Inactive Domain Research Articles

In-silico molecular docking, ADME study, and molecular dynamic simulation of new azetidin-2-one derivatives with antiproliferative activity

Over the past two decades, protein kinase has been a heavily studied target in the development of new anti-proliferative medications. Heterocyclic systems have been identified as the preferred scaffold because of their wide range of biological properties. In this research, the objective was to design and develop fifteen novel azetidin-2-one derivatives and assess their cytotoxic potential as inhibitors of the epidermal growth factor receptor, which is considered one of the key factors influencing cell growth and proliferation. The crystal structure of inactive EGFR tyrosine kinase domain ligand erlotinib from protein data bank was retrieved in order to be docked with our proposed azetidine-2-one derivatives to evaluate their activity as anti-proliferative agents. In this article, an in-silico molecular docking approach proposes that these azetidine-2-one derivatives have satisfactory binding contact with the erlotinib binding site. Although, three compounds have been identified as the most powerful as they have PLP fitness scores of (77.79, 76.68 and 71.46), respectively, while the reference ligand’s fitness score was (71.94). Additionally, all of our derivatives have satisfied the Swiss-ADME parameters, indicating that they may be orally active compounds. In conclusion, two compounds (A-2 and A-8) have better PLP fitness, and one (A-14) has a comparable score in comparison to the reference ligand, at the active site of epidermal growth factor receptor. indicating that the novel azetidine-2-one derivatives have shown interesting results and could be used as model compounds to create novel anti-proliferative drugs. However, more pharmacological evaluation is needed.

A eukaryotic-like ubiquitination system in bacterial antiviral defence.

Ubiquitination pathways have crucial roles in protein homeostasis, signalling and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2 and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6, but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here we demonstrate that a bacterial operon associated with phage defence islands encodes a complete ubiquitination pathway. Two structures of a bacterial E1-E2-Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 possesses an amino-terminal inactive adenylation domain and a carboxy-terminal active adenylation domain with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C terminus positioned for adenylation, and a second structure mimics an E1-to-E2 transthioesterification state with the E1 CYS domain adjacent to the bound E2. We show that a deubiquitinase in the same pathway preprocesses the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.

Selectively superior production of docosahexaenoic acid in Schizochytrium sp. through engineering the fatty acid biosynthetic pathways

BackgroundSchizochytrium sp. is commercially used for production of docosahexaenoic acid (DHA). Schizochytrium sp. utilizes the polyketide synthase complex (PKS) and a single type I fatty acid synthase (FAS) to synthesize polyunsaturated fatty acids and saturated fatty acids, respectively. The acyl carrier protein (ACP) domains of FAS or PKS are used to load acyl groups during fatty acids biosynthesis. Phosphopantetheinyl transferase (PPTase) transfers the pantetheine moiety from Coenzyme A to the conserved serine residue of an inactive ACP domain to produce its active form.ResultsIn this study, in order to improve production and content of DHA, we decreased the expression of fas, strengthened the expression of the PKS pathway, and enhanced the supply of active ACP in Schizochytrium sp. ATCC20888. Weakening the expression of fas or disruption of orfA both led to growth defect and reduction of lipid yields in the resulting strains WFAS and DPKSA, indicating that both FAS and PKS were indispensable for growth and lipid accumulation. Although WFAS had a higher DHA content in total fatty acids than the wild-type strain (WT), its growth defect and low DHA yield hinders its use for DHA production. Overexpression of the orfAB, orfC, orfC-DH (truncated orfC), or ppt promoted DHA and lipid production, respectively. The yields and contents of DHA were further increased by combined overexpression of these genes. Highest values of DHA yield (7.2 g/L) and DHA content (40.6%) were achieved in a recombinant OPKSABC-PPT, ⁓56.5% and 15.3% higher than the WT values, respectively.ConclusionsThis study demonstrates that genetic engineering of the fatty acid biosynthetic pathways provides a new strategy to enhance DHA production in Schizochytrium.

Deciphering the functional role of clinical mutations in ABCB1, ABCC1, and ABCG2 ABC transporters in endometrial cancer.

ATP-binding cassette transporters represent a superfamily of dynamic membrane-based proteins with diverse yet common functions such as use of ATP hydrolysis to efflux substrates across cellular membranes. Three major transporters-P-glycoprotein (P-gp or ABCB1), multidrug resistance protein 1 (MRP1 or ABCC1), and breast cancer resistance protein (BCRP or ABCG2) are notoriously involved in therapy resistance in cancer patients. Despite exhaustive individual characterizations of each of these transporters, there is a lack of understanding in terms of the functional role of mutations in substrate binding and efflux, leading to drug resistance. We analyzed clinical variations reported in endometrial cancers for these transporters. For ABCB1, the majority of key mutations were present in the membrane-facing region, followed by the drug transport channel and ATP-binding regions. Similarly, for ABCG2, the majority of key mutations were located in the membrane-facing region, followed by the ATP-binding region and drug transport channel, thus highlighting the importance of membrane-mediated drug recruitment and efflux in ABCB1 and ABCG2. On the other hand, for ABCC1, the majority of key mutations were present in the inactive nucleotide-binding domain, followed by the drug transport channel and membrane-facing regions, highlighting the importance of the inactive nucleotide-binding domain in facilitating indirect drug efflux in ABCC1. The identified key mutations in endometrial cancer and mapped common mutations present across different types of cancers in ABCB1, ABCC1, and ABCG2 will facilitate the design and discovery of inhibitors targeting unexplored structural regions of these transporters and re-engineering of these transporters to tackle chemoresistance.

Abstract LB210: Identification of histone deacetylase 10 as a repressor of cancer cell migration

Abstract Histone deacetylases (HDACs) are a family of 18 enzymes with the potential to be epigenetic modifiers. Of the 4 classes of HDACs, HDAC6 and HDAC10 belong to the class IIb subgroup. Unlike HDAC6, HDAC10 contains two deacetylase-like domains, with one active deacetylase domain and one inactive leucine-rich domain, and a preference for a specific acetylated polyamine over other substrates. Aberrant expression of HDAC10 has been implicated in cancer and other diseases. However, the deacetylation targets and biological functions of HDAC10 have not been well characterized due to a lack of specific and potent HDAC10 inhibitors with cellular activity. We previously established CRISPR/Cas9-mediated HDAC10 knockout (KO) cell lines in HCT116 and HeLa S3 cells. In the current study, we used the HCT116 wildtype and KO pair for bulk RNA sequencing, differential gene expression, and gene set enrichment analyses (GSEA), which revealed an upregulation of gene markers and pathways potentially associated with metastasis and invasion in the HDAC10-KO cells, including S100A14, LAMA4, and TGF-beta. As the role of HDAC10 expression on cell migration and invasion has not been previously reported, we performed cell migration assays using our HDAC10 parental and knockout cell line pairs. In both HCT116 and HeLa systems, HDAC10 KO cells exhibited dramatically higher rates of cell migration in Boyden chamber and scratch assays. These same assays were performed in HDAC10-expressing cells in the presence of the HDAC10-specific inhibitor DKFZ-728, which similarly caused a dose-dependent increase in migration, strongly supporting a catalytic role for HDAC10 in repressing migration. In addition to these results, we present data investigating the role of HDAC10 in cell migration/invasion in representative prostate cancer cell lines (LNCAP and LAPC4), as the prostate tumor microenvironment is particularly rich in polyamines, including N 8-acetylated spermidine, the substrate of HDAC10. Finally, using our panel of HDAC10-KO cell lines and inhibitors, we analyze the contribution of commonly perturbed genes to the migratory phenotype with the goal of defining the precise mechanism of action. Overall, our studies provide functional evidence that loss of HDAC10 can increase cell migration, and thus, may lead to increased metastatic potential. These results emphasize the need for isoform-specific inhibitors and may have important clinical implications, as commonly utilized HDAC inhibitors, including vorinostat (SAHA), tubastatin A, and trichostatin A (TSA), also inhibit HDAC10. Our ongoing studies include incorporating HDAC10 proteolysis targeting chimeras (PROTACs) into our functional assays to examine differences between HDAC10 inhibition and PROTAC-mediated degradation, providing further rationale for modulating HDAC10 activity as an antineoplastic strategy. Citation Format: Ting-Ann Liu, Ashley Nwafor, Rajendra Kumar, Laura Sena, Raphael R. Steimbach, Aubry K. Miller, Robert A. Casero, Tracy Murray Stewart. Identification of histone deacetylase 10 as a repressor of cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB210.

Binding of GTP to BifA is required for the production of Pel-dependent biofilms in Pseudomonas aeruginosa.

The Pel exopolysaccharide is one of the most mechanistically conserved and phylogenetically diverse bacterial biofilm matrix determinants. Pel is a major contributor to the structural integrity of Pseudomonas aeruginosa biofilms, and its biosynthesis is regulated by the binding of cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP) to the PelD receptor. c-di-GMP is synthesized from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases with GGDEF domains and degraded by phosphodiesterases with EAL or HD-GYP domains. As the P. aeruginosa genome encodes 43 c-di-GMP metabolic enzymes, one way signaling specificity can be achieved is through direct interaction between specific enzyme-receptor pairs. Here, we show that the inner membrane hybrid GGDEF-EAL enzyme, BifA, directly interacts with PelD via its cytoplasmic HAMP, GGDEF, and EAL domains. Despite having no catalytic function, the degenerate active site motif of the BifA GGDEF domain (GGDQF) has retained the ability to bind GTP with micromolar affinity. Mutations that abolish GTP binding result in increased biofilm formation but stable global c-di-GMP levels. Our data suggest that BifA forms a dimer in solution and that GTP binding induces conformational changes in dimeric BifA that enhance the BifA-PelD interaction and stimulate its phosphodiesterase activity, thus reducing c-di-GMP levels and downregulating Pel biosynthesis. Structural comparisons between the dimeric AlphaFold2 model of BifA and the structures of other hybrid GGDEF-EAL proteins suggest that the regulation of BifA by GTP may occur through a novel mechanism.IMPORTANCEc-di-GMP is the most common cyclic dinucleotide used by bacteria to regulate phenotypes such as motility, biofilm formation, virulence factor production, cell cycle progression, and cell differentiation. While the identification and initial characterization of c-di-GMP metabolic enzymes are well established, our understanding of how these enzymes are regulated to provide signaling specificity remains understudied. Here we demonstrate that the inactive GGDEF domain of BifA binds GTP and regulates the adjacent phosphodiesterase EAL domain, ultimately downregulating Pel-dependent P. aeruginosa biofilm formation through an interaction with PelD. This discovery adds to the growing body of literature regarding how hybrid GGDEF-EAL enzymes are regulated and provides additional precedence for studying how direct interactions between c-di-GMP metabolic enzymes and effectors result in signaling specificity.

In silico validation of allosteric inhibitors targeting Zika virus NS2B-NS3 protease.

The Zika virus (ZIKV), a member of the Flaviviridae family, poses a major threat to human health because of the lack of effective antiviral drugs. Although the NS2B-NS3 protease of ZIKV (NS2B-NS3pro) is regarded as a major target for antiviral inhibitors, viral mutations can lead to ineffective competitive inhibitors. Allosteric inhibitors bind to highly conserved nonprotease active sites, induce conformational changes in the protease active site, and prevent substrate binding. Currently, no molecular simulation techniques are available for accurately predicting and analysing conformational changes in the protease catalytic domain. In this study, we developed a combined approach that involves blind docking, Gaussian accelerated molecular dynamics, two-dimensional potential of mean force profiling, density functional theory (DFT) calculations, and interaction region indicator (IRI) analysis and employed it to examine the allosteric inhibitor-01 molecule and its interaction with ZIKV NS2B-NS3pro. Our results indicated that the binding of inhibitor-01 to NS2B-NS3pro resulted in two major conformational states, state I and state II, which in turn changed the volume of the protease active site from 1014 Å3 to 710 and 820 Å3, respectively. These two states had an inactive catalytic domain (residues His116, Asp140, and Ser200). DFT and IRI analyses revealed that, in state I, Lys138 and Gln139 formed hydrogen bonds with inhibitor-01, whereas Lys138, Leu214, Asn217, Val220, and Ile221 engaged in van der Waals interactions with inhibitor-01. Advancements in computational techniques and power are expected to facilitate further progress in overcoming challenges associated with designing allosteric inhibitors for viral proteases.

TLR3 agonism augments CD47 inhibition in acute myeloid leukemia.

CD47-SIRPa is a myeloid check point pathway that promotes phagocytosis of cells lacking markers for self-recognition. Tumor cells can overexpress CD47 and bind to SIRPa on macrophages, preventing phagocytosis. CD47 expression is enhanced and correlated with a negative prognosis in acute myeloid leukemia (AML), with its blockade leading to cell clearance. ALX90 is an engineered fusion protein with high affinity for CD47. Composed of the N-terminal D1 domain of SIRPα genetically linked to an inactive Fc domain from human immunoglobulin (Ig) G, ALX90 is designed to avoid potential toxicity of CD47-expressing red blood cells. Venetoclax (VEN) is a specific B-cell lymphoma-2 (BCL-2) inhibitor that can restore apoptosis in malignant cells. In AML, VEN is combined with azanucleosides to induce superior remission rates, however treatment for refractory/relapse is an unmet need. We questioned whether the anti-tumor activity of a VENbased regimen can be augmented through CD47 inhibition (CD47i) in AML and how this triplet may be enhanced. Human AML cell lines were sensitive to ALX90 and its addition increased efficacy of a VEN plus azacitidin (VEN+AZA) regimen in vivo. However, CD47i failed to clear bone marrow tumor burden in PDX models. We hypothesized that the loss of resident macrophages in the bone marrow in AML reduced efficiency of CD47i. Therefore, we attempted to enhance this medullary macrophage population with agonism of TLR3 via polyinosinic:polycytidylic acid (poly(I:C)), which led to expansion and activation of medullary macrophages in in vivo AML PDX models and potentiated CD47i. In summary, the addition of poly(I:C) can enhance medullary macrophage populations to potentiate the phagocytosis merited by therapeutic inhibition of CD47.

Deubiquitinase USP39 promotes SARS-CoV-2 replication by deubiquitinating and stabilizing the envelope protein

The SARS-CoV-2 envelope (E) protein is highly conserved among different viral variants and important for viral assembly and production. Our recent study found that the E protein is ubiquitinated and degraded by the E3 ligase RNF5 through the proteasome pathway. However, whether E ubiquitination can be reversed by host deubiquitinase has not yet been determined. Here, we identify by mass spectrum analysis that the deubiquitinases USP14 and USP39 specifically interact with E, while USP39 potently reverses E polyubiquitination. USP39 interacts with E via the arginine-rich motif (AR) and deubiquitinates E polyubiquitination via the inactive ubiquitin-specific protease domain. Therefore, USP39 protects E from RNF5-mediated degradation, resulting in the enhancement of E stability and E-induced cytokine storms. Moreover, loss-and-gain assays demonstrated that USP39 promotes the replication of various SARS-CoV-2 strains by stabilizing protein level of E that can be ubiquitinated but not other viral proteins. Our findings provide useful targets for the development of novel anti-SARS-CoV-2 strategies.

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo.

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.

DipM controls multiple autolysins and mediates a regulatory feedback loop promoting cell constriction in Caulobacter crescentus

Proteins with a catalytically inactive LytM-type endopeptidase domain are important regulators of cell wall-degrading enzymes in bacteria. Here, we study their representative DipM, a factor promoting cell division in Caulobacter crescentus. We show that the LytM domain of DipM interacts with multiple autolysins, including the soluble lytic transglycosylases SdpA and SdpB, the amidase AmiC and the putative carboxypeptidase CrbA, and stimulates the activities of SdpA and AmiC. Its crystal structure reveals a conserved groove, which is predicted to represent the docking site for autolysins by modeling studies. Mutations in this groove indeed abolish the function of DipM in vivo and its interaction with AmiC and SdpA in vitro. Notably, DipM and its targets SdpA and SdpB stimulate each other’s recruitment to midcell, establishing a self-reinforcing cycle that gradually increases autolytic activity as cytokinesis progresses. DipM thus coordinates different peptidoglycan-remodeling pathways to ensure proper cell constriction and daughter cell separation.

Bifurcate evolution of quinone synthetases in basidiomycetes

BackgroundThe terphenylquinones represent an ecologically remarkable class of basidiomycete natural products as they serve as central precursors of pigments and compounds that impact on microbial consortia by modulating bacterial biofilms and motility. This study addressed the phylogenetic origin of the quinone synthetases that assemble the key terphenylquinones polyporic acid and atromentin.ResultsThe activity of the Hapalopilus rutilans synthetases HapA1, HapA2 and of Psilocybe cubensis PpaA1 were reconstituted in Aspergilli. Liquid chromatography and mass spectrometry of the culture extracts identified all three enzymes as polyporic acid synthetases. PpaA1 is unique in that it features a C-terminal, yet catalytically inactive dioxygenase domain. Combined with bioinformatics to reconstruct the phylogeny, our results demonstrate that basidiomycete polyporic acid and atromentin synthetases evolved independently, although they share an identical catalytic mechanism and release structurally very closely related products. A targeted amino acid replacement in the substrate binding pocket of the adenylation domains resulted in bifunctional synthetases producing both polyporic acid and atromentin.ConclusionsOur results imply that quinone synthetases evolved twice independently in basidiomycetes, depending on the aromatic α-keto acid substrate. Furthermore, key amino acid residues for substrate specificity were identified and changed which led to a relaxed substrate profile. Therefore, our work lays the foundation for future targeted enzyme engineering.

A unique AA5 alcohol oxidase fused with a catalytically inactive CE3 domain from the bacterium Burkholderia pseudomallei.

Copper radical oxidases (CROs) are redox enzymes able to oxidize alcohols or aldehydes, while only requiring a single copper atom as cofactor. Studied CROs are found in one of two subfamilies within the Auxiliary Activities family 5 (AA5) in the carbohydrate-active enzymes database. We here characterize an AA5 enzyme outside the subfamily classification from the opportunistic bacterial pathogen Burkholderia pseudomallei, which curiously was fused to a carbohydrate esterase family 3 domain. The enzyme was shown to be a promiscuous primary alcohol oxidase, with an activity profile similar to enzymes from subfamily 2. The esterase domain was inactive on all tested substrates, and structural predictions revealed this being an effect of crippling substitutions in the expected active site residues.

Targeting the NKG2A axis with a TCR mimic antibody containing an active Fc domain promotes anti-tumor immunity

Abstract The NKG2A axis is an emerging immune checkpoint. Blocking antibodies to the NKG2A receptor in combination with cancer vaccines and PD-(L)1 antibodies have been found to enhance anti-tumor immunity in murine models and cancer patients. The ligand for the NKG2A receptor is the non-classical major histocompatibility (MHC) class Ib molecule, Qa-1 bin mice (HLA-E in humans), loaded with leader peptides from classical MHC class Ia molecules to form Qa-1 b/Qdm peptide complexes. Here, we explore the therapeutic value of targeting the NKG2A ligand, not done previously, using our recently described single-domain T-cell receptor mimic (TCRm) antibody, EXX-1. Two syngeneic murine tumor models (A20 and CT26) were used to evaluate the therapeutic potential of EXX-1 and its mode(s) of action. The initial study done showed EXX-1 Fc active (mIgG2a) displayed optimal tumor growth inhibition and prolonged survival time in mice compared to a modified form of EXX-1 containing an inactive Fc domain. These findings suggested that in addition to blocking the NKG2A pathway, EXX-1 Fc active antibody likely induces tumor cell death via antibody dependent cellular cytotoxicity (ADCC). Moreover, mice treated with EXX-1 antibody as single agent therapy showed significantly better survival outcomes compared to the cohort of mice treated with blocking antibody to the NKG2A receptor. This implies that there might be potential benefits of targeting the NKG2a ligand over the NKG2A receptor though this will require further investigation. Additionally, combination therapy using EXX-1 Fc active and PD-(L)1 blocking antibodies revealed markedly improved rates of tumor-free mice compared to mice receiving treatment with EXX-1 Fc active or anti-PD-(L)1 antibody alone.

Abstract 12528: Variants in Solute Cation Transporters 3 Associate with High Lp(a) Levels Independent of Apo(a) Isoform Size

Introduction: Lipoprotein (a) [Lp(a)] consists of a low-density lipoprotein (LDL) molecule attached to apolipoprotein(a) [apo(a)]. Apo(a) comprises two kringle domains (KIV and KV) derived from the plasminogen gene and an inactive protease domain. The KIV domain is subdivided into 10 subtypes (KIV1-10), with KIV2 occurring as a copy number variation (CNV) that varies among individuals. Elevated Lp(a) levels are a major risk factor for CVDs and are inversely correlated to the KIV2 CNV determining apo(a) isoform size. Objectives: This study aimed to identify variants associated with a high Lp(a) phenotype that were not linked to apo(a) isoform size. Methods: Illumina sequencing of 13 candidate genes was performed on a discovery set comprising 54 high Lp(a) European samples (>50mg/dL). The sequenced genes included LPA, adjacent genes, and those coding for receptors involved in Lp(a) uptake. Variants commonly found in high samples were genotyped in the Otago LPA European cohort (n=589) and their association with Lp(a) levels was checked via Kruskal-Wallis and linear regression statistical models. The associations were then replicated in the much bigger German Chronic Kidney Disease (GCKD) cohort (n=4930). Results: Nine variants were significantly associated with high Lp(a) levels. Two variants were in the LPA gene (rs3124784 and rs3798220) and seven were in neighbouring genes (rs2282143 in SLC22A1, rs3127592, rs3127593, rs3103353 and rs3127594 in SLC22A2 and rs1810126 and rs3088442 in SLC22A3). Interestingly, the minor allele of all nine variants was significantly associated with increased LPA expression in liver tissues in the GTEx database. Seven of the variants were significantly associated with small apo(a) isoform size (P=1x10-4 to 2.9x10-18), while rs1810126 and rs3088442 in SLC22A3 were not associated with the small isoform size (P=0.43 and 0.42). Conclusion: Nine variants in the LPA and nearby genes were significantly associated with elevated Lp(a) levels and increased LPA expression, with the majority being linked to a short apo(a) isoform. The association of two SLC22A3 gene variants with Lp(a) levels independent of apo(a) isoform size warrants further investigation to establish any functional connection between LPA and this gene.

Deucravacitinib: First Approval.

Deucravacitinib (SOTYKTU™) is a first-in-class, highly selective, oral tyrosine kinase 2 (TYK2) inhibitor. It acts via an allosteric mechanism, binding to the catalytically inactive pseudokinase regulatory domain of TYK2 and stabilizing an inhibitory interaction between the regulatory and catalytic domains. Deucravacitinib is being developed by Bristol Myers Squibb for the treatment of multiple immune-mediated diseases, including psoriasis, psoriatic arthritis, lupus and inflammatory bowel disease. It received its first approval (in the USA on 9September 2022) for the treatment of adults with moderate-to-severe plaque psoriasis who are candidates for systemic therapy or phototherapy. On 26 September 2022, it was subsequently approved in Japan for the treatment of plaque psoriasis, generalized pustular psoriasis and erythrodermic psoriasis. The Marketing Authorisation Application for deucravacitinib for the treatment of adults with moderate to severe plaque psoriasis has been validated in the EU, and clinical development of the drug for the treatment of multiple immune-mediated diseases is underway in numerous countries worldwide. This article summarizes the milestones in the development of deucravacitinib leading to this first approval for the treatment of adults with moderate-to-severe plaque psoriasis who are candidates for systemic therapy or phototherapy.

Mining of gene clusters for biosynthesis of secondary metabolites and analysis of genes encoding antibiotic resistance and virulence in 4 644 representative human gut strains

Genome sequences of 4 644 representative strains from human gut microbiota were analyzed to mine gene clusters for biosynthesis of novel secondary metabolites, as well as genes encoding antibiotic resistance and virulence factors. AntiSMASH analysis showed that more than 60% of the representative strains encoded at least one secondary metabolite gene cluster, and 8 potential novel secondary metabolite gene clusters were identified from 8 unculturable bacteria. The secondary metabolite gene clusters in human intestine are mainly composed of nonribosomal peptide synthetase (NRPS), bacteriocin, arylpolyene, terpene, betalactone and NRPS like gene clusters distributed in Clostridia, Bacilli, Gammaproteobacteria, Bacteroidia, Actinobacteria and Negativicutes. PathoFact analysis showed that genes encoding antibiotic resistance and virulence factors are widely distributed in representative strains, but the frequency encoded by potential pathogens is significantly higher than that of non-potential pathogens. The frequency of genes encoding secretory toxins such as outer membrane protein, PapC N-terminal domain, PapC C-terminal domain, peptidase M16 inactive domain, and non-secretory toxins such as nitroreductase family, AcrB/AcrD/AcrF family, PLD-like domain, Cupin domain, putative hemolysin, S24-like peptidase, phosphotransferase enzyme family, endonuclease/ exonuclease/ phosphatase family, glyoxalase/ bleomycin resistance was high in potential pathogens. This study may facilitate mining new microbial natural products from the intestinal microbiome, understanding the colonization and infection mechanism of intestinal microorganisms, and providing targeted prevention and treatment of intestinal microbial related diseases.

Investigation on conformational variation and enzymatic activity of trypsin affected by Ti3C2 QDs via spectroscopic technique and molecular modeling

In this paper, Ti3C2 quantum dots (Ti3C2 QDs) were synthesized by simply treating Ti3C2 MXene powder with acid and base via hydrothermal method. Ti3C2 QDs exhibited superior fluorescence property and were used for the fluorescent imaging of living HeLa cells successfully. In order to evaluate the influence of Ti3C2 QDs on protease with specific biological functions, binding interaction of Ti3C2 QDs with trypsin was studied comprehensively and deeply through spectroscopic strategies and molecular modeling technique. The intrinsic fluorescence of trypsin was spontaneously quenched by Ti3C2 QDs through static quenching mode under van der Waals interaction force, and Ti3C2 QDs bound with the inactive residue domain of trypsin firmly with stoichiometric ratio of 1:1. Ti3C2 QDs induced the microenvironmental variation of the amino acid residues in trypsin, reducing the thermal stability of trypsin significantly. Gel electrophoresis experiments and microscopic imaging experiments demonstrated that Ti3C2 QDs inhibited the enzymatic activity of trypsin on the digestion of human serum albumin and HeLa cells obviously. These results revealed not only the deep interaction mechanism between Ti3C2 QDs and protease but also the influence of Ti3C2 QDs on the enzymatic activity of trypsin, paving the way for the safe biological application of Ti3C2 QDs in the diagnosis and the therapy of protease-related diseases.

Kinetoplastid-specific X2-family kinesins interact with a kinesin-like pleckstrin homology domain protein that localizes to the trypanosomal microtubule quartet.

Kinesins are motor proteins found in all eukaryotic lineages that move along microtubules to mediate cellular processes such as mitosis and intracellular transport. In trypanosomatids, the kinesin superfamily has undergone a prominent expansion, resulting in one of the most diverse kinesin repertoires that includes the two kinetoplastid-restricted families X1 and X2. Here, we characterize in Trypanosoma brucei TbKifX2A, an orphaned X2 kinesin. TbKifX2A tightly interacts with TbPH1, a kinesin-like protein with a likely inactive motor domain, a rarely reported occurrence. Both TbKifX2A and TbPH1 localize to the microtubule quartet (MtQ), a characteristic but poorly understood cytoskeletal structure that wraps around the flagellar pocket as it extends to the cell body anterior. The proximal proteome of TbPH1 revealed two other interacting proteins, the flagellar pocket protein FP45 and intriguingly another X2 kinesin, TbKifX2C. Simultaneous ablation of TbKifX2A/TbPH1 results in the depletion of FP45 and TbKifX2C and also an expansion of the flagellar pocket, among other morphological defects. TbKifX2A is the first motor protein to be localized to the MtQ. The observation that TbKifX2C also associates with the MtQ suggests that the X2 kinesin family may have co-evolved with the MtQ, both kinetoplastid-specific traits.

Molecular analysis and essentiality of Aro1 shikimate biosynthesis multi-enzyme in Candida albicans.

In the human fungal pathogen Candida albicans, ARO1 encodes an essential multi-enzyme that catalyses consecutive steps in the shikimate pathway for biosynthesis of chorismate, a precursor to folate and the aromatic amino acids. We obtained the first molecular image of C. albicans Aro1 that reveals the architecture of all five enzymatic domains and their arrangement in the context of the full-length protein. Aro1 forms a flexible dimer allowing relative autonomy of enzymatic function of the individual domains. Our activity and in cellulo data suggest that only four of Aro1's enzymatic domains are functional and essential for viability of C. albicans, whereas the 3-dehydroquinate dehydratase (DHQase) domain is inactive because of active site substitutions. We further demonstrate that in C. albicans, the type II DHQase Dqd1 can compensate for the inactive DHQase domain of Aro1, suggesting an unrecognized essential role for this enzyme in shikimate biosynthesis. In contrast, in Candida glabrata and Candida parapsilosis, which do not encode a Dqd1 homolog, Aro1 DHQase domains are enzymatically active, highlighting diversity across Candida species.