Discovery of a Novel Thienopyrimidine Compound as a Urate Transporter 1 and Glucose Transporter 9 Dual Inhibitor with Improved Efficacy and Favorable Druggability.

Gout and hyperuricemia are metabolic diseases characterized with high serum uric acid (SUA) levels that significantly impact human health. Lesinurad, a uricosuric agent, is limited to concurrent use with xanthine oxidase inhibitors (XOIs) in clinical practice due to its restricted efficacy and potential nephrotoxicity. Herein, extensive structural modifications of lesinurad were conducted through scaffold hopping and substituent modification strategies, affording 54 novel derivatives containing pyrimidine-fused cyclic structures. Notably, the thienopyrimidine compound 29 demonstrated a remarkable 2-fold increase in SUA-lowering in vivo activity compared to lesinurad, while exhibiting potent inhibitory activity against the urate transporter 1 (URAT1, IC50 = 2.01 µM) and glucose transporter 9 (GLUT9, IC50 = 18.21 µM). Furthermore, it possessed a lower effective dosage of 0.5 mg/kg, favorable safety profile without any apparent acute toxicity at doses of 1000 mg/kg, and improved pharmacokinetic properties. Overall, we have discovered an efficacious URAT1/GLUT9 dual inhibitor for inhibiting urate reabsorption with favorable pharmacokinetic profiles.

Anatomical Prior and Inter-Slice Consistency for Semi-Supervised Vertebral Structure Detection in 3D Ultrasound Volume.

Three-dimensional (3D) ultrasound imaging technique has been applied for scoliosis assessment, but the current assessment method only uses coronal projection images and cannot illustrate the 3D deformity and vertebra rotation. The vertebra detection is essential to reveal 3D spine information, but the detection task is challenging due to complex data and limited annotations. We propose VertMatch to detect vertebral structures in 3D ultrasound volume containing a detector and classifier. The detector network finds the potential positions of structures on transverse slice globally, and then the local patches are cropped based on detected positions. The classifier is used to distinguish whether the patches contain real vertebral structures and screen the predicted positions from the detector. VertMatch utilizes unlabeled data in a semi-supervised manner, and we develop two novel techniques for semi-supervised learning: 1) anatomical prior is used to acquire high-quality pseudo labels; 2) inter-slice consistency is used to utilize more unlabeled data by inputting multiple adjacent slices. Experimental results demonstrate that VertMatch can detect vertebra accurately in ultrasound volume and outperforms state-of-the-art methods. Moreover, VertMatch is also validated in automatic spinous process angle measurement on forty subjects with scoliosis, and the results illustrate that it can be a promising approach for the 3D assessment of scoliosis.

Genome-wide analysis of the TIFY family and function of CaTIFY7 and CaTIFY10b under cold stress in pepper (Capsicum annuum L.).

TIFY [TIF(F/Y)XG] proteins are a plant particular transcription factor family that regulates plant stress responses. Therefore, to fill this gap, we investigated CaTIFY genes in pepper. Gene structure and conserved motifs of the pepper TIFY gene family were systematically analyzed using sequence alignment analysis, Cis-acting element analysis, transcriptomic data, and RT-qPCR analysis, and their expression patterns were further analyzed using Virus-Induced Gene Silencing (VIGS) and cold stress reactive oxygen species (ROS) response. We identified 16 CaTIFY genes in pepper, which were dispersed among seven subgroups (JAZI, JAZII, JAZIII, PPD, TIFY, and ZIM/ZML). Several CaTIFY members had stress-related harmonic-responsive elements, and four (CaTIFY7, CaTIFY10b, CaTIFY1b, and CaTIFY6b) had low-temperature-responsive elements. Transcriptomic data and RT-qPCR analysis revealed that the TIFY genes in pepper displayed different expression patterns in the roots, stems, leaves, flower fruits, and seeds. In particular, CaTIFY7 was highly expressed in young leaves, and CaTIFY10b was highly expressed in roots. CaTIFYs participated in the regulation of several different abiotic stresses and CaTIFY7 and CaTIFY10b were significantly induced by cold stress. Additionally, Virus-Induced Gene Silencing (targeting CaTIFY7 and CaTIFY10b) resulted in plants that were sensitive to cold stress. Conversely, overexpression of CaTIFY7 and CaTIFY10b enhanced plant cold tolerance by promoting the expression of genes related to cold stress and the ROS response. CaTIFY7 and CaTIFY10b interacted with themselves and CaTIFY7 also interacted with CaTIFY10b in the yeast two-hybrid (Y2H) system. Our data provide a basis for further analysis of the role of pepper TIFY genes in cold-stress responses in the future.

Genome-Wide Identification and Expression Analysis of the SWEET Gene Family in Capsicum annuum L.

Sugars will eventually be exported transporters (SWEETs) are a novel class of sugar transport proteins that play a crucial role in plant growth, development, and response to stress. However, there is a lack of systematic research on SWEETs in Capsicum annuum L. In this study, 33 CaSWEET genes were identified through bioinformatics analysis. The Ka/Ks analysis indicated that SWEET genes are highly conserved not only among peppers but also among Solanaceae species and have experienced strong purifying selection during evolution. The Cis-elements analysis showed that the light-responsive element, abscisic-acid-responsive element, jasmonic-acid-responsive element, and anaerobic-induction-responsive element are widely distributed in the promoter regions of CaSWEETs. The expression pattern analysis revealed that CaSWEETs exhibit tissue specificity and are widely involved in pepper growth, development, and stress responses. The post-transcription regulation analysis revealed that 20 pepper miRNAs target and regulate 16 CaSWEETs through cleavage and translation inhibition mechanisms. The pathogen inoculation assay showed that CaSWEET16 and CaSWEET22 function as susceptibility genes, as the overexpression of these genes promotes the colonization of pathogens, whereas CaSWEET31 functions as a resistance gene. In conclusion, through systematic identification and characteristic analysis, a comprehensive understanding of CaSWEET was obtained, which lays the foundation for further studies on the biological functions of SWEET genes.

Identification of Ebselen derivatives as novel SARS-CoV-2 main protease inhibitors: Design, synthesis, biological evaluation, and structure-activity relationships exploration.

The main protease (Mpro) represents one of the most effective and attractive targets for designing anti-SARS-CoV-2 drugs. In this study, we designed and synthesized a novel series of Ebselen derivatives by incorporating privileged fragments from different pockets of the Mpro active site. Among these compounds, 11 compounds showed submicromolar activity in the FRET-based SARS-CoV-2 Mpro inhibition assay, with IC50 values ranging from 233 nM to 550 nM. Notably, compound 3a displayed submicromolar Mpro activity (IC50 = 364 nM) and low micromolar antiviral activity (EC50 = 8.01 µM), comparable to that of Ebselen (IC50 = 339 nM, EC50 = 3.78 µM). Time-dependent inhibition assay confirmed that these compounds acted as covalent inhibitors. Taken together, our optimization campaigns thoroughly explored the structural diversity of Ebselen and verified the impact of specific modifications on potency against Mpro.

Design, Synthesis, and Biological Evaluation of Trisubstituted Piperazine Derivatives as Noncovalent Severe Acute Respiratory Syndrome Coronavirus 2 Main Protease Inhibitors with Improved Antiviral Activity and Favorable Druggability.

The ongoing transmission of SARS-CoV-2 necessitates the development of additional potent antiviral agents capable of combating the current highly infectious variants and future coronaviruses. Here, we present the discovery of potent nonpeptide main protease (Mpro) inhibitors with prominent antiviral activity and improved pharmacokinetic properties. Three series of 1,2,4-trisubstituted piperazine derivatives were designed and synthesized, and the optimal GC-78-HCl demonstrated high enzyme-inhibitory potency (IC50 = 0.19 µM) and exhibited excellent antiviral activity (EC50 = 0.40 µM), reaching the same level as Nirmatrelvir (EC50 = 0.38 µM). Additionally, GC-78-HCl displayed potent antiviral activities against various SARS-CoV-2 variants as well as HCoV-OC43 and HCoV-229E, indicating its potential broad-spectrum anticoronaviral activity. Notably, the pharmacokinetic properties of GC-78-HCl were somewhat enhanced compared to those of the lead compound. Furthermore, the cocrystal and molecular docking elucidated the mechanism of action. In conclusion, we discovered a novel nonpeptidic Mpro inhibitor with promising antiviral activity and a favorable pharmacokinetic profile.

Identification of novel 1,2,3-triazole isatin derivatives as potent SARS-CoV-2 3CLpro inhibitors via click-chemistry-based rapid screening.

SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) is considered an attractive target for the development of anti-COVID-19 agents due to its vital function. The N-substituted isatin derivative L-26 is a potential SARS-CoV-2 3CLpro inhibitor, but it has poor cell-based antiviral activity and high cytotoxicity. With L-26 as the lead compound, 58 isatin derivatives were prepared using click-chemistry-based miniaturized synthesis and their 3CLpro inhibitory activities were determined by a fluorescence resonance energy transfer-based enzymatic assay. Compounds D1N8 (IC50 = 0.44 ± 0.12 µM) and D1N52 (IC50 = 0.53 ± 0.21 µM) displayed excellent inhibitory potency against SARS-CoV-2 3CLpro, being equivalent to that of L-26 (IC50 = 0.30 ± 0.14 µM). In addition, the cytotoxicity of D1N8 (CC50 >20 µM) and D1N52 (CC50 >20 µM) decreased significantly compared with L-26 (CC50 <2.6 µM). Further molecular dynamics simulations revealed the potential binding interactions between D1N52 and SARS-CoV-2 3CLpro. These efforts lay a solid foundation for the research of novel anti-SARS-CoV-2 agents targeting 3CLpro.

Discovery of novel sulfonamide substituted indolylarylsulfones as potent HIV-1 inhibitors with better safety profiles.

Indolylarylsulfones (IASs) are classical HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a unique scaffold and possess potent antiviral activity. To address the high cytotoxicity and improve safety profiles of IASs, we introduced various sulfonamide groups linked by alkyl diamine chain to explore the entrance channel of non-nucleoside inhibitors binding pocket. 48 compounds were designed and synthesized to evaluate their anti-HIV-1 activities and reverse transcriptase inhibition activities. Especially, compound R10L4 was endowed with significant inhibitory activity towards wild-type HIV-1 (EC50(WT) = 0.007 µmol/L, SI = 30,930) as well as a panel of single-mutant strains exemplified by L100I (EC50 = 0.017 µmol/L, SI = 13,055), E138K (EC50 = 0.017 µmol/L, SI = 13,123) and Y181C (EC50 = 0.045 µmol/L, SI = 4753) which were superior to Nevirapine and Etravirine. Notably, R10L4 was characterized with significantly reduced cytotoxicity (CC50 = 216.51 µmol/L) and showed no remarkable in vivo toxic effects (acute and subacute toxicity). Moreover, the computer-based docking study was also employed to characterize the binding mode between R10L4 and HIV-1 RT. Additionally, R10L4 presented an acceptable pharmacokinetic profile. Collectively, these results deliver precious insights for next optimization and indicate that the sulfonamide IAS derivatives are promising NNRTIs for further development.

Identification of CaPs locus involving in purple stripe formation on unripe fruit, reveals allelic variation and alternative splicing of R2R3-MYB transcription factor in pepper (Capsicum annuum L.).

The purple color of unripe pepper fruit is attributed to the accumulation of anthocyanins. Only a few genes controlling the biosynthesis and regulation of anthocyanins have been cloned in Capsicum. In this study, we performed a bulked segregant analysis of the purple striped trait using an F2 population derived from a cross between the immature purple striped fruit line Chen12-4-1-1-1-1 and the normal green fruit line Zhongxian101-M-F9. We mapped the CaPs locus to an 841.39 kb region between markers M-CA690-Xba and MCA710-03 on chromosome 10. CA10g11690 encodes an R2R3-MYB transcription factor that is involved in the biosynthesis of anthocyanins as the best candidate gene. Overexpression and silencing in transformed tobacco (Nicotiana tabacum) lines indicated that CA10g11690 is involved in the formation of purple stripes in the exocarp. A comparison of parental sequences identified an insertion fragment of 1,926 bp in the second intron region of Chen12-4, and eight SNPs were detected between the two parents. Additionally, there were 49 single nucleotide polymorphic variations, two sequence deletions, and four sequence insertions in the promoter region. We found that CA10g11690 undergoes alternative splicing and generates different transcripts. Thus, the functional transcript of CA10g11690 appeared to be primarily involved in the development of purple phenotype in the exocarp. Our data provide new insight into the mechanism of anthocyanin biosynthesis and a theoretical basis for the future breeding of purple striped pepper varieties.

Silencing of Pepper CaFtsH1 or CaFtsH8 Genes Alters Normal Leaf Development.

Filamentation temperature-sensitive H (FtsH) is a proteolytic enzyme that plays an important role in plant photomorphogenesis and stress resistance. However, information regarding the FtsH family genes in pepper is limited. In our research, through genome-wide identification, 18 members of the pepper FtsH family (including five FtsHi members) were identified and renamed based on phylogenetic analysis. CaFtsH1 and CaFtsH8 were found to be essential for pepper chloroplast development and photosynthesis because FtsH5 and FtsH2 were lost in Solanaceae diploids. We found that the CaFtsH1 and CaFtsH8 proteins were located in the chloroplasts and specifically expressed in pepper green tissues. Meanwhile, CaFtsH1 and CaFtsH8-silenced plants created by virus-induced gene silencing exhibited albino leaf phenotypes. In addition, CaFtsH1-silenced plants were observed to contain very few dysplastic chloroplasts and lost the capacity for photoautotrophic growth. Transcriptome analysis revealed that the expression of chloroplast-related genes such as those coding the photosynthesis-antenna protein and structural proteins was downregulated in CaFtsH1-silenced plants, resulting in the inability to form normal chloroplasts. This study improves our understanding of pepper chloroplast formation and photosynthesis through the identification and functional study of CaFtsH genes.

Covalently Targeted Highly Conserved Tyr318 to Improve the Drug Resistance Profiles of HIV-1 NNRTIs: A Proof-of-Concept Study.

This study presents proof of concept for designing a novel HIV-1 covalent inhibitor targeting the highly conserved Tyr318 in the HIV-1 non-nucleoside reverse transcriptase inhibitors binding pocket to improve the drug resistance profiles. The target inhibitor ZA-2 with a fluorosulfate warhead in the structure was found to be a potent inhibitor (EC50 = 11-246 nM) against HIV-1 IIIB and a panel of NNRTIs-resistant strains, being far superior to those of NVP and EFV. Moreover, ZA-2 was demonstrated with lower cytotoxicity (CC50 = 125 µM). In the reverse transcriptase inhibitory assay, ZA-2 exhibited an IC50 value of 0.057 µM with the ELISA method, and the MALDI-TOF MS data demonstrated the covalent binding mode of ZA-2 with the enzyme. Additionally, the molecular simulations have also demonstrated that compounds can form covalent binding to the Tyr318.

Structure-Based Optimization of 2,4,5-Trisubstituted Pyrimidines as Potent HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors: Exploiting the Tolerant Regions of the Non-Nucleoside Reverse Transcriptase Inhibitors' Binding Pocket.

Although non-nucleoside reverse transcriptase inhibitors (NNRTIs) exhibit potent anti-HIV-1 activity and play an important role in the active antiretroviral therapy of AIDS, the emergence of drug-resistant strains has seriously reduced their clinical efficacy. Here, we report a series of 2,4,5-trisubstituted pyrimidines as potent HIV-1 NNRTIs by exploiting the tolerant regions of the NNRTI binding pocket. Compounds 16b and 16c were demonstrated to have excellent activity (EC50 = 3.14-22.1 nM) against wild-type and a panel of mutant HIV-1 strains, being much superior to that of etravirine (EC50 = 3.53-52.2 nM). Molecular modeling studies were performed to illustrate the detailed interactions between RT and 16b, which shed light on the improvement of the drug resistance profiles. Moreover, 16b possessed favorable pharmacokinetic (T1/2 = 1.33 h, F = 31.8%) and safety profiles (LD50 > 2000 mg/kg), making it a promising anti-HIV-1 drug candidate for further development.

Discovery and Crystallographic Studies of Nonpeptidic Piperazine Derivatives as Covalent SARS-CoV-2 Main Protease Inhibitors.

The spread of SARS-CoV-2 keeps threatening human life and health, and small-molecule antivirals are in demand. The main protease (Mpro) is an effective and highly conserved target for anti-SARS-CoV-2 drug design. Herein, we report the discovery of potent covalent non-peptide-derived Mpro inhibitors. A series of covalent compounds with a piperazine scaffold containing different warheads were designed and synthesized. Among them, GD-9 was identified as the most potent compound with a significant enzymatic inhibition of Mpro (IC50 = 0.18 µM) and good antiviral potency against SARS-CoV-2 (EC50 = 2.64 µM), similar to that of remdesivir (EC50 = 2.27 µM). Additionally, GD-9 presented favorable target selectivity for SARS-CoV-2 Mpro versus human cysteine proteases. The X-ray co-crystal structure confirmed our original design concept showing that GD-9 covalently binds to the active site of Mpro. Our nonpeptidic covalent inhibitors provide a basis for the future development of more efficient COVID-19 therapeutics.

DeepPROTACs is a deep learning-based targeted degradation predictor for PROTACs.

The rational design of PROTACs is difficult due to their obscure structure-activity relationship. This study introduces a deep neural network model - DeepPROTACs to help design potent PROTACs molecules. It can predict the degradation capacity of a proposed PROTAC molecule based on structures of given target protein and E3 ligase. The experimental dataset is mainly collected from PROTAC-DB and appropriately labeled according to the DC50 and Dmax values. In the model of DeepPROTACs, the ligands as well as the ligand binding pockets are generated and represented with graphs and fed into Graph Convolutional Networks for feature extraction. While SMILES representations of linkers are fed into a Bidirectional Long Short-Term Memory layer to generate the features. Experiments show that DeepPROTACs model achieves 77.95% average prediction accuracy and 0.8470 area under receiver operating characteristic curve on the test set. DeepPROTACs is available online at a web server ( https://bailab.siais.shanghaitech.edu.cn/services/deepprotacs/ ) and at github ( https://github.com/fenglei104/DeepPROTACs ).

Design, synthesis and activity evaluation of novel lesinurad analogues containing thienopyrimidinone or pyridine substructure as human urate transporter 1 inhibitors.

Urate Transporter 1 (URAT1) plays a crucial role in uric acid transport, making it an attractive target for the treatment of gout and hyperuricemia. As a representative URAT1 inhibitor, Lesinurad treat gout by promoting the uric acid excretion. However, its lower in vitro and in vivo activity should be highly attracted attention. Herein, the bioisosterism, molecular hybridization and scaffold hopping strategies were exploited to modify all the structural components of Lesinurad and finally thirty novel compounds bearing thienopyrimidinone or pyridine core were obtained. Most of the compounds displayed certain URAT1 inhibitory activity in vitro. Among them, thienopyrimidinones 6 (IC50 = 7.68 µM), 10 (IC50 = 7.56 µM), 14 (IC50 = 7.31 µM) and 15 (IC50 = 7.90 µM) showed slightly better potency than positive control Lesinurad (IC50 = 9.38 µM). Notably, 10 also displayed inhibitory activity (IC50 = 55.96 µM) against GLUT9. Additionally, in vivo serum uric acid (SUA)-lowering experiments were performed on some representative compounds and it was revealed that all the selected compounds could decrease the SUA level in mice, of which the decrease rate of SUA was 73.29% for the most promising compound 10, significantly greater than that of Lesinurad (26.89%). Meanwhile, the preliminary SARs based on the URAT1 inhibitory activity were discussed in detail, which pointed out the direction for further structural optimization. Overall, the thienopyrimidinone and pyridine are prospective skeletons for the developing novel URAT1 inhibitors with considerable potential for optimization.

Discovery and Crystallographic Studies of Trisubstituted Piperazine Derivatives as Non-Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity and Low Toxicity.

The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (Mpro) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide Mpro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits Mpro with high potency (IC50 = 0.40 µM) and displays excellent antiviral activity (EC50 = 1.1 µM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC50 > 100 µM) and excellent target selectivity for SARS-CoV-2 Mpro (IC50 > 50 µM for cathepsins B, F, K, L, and caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.

Design, synthesis, and biological evaluation of novel double-winged galloyl derivatives as HIV-1 RNase H inhibitors.

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains as the only enzyme encoded within the viral genome not clinically validated as an antiviral target. We have previously reported that the galloyl derivative II-25 had RNase H inhibitory activity in enzymatic assays but showed weak antiviral activity in phenotypic assays due its large polarity and poor membrane permeability. In this report, we report on a series of II-25 derivatives, obtained by addition of different hydrophobic moieties ("the wings") at the C-2 and C-3 positions of the piperazine ring that showed improved RNase H inhibitory activity. Six compounds showed strong inhibitory activity and were found to be more potent than ß-thujaplicinol in enzymatic assays. The most potent compound was IA-6 and exhibited the best inhibitory activity (IC50 = 0.067 ± 0.02 µM). IA-6 was around 11 and 30 times more potent than II-25 and ß-thujaplicinol, respectively. Molecular modeling studies predict a strong hydrophobic interaction between the furylmethylaminyl group of IA-6 and the side chain of His539, explaining the potent HIV-1 RNase H inhibition. Unfortunately, none of the derivatives showed significant antiviral activity in cell culture. It is worth emphasizing that most of the obtained compounds show low cytotoxicity (CC50 > 20 µM), which confirms the significance of identifying galloyl derivatives as valuable leads for further optimization.

Chemical space exploration around indolylarylsulfone scaffold led to a novel class of highly active HIV-1 NNRTIs with spiro structural features.

To thoroughly investigate the uncharted chemical space around the entrance channel of HIV-1 reverse transcriptase (RT) and to improve the physicochemical properties, we introduced different spiro ring structures with high Fsp3 values as linkers at indole-2-carboxamide, attaching to various terminal substituents to enhance the interactions with the entrance channel. All the newly designed and synthesized indolylarylsulfone (IAS) derivatives exhibited moderate to excellent potency against wild-type HIV-1 with EC50 values ranging from 0.0053 to 0.19 µM. Among them, compounds SO-7g (EC50 = 0.0053 µM) and SO-7h (EC50 = 0.009 µM, SI > 21552) were identified as the most two potent compounds, which displayed 30- and 16-fold improvement than nevirapine and zidovudine and comparable potency to efavirenz and etravirine. Moreover, SO-7g maintained the promising activity against a variety of mutant strains, especially for L100I (EC50 = 0.047 µM), K103 N (EC50 = 0.056 µM), and E138K (EC50 = 0.040 µM). Notably, the introduction of spiro rings could effectively reduce the cytotoxicity (CC50) and greatly improve the selectivity index compared to lead compound, exemplified by SO-7h (CC50 > 214.4 µM, SI > 21552) and SO-7a (CC50 > 233.2 µM, SI > 20933). Additionally, the preliminary SARs based on antiviral activity and molecular simulation perspective were analyzed with a detailed description, which could point out the direction for further structural optimization.