resonance (NMR) analysis, focusing on the strong binding between the 5'-terminal phosphate of RNA and the AGO2 middle (MID) domain. By combining SPR and NMR, we identified binding modes of amino acid residues binding to AGO2. First, using a large chemical library (over 6,000,000 compounds), 171 compounds with acidic functional groups were screened using in silico SCR. Next, we constructed an SPR inhibition system that could analyze only the 5'-terminal binding site of RNA, and nine molecules that strongly bound to the AGO2 MID domain were selected. Finally, using NMR, three molecules that bound to the desired site were identified. The RISC inhibitory ability of the "hit" compounds was analyzed in human cell lysate, and all three hit compounds strongly inhibited the binding between double-stranded RNA and AGO2.

The discovery of a new antibody for BRIL-fused GPCR structure determination.

G-protein-coupled receptors (GPCRs)-the largest family of cell-surface membrane proteins-mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them.

Intestinal Clostridium species lower host susceptibility to enterohemorrhagic Escherichia coli O157:H7 infection.

Susceptibility to enterohemorrhagic Escherichia coli (EHEC) infection varies among humans. The intestinal microbiota seems to play an essential role in host defense against EHEC; thus, we hypothesized that indigenous bacteria, such as Clostridium ramosum and Clostridium perfringens, could influence the susceptibility to EHEC infection. To evaluate the effect of indigenous bacteria on EHEC infection, germ-free mice were precolonized with each indigenous bacterium, and then infected with EHEC O157:H7. Precolonization with C. ramosum or C. perfringens completely prevented death from EHEC infection througout a test period. Precolonization with C. ramosum also reduced the level of secreted Shiga toxin (Stx) 2 and prevented histopathological changes in the kidneys in a similar way to precolonization with Bifidobacterium longum, which is used as a model for preventing EHEC infection. In contrast, the mice precolonized with C. perfringens showed mild renal injuries. When evaluated using an in vitro co-culturing system, again C. ramosum inhibited the growth and Stx production of EHEC more potently than C. perfringens. These results indicate that C. ramosum and C. perfringens suppressed EHEC infection; however, the extent of their preventive effects differed. Therefore, the susceptibility to EHEC infection and its severity can depend on the functional bacteria present in the intestinal microbiota of individuals.

A bovine myeloid antimicrobial peptide (BMAP-28) kills methicillin-resistant Staphylococcus aureus but promotes adherence of the bacteria.

The cathelicidin family is one of the several families of antimicrobial peptides (AMPs). A bovine myeloid antimicrobial peptide (BMAP-28) belongs to this family. Recently, the emergence of drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) has become a big problem. AMPs are expected to be leading compounds of new antibiotics against drug-resistant bacteria. In this study, we focused on the activity of BMAP-28 against bacterial cell surfaces. First, we observed morphological change of MRSA caused by BMAP-28 using a scanning probe microscope. We also studied activities of BMAP-28 against adherence of S. aureus to fibronectin, collagen type I, collagen type IV. We confirmed whether BMAP-28 can bind to lipoteichoic acid (LTA) of S. aureus. BMAP-28 was indicated as damaging the cell surface of MRSA. In a particular range of concentrations, BMAP-28 promoted adherence of S. aureus against fibronectin and collagens. It was revealed that BMAP-28 and LTA of S. aureus bound with each other. Our study showed the potential of BMAP-28 which can damage MRSA and interact with LTA of S. aureus but promote its adherence in some concentrations. This study provides new points of which to take notice when we use AMPs as medicines.

Susceptibility difference between methicillin-susceptible and methicillin-resistant Staphylococcus aureus to a bovine myeloid antimicrobial peptide (BMAP-28).

A bovine myeloid antimicrobial peptide antimicrobial peptide (BMAP-28) is a member of the cathelicidin family and acts as a component of innate immunity. There are few reports of susceptibility difference of methicillin-resistant Staphylococcus aureus (MRSA) and susceptible strains (MSSA) against BMAP-28. This study aims to clarify how a few amino acid substitutions of BMAP-28 are related to its antimicrobial activity using four analog peptides of BMAP-28. We also compared cellular fatty acid components of MSSA and MRSA using gas chromatography. We found that a few amino acid substitutions of BMAP-28 do not change antimicrobial activity. It was also revealed that the percentage of cis-11-eicosenoic acid in total detected fatty acids of MRSA was significantly higher than that of MSSA. In addition, the percentage of palmitic acid in total detected fatty acids of MRSA tended to be lower than that of MSSA. Our results will provide new information to deal with the question of differences in bacterial susceptibility against BMAP-28.

Two sources of mitochondrial NADPH in the yeast Saccharomyces cerevisiae.

Cells of the yeast Saccharomyces cerevisiae contain three NAD kinases; namely, cytosolic Utr1p, cytosolic Yef1p, and mitochondrial Pos5p. Previously, the NADH kinase reaction catalyzed by Pos5p, rather than the NAD kinase reaction followed by the NADP(+)-dependent dehydrogenase reaction, had been regarded as a critical source of mitochondrial NADPH, which plays vital roles in various mitochondrial functions. This study demonstrates that the mitochondrial NADH kinase reaction is dispensable as a source of mitochondrial NADPH and emphasizes the importance of the NAD kinase reaction, followed by the mitochondrial NADP(+)-dependent dehydrogenase reaction. Of the potential dehydrogenases (malic enzyme, Mae1p; isocitrate dehydrogenase, Idp1p; and acetaldehyde dehydrogenases, Ald4/5p), evidence is presented that acetaldehyde dehydrogenases, and in particular Ald4p, play a prominent role in generating mitochondrial NADPH in the absence of the NADH kinase reaction. The physiological significance of the mitochondrial NADH kinase reaction in the absence of Ald4p is also demonstrated. In addition, Pos5p is confirmed to have a considerably higher NADH kinase activity than NAD kinase activity. Taking these results together, it is proposed that there are two sources of mitochondrial NADPH in yeast: one is the mitochondrial Pos5p-NADH kinase reaction and the other is the mitochondrial Pos5p-NAD kinase reaction followed by the mitochondrial NADP(+)-dependent acetaldehyde dehydrogenase reaction.