The Janus kinase inhibitor (baricitinib) suppresses the rheumatoid arthritis active marker gliostatin/thymidine phosphorylase in human fibroblast-like synoviocytes

Gliostatin/thymidine phosphorylase (GLS/TP) is known to have angiogenic and arthritogenic activities in the pathogenesis of rheumatoid arthritis (RA). The novel oral Janus kinase (JAK) inhibitor baricitinib has demonstrated high efficacy in RA. However, the effect of baricitinib on fibroblast-like synoviocytes (FLSs), a key component of invasive synovitis, has not been still elucidated. This study investigated whether GLS/TP production could be regulated by JAK/signal transducers and activators of transcription (STAT) signaling in FLSs derived from patients with RA. FLSs were cultured and stimulated by interferon (IFN)γ in the presence of baricitinib. Expression levels of GLS/TP were determined using reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunocytochemistry. Phosphorylation of STAT proteins was investigated by Western blot. In cultured FLSs, GLS/TP mRNA and protein levels were significantly induced by treatment with IFNγ and these inductions were suppressed by baricitinib treatment. Baricitinib inhibited IFNγ-induced STAT1 phosphorylation, while JAK/STAT activation played a pivotal role in IFNγ-mediated GLS/TP upregulation in RA. These results suggested that baricitinib suppressed IFNγ-induced GLS/TP expression by inhibiting JAK/STAT signaling, resulting in the attenuation of neovascularization, synovial inflammation, and cartilage destruction.

Engineering of the Recombinant Expression and PEGylation Efficiency of the Therapeutic Enzyme Human Thymidine Phosphorylase

Human thymidine phosphorylase (HsTP) is an enzyme with important implications in the field of rare metabolic diseases. Defective mutations of HsTP lead to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a disease with a high unmet medical need that is associated with severe neurological and gastrointestinal complications. Current efforts focus on the development of an enzyme replacement therapy (ERT) using the Escherichia coli ortholog (EcTP). However, bacterial enzymes are counter-indicated for human therapeutic applications because they are recognized as foreign by the human immune system, thereby eliciting adverse immune responses and raising significant safety and efficacy risks. Thus, it is critical to utilize the HsTP enzyme as starting scaffold for pre-clinical drug development, thus de-risking the safety concerns associated with the use of bacterial enzymes. However, HsTP expresses very poorly in E. coli, whereas its PEGylation, a crucial chemical modification for achieving long serum persistence of therapeutic enzymes, is highly inefficient and negatively affects its catalytic activity. Here we focused on the engineering of the recombinant expression profile of HsTP in E. coli cells, as well as on the optimization of its PEGylation efficiency aiming at the development of an alternative therapeutic approach for MNGIE. We show that phylogenetic and structural analysis of proteins can provide important insights for the rational design of N’-terminus-truncation constructs which exhibit significantly improved recombinant expression levels. In addition, we developed and implemented a criteria-driven rational surface engineering strategy for the substitution of arginine-to-lysine and lysine-to-arginine residues to achieve more efficient, homogeneous and reproducible PEGylation without negatively affecting the enzymatic catalytic activity upon PEGylation. Collectively, our proposed strategies provide an effective way to optimize enzyme PEGylation and E. coli recombinant expression and are likely applicable for other proteins and enzymes.

Molecular Dynamics Study of Escherichia coli Thymidine Phosphorylase in a Complex with 3'-Azidothymidine Inhibitor and Phosphate

Abstract— Using a molecular dynamics method, the state of the dimeric thymidine phosphorylase molecule from Escherichia coli in a complex with noncompetitive enzyme inhibitor 3'-azidothymidine and phosphate ion was studied on a trajectory of 50 ns. Previously obtained atomic coordinates of a complex of thymidine phosphorylase with azidothymidine and sulfate at a resolution of 1.52 Å were used as a starting model. It was demonstrated that both subunits of a dimeric enzyme molecule function asynchronously in a given time interval; moreover, each subunit maintains an open conformation. It was found that the nature of ligand at the nucleoside center affects the binding strength of phosphate in the phosphate center. In a complex with an inhibitor, both ligands over the entire time interval remain bound to the enzyme, while the release of phosphate from the active center is observed when simulating the behavior of thymidine phosphorylase in the presence of phosphate and thymidine substrate. The stabilizing effect of azidothymidine on phosphate binding is consistent with the behavior of azidothymidine as a noncompetitive inhibitor of thymidine phosphorylase.

Bioinformatics of thymidine metabolism in Trypanosoma evansi: exploring nucleoside deoxyribosyltransferase (NDRT) as a drug target

Trypanosoma evansi, the causative agent of surra or camel trypanosomiasis, is characterized by the widest geographic distribution and host range among the known trypanosomes. Its zoonotic importance and increasing evidence of drug resistance necessitate the discovery of new drug targets. The drug discovery process entails finding an exploitable difference between the host and the parasite. In this study, the thymidine metabolic pathways in camel and T. evansi were compared by analyzing their metabolic maps, protein sequences, domain and motif contents, phylogenetic relationships, and 3D structure models. The two organisms were revealed to recycle thymidine differently: performed by thymidine phosphorylase in camels (Camelus genus), this role in T. evansi was associated with nucleoside deoxyribosyltransferase (NDRT), a unique trypanosomal enzyme absent in camels. Thymidine in T. evansi seems to be governed by thymine through NDRT, whereas in camels, thymidine can be produced from thymidylate via 5'-nucleotidase. As a result, NDRT may be a promising drug target against T. evansi.

Thymidine Phosphorylase is Increased in Patients with Infection and Could be a Prognostic Marker for Sepsis and Disseminated Intravascular Coagulation

Background: Sepsis, a serious condition caused by infection, can ultimately progress to life-threatening septic shock and multiple organ failure. Hitherto, no reliable markers could be used for the early diagnosis and sepsis is still a challenge for clinicians. We investigated the potential role of thymidine phosphorylase (TYMP) in infection and delved its value in diagnosing sepsis and disseminated intravascular coagulation (DIC). Methods: This prospective study enrolled infection patients consecutively admitted to Beijing Friendship Hospital from November 2019 to January 2021. Non-infection volunteers were used as control (NIC). Blood sample were obtained on day 1, 2, 4, and 7 after admission. TYMP serum levels and its correlation with DIC scores, Sequential Organ Failure Assessment (SOFA) scores, and other laboratory parameters were analyzed using the non-parametric Mann-Whitney U test and the Spearman's Rank-Order Correlation analyses. Results: Eighty-three infectious patients were enrolled. Forty-two patients were diagnosed as sepsis (SP) and 41 were non-septic infection (NSP). NIC group included 20 individuals without any infection. Serum TYMP levels were significantly higher in both SP and NSP groups when compared with the NIC group. TYMP serum levels was further higher in the SP than in the NSP group. The increase of TYMP was positively correlated with DIC scores. TYMP was higher in patients with DIC than those patients without DIC. A positive correlation presented between serum TYMP and plasma D-dimer levels. No positive correlations were found between TYMP and inflammatory markers. TYMP was higher in patients with SOFA score > 1 than those with SOFA score zero. The increase of TYMP is also positively correlated with SOFA score and death risk coefficient, and negatively correlated with oxygen index. By conducting receiver operating characteristic curve analyses, we found TYMP has a specific and sensitive predictive value for diagnosing sepsis and a modest value for DIC. Conclusions: This study is the first time to determine the role of TYMP in infectious disease. TYMP is significantly increased in infectious patients and is further higher in patients with sepsis or DIC. TYMP is positively correlated with acuity markers and has a diagnostic and prognostic value for sepsis and DIC.