Antiviral activity of curcumin and its analogs selected by an artificial intelligence-supported activity prediction system in SARS-CoV-2-infected VeroE6 cells.

Curcumin has been reported to exert its anti-SARS-CoV-2 activity by inhibiting the binding of spike receptor-binding domain (RBD) to angiotensin-converting enzyme-2 (ACE2). To identify more potent compounds, we evaluated the antiviral activities of curcumin and its analogs in SARS-CoV-2-infected cells. An artificial intelligence-supported activity prediction system was used to select the compounds, and 116 of the 334 curcumin analogs were proposed to have spike RBD-ACE2 binding inhibitory activity. These compounds were narrowed down to eight compounds for confirmatory studies. Six out of the eight compounds showed antiviral activity with EC50 values of less than 30 µM and binding inhibitory activity with IC20 values of less than 30 µM. Structure-activity relationship analyses revealed that the double bonds in the carbon chain connecting the two phenolic groups were essential for both activities. X-ray co-crystallography studies are needed to clarify the true binding pose and design more potent derivatives.

Magnetic Resonance Imaging Scan of the Brain After Mild COVID-19 Infection.

PURPOSE: There have been several reports of central nervous system impairments associated with severe coronavirus disease 2019 (COVID-19) infection on head magnetic resonance imaging and angiography (MRI/A). However, head MRI/A is rarely performed in mild cases, and there have been few reports on intracranial changes after COVID-19 infection in these cases. Here, we report a comparative examination of the findings seen in common head MRI/A sequences in mild cases of COVID-19. METHODS: Of the 15,376 patients who underwent head MRI/A examination called "Brain Dock" between June 2020 and June 2021, 746 patients who received a COVID-19 antibody test were evaluated. Positive and negative patients were comparatively examined for head MRI/A findings such as cerebral white matter lesions, ischemic changes, cerebral microbleeds, cerebral aneurysms, arterial stenosis, sinusitis, and other abnormal findings. RESULTS: Overall, 31 (4.2%) patients were COVID-19 positive, and all of them had mild infections not requiring hospitalization. There was no significant difference in patient characteristics and head MRI/A findings between positive and negative patients. All positive patients showed no particular abnormalities in the nasal findings such as olfactory bulb atrophy or thickening of the olfactory mucosa. CONCLUSION: Intracranial lesions in mild patients do not show a clear difference from those in negative patients. This indicates that findings seen in common MRI/A sequences of severe patients are not likely in mild patients, supporting that there is relatively no damage to the central nervous system in mild patients.

Structure-activity relationship of novel DAPK inhibitors identified by structure-based virtual screening.

Death-associated protein kinase (DAPK) is a serine/threonine protein kinase implicated in diverse programmed cell death pathways. DAPK is a promising target protein for the treatment of ischemic diseases. We identified novel potent and selective DAPK inhibitors efficiently by structure-based virtual screening, then further developed the hit compounds. In this paper, we describe the development of the hit compounds and the structure-activity relationship studies of the DAPK inhibitors in detail, including calculation of the solvated interaction energy (SIE), and verification of selectivity using a kinase panel.

Functional characterization of LePGT1, a membrane-bound prenyltransferase involved in the geranylation of p-hydroxybenzoic acid.

The AS-PT (aromatic substrate prenyltransferase) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane alpha-helices. PPTs [PHB (p-hydroxybenzoic acid) prenyltransferases] are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 [Lithospermum erythrorhizon PHB geranyltransferase] is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in the present study as a representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level. Site-directed mutagenesis of LePGT1 with a yeast expression system indicated three out of six conserved aspartate residues to be critical to the enzymatic activity. A detailed kinetic analysis of mutant enzymes revealed the amino acid residues responsible for substrate binding were also identified. Contrary to ubiquinone biosynthetic PPTs, such as UBIA in Escherichia coli which accepts many prenyl substrates of different chain lengths, LePGT1 can utilize only geranyl diphosphate as its prenyl substrate. Thus the substrate specificity was analysed using chimeric enzymes derived from LePGT1 and UBIA. In vitro and in vivo analyses of the chimeras suggested that the determinant region for this specificity was within 130 amino acids of the N-terminal. A 3D (three-dimensional) molecular model of the substrate-binding site consistent with these biochemical findings was generated.

Evaluation of docking calculations on X-ray structures using CONSENSUS-DOCK.

We are participating in the challenge of identifying active compounds for target proteins using structure-based virtual screening (SBVS). We use an in-house customized docking program, CONSENSUS-DOCK, which is a customized version of the DOCK4 program in which three scoring functions (DOCK4, FlexX and PMF) and consensus scoring have been implemented. This paper compares the docking calculation results obtained using CONSENSUS-DOCK and DOCK4, and demonstrates that CONSENSUS-DOCK produces better results than DOCK4 for major X-ray structures obtained from the Protein Data Bank (PDB).

[In-silico approaches for fragment-based drug design].

An important step to promote fragment-based drug design (FBDD) is to find high-quality fragment molecules. Therefore the design of the fragment library is the most crucial stage. In our fragment library, the main considerations are ligand efficiency (LE), diversity, and solubility with drug-like properties. We especially considered LE to raise hit probability in screening. We estimated LE of the fragment molecule based on known LE values of the active compounds. We also developed a docking program suitable for screening fragments rather than drug compounds. Furthermore, we explored fragment-linking program, which links together fragments that bind to adjacent sites on a target protein so as to promote FBDD in silico.

Crystallization and preliminary X-ray crystallographic analysis of the receptor-uncoupled mutant of Galphai1.

In order to understand the molecular mechanisms by which G-protein-coupled receptors (GPCRs) activate G proteins, the K349P mutant of Galpha(i1) (K349P), which is unable to couple to the muscarinic acetylcholine receptor, was prepared and its crystals were grown along with those of wild-type Galpha(i1) protein (WT). The two proteins were crystallized under almost identical conditions, thus enabling a detailed structural comparison. The crystallization conditions performed well irrespective of the identity of the bound nucleotide (GDP or GTPgammaS) and the crystals diffracted to resolutions of 2.2 A (WT.GDP), 2.8 A (WT.GTPgammaS), 2.6 A (K349P.GDP) and 3.2 A (K349P.GTPgammaS).

Importance of an N-terminal extension in ribonuclease HII from Bacillus stearothermophilus for substrate binding.

The gene encoding ribonuclease HII from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The overproduced protein, Bst-RNase HII, was purified and biochemically characterized. Bst-RNase HII, which consists of 259 amino acid residues, showed the highest amino acid sequence identity (50.2%) to Bacillus subtilis RNase HII. Like B. subtilis RNase HII, it exhibited Mn2+-dependent RNase H activity. It was, however, more thermostable than B. subtilis RNase HII. When the Bst-RNase HII amino acid sequence is compared with that of Thermococcus kodakaraensis RNase HII, to which it shows 29.8% identity, 30 residues are observed to be truncated from the C-terminus and there is an extension of 71 residues at the N-terminus. The C-terminal truncation results in the loss of the alpha9 helix, which is rich in basic amino acid residues and is therefore important for substrate binding. A truncated protein, Delta59-Bst-RNase HII, in which most of the N-terminal extension was removed, completely lost its RNase H activity. Surface plasmon resonance analysis indicated that this truncated protein did not bind to the substrate. These results suggest that the N-terminal extension of Bst-RNase HII is important for substrate binding. Because B. subtilis RNase HII has an N-terminal extension of the same length and these extensions contain a region in which basic amino acid residues are clustered, the Bacillus enzymes may represent a novel type of RNase H which possesses a substrate-binding domain at the N-terminus.

Identification of a New Series of STAT3 Inhibitors by Virtual Screening.

The signal transducer and activator of transcription 3 (STAT3) is considered to be an attractive therapeutic target for oncology drug development. We identified a N-[2-(1,3,4-oxadiazolyl)]-4-quinolinecarboxamide derivative, STX-0119, as a novel STAT3 dimerization inhibitor by a virtual screen using a customized version of the DOCK4 program with the crystal structure of STAT3. In addition, we used in vitro cell-based assays such as the luciferase reporter gene assay and the fluorescence resonance energy transfer-based STAT3 dimerization assay. STX-0119 selectively abrogated the DNA binding activity of STAT3 and suppressed the expression of STAT3-regulated oncoproteins such as c-myc and survivin in cancer cells. In contrast, a truncated inactive analogue, STX-0872, did not exhibit those activities. Oral administration of STX-0119 effectively abrogated the growth of human lymphoma cells in a SCC-3 subcutaneous xenograft model without visible toxicity. Structure-activity relationships of STX-0119 derivatives were investigated using the docking model of the STAT3-SH2 domain/STX-0119.