First-in-human, double-blind, randomized phase 1b study of peptide immunotherapy IMCY-0098 in new-onset type 1 diabetes

: Background : Type 1 diabetes (T1D) is a CD4+ T cell-driven autoimmune disease characterized by the destruction of insulin-producing pancreatic β-cells by CD8+ T cells. Achieving glycemic targets in T1D remains challenging in clinical practice; new treatments aim to halt autoimmunity and prolong β-cell survival. IMCY-0098 is a peptide derived from human proinsulin that contains a thiol-disulfide oxidoreductase motif at the N-terminus and was developed to halt disease progression by promoting the specific elimination of pathogenic T cells. Methods: This first-in-human, 24-week, double-blind phase 1b study evaluated the safety of three dosages of IMCY-0098 in adults diagnosed with T1D < 6 months before study start. Forty-one participants were randomized to receive four bi-weekly injections of placebo or increasing doses of IMCY-0098 (dose groups A/B/C received 50/150/450 μg for priming followed by three further administrations of 25/75/225 μg, respectively). Multiple T1D-related clinical parameters were also assessed to monitor disease progression and inform future development. Long-term follow-up to 48 weeks was also conducted in a subset of patients. Results: Treatment with IMCY-0098 was well tolerated with no systemic reactions; a total of 315 adverse events (AEs) were reported in 40 patients (97.6%) and were related to study treatment in 29 patients (68.3%). AEs were generally mild; no AE led to discontinuation of the study or death. No significant decline in C-peptide was noted from baseline to Week 24 for dose A, B, C, or placebo (mean change − 0.108, − 0.041, − 0.040, and − 0.012, respectively), suggesting no disease progression. Conclusions: Promising safety profile and preliminary clinical response data support the design of a phase 2 study of IMCY-0098 in patients with recent-onset T1D. Trial registration: IMCY-T1D-001: ClinicalTrials.gov NCT03272269; EudraCT: 2016–003514-27; and IMCY-T1D-002: ClinicalTrials.gov NCT04190693; EudraCT: 2018–003728-35

Identification of a novel small-molecule inducer of ectopic tail formation suggestive of alterations in Wnt/PCP signaling using a zebrafish chemical genetics screen for BMP and Wnt signaling modulators

Over the last decade, zebrafish have proven increasingly useful as an animal model in theearly drug discovery process. As an alternative to target-based drug discovery approaches,chemical genetics in zebrafish enables the identification of bioactive small molecules basedon their ability to induce specific developmental dysmorphologies or behaviors. These in vivophenotypes can be associated with the modulation of key signaling pathways and ultimatelylead to the elucidation of the compound s biological target.Because of multiple genetic screens carried out to date, a wide range of mutant phenotypesin zebrafish have been thoroughly characterized and reported, including genes and pathwaycomponents of the Bone Morphogenetic Protein (BMP) and Wingless/Int (Wnt) signalingpathways for which the biochemistry and/or mechanisms of action have been well-studied.Dorsoventral (DV) and anterior-posterior (AP) axis defects as well as ectopic tail formationhave been reported in BMP/Wnt mutant or transgenic zebrafish. The recent identification ofdorsomorphin, a novel small-molecule inhibitor of BMP receptor signaling, proved theusefulness of phenotype-based compound screening in zebrafish embryos. This moleculeinduced severe dorsalization (as a result of DV patterning defects) in developing embryos. Todate, this molecule and some of its derivatives have been extensively studied as modifiers ofiron homeostasis, bone formation, metabolism and stem cell differentiation.Molecules that can modulate BMP or Wnt signaling pathways are of pharmacologicalinterest, as these conserved pathways are not only crucial for embryonic development, butare also important in adult homeostasis, tissue repair and disease. Aberrant signaling islinked with several major diseases such as cancer and arthritic diseases. New drug-like leadcompounds that target and modulate BMP or Wnt signaling could therefore be oftherapeutic utility.A zebrafish-based phenotypic screen for BMP and Wnt signaling modulators was carried outusing the Strathclyde Natural Products Library as a potential source of novel, bioactivecompounds. The library consists of 5000 methanolic plant extracts representing 90% of plantfamilies worldwide. One of the bioactive plant extracts identified in this screen Jasminumgilgianum, an Oleaceae species native to Papua New Guinea induced ectopic tails duringzebrafish embryonic development. As ectopic tail formation occurs when BMP or noncanonicalWnt signaling is inhibited during the tail protrusion process, we suspected aconstituent of this extract to act as a modulator of either one or both of these pathways. Abioassay-guided isolation was carried out on the basis of this zebrafish phenotype,identifying para-coumaric acid methyl ester (pCAME) as the active compound.We performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos,including a tissue-specific marker analysis of the secondary tails. We found pCAME tosynergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails,and cause convergence-extension defects in compound-treated embryos. Taken together,our results indicated that pCAME may interfere with non-canonical Wnt signaling.Structurally-related compounds of pCAME were reported to inhibit both MAP kinases andtyrosine kinases. Therefore, we looked for possible kinase targets present in the Wnt/PCPpathway. C-jun N-terminal kinase (Jnk) belongs to the family of MAPKs, and is downstreamof the receptor complex of the Wnt/PCP pathway. Antisense morpholino knockdown of jnk2and pulsing of embryos with the small-molecule inhibitor SP600125, suggested pCAME totarget Jnk kinase. Although SP600125 is generally used as specific Jnk inhibitor thecompound is able to inhibit more kinases than only Jnk isoforms. Immunoblottingexperiments however, revealed that pCAME does not directly inhibit Jnk-mediatedphosphorylation of C-jun. These results suggested the modulation of other targets/pathwaysin ectopic tail formation by pCAME. Further investigation of pCAME s mechanism of actionwill help determine this compound s pharmacological utility.status: publishe

PHÂN LẬP VÀ XÁC ĐỊNH MỘT SỐ ĐẶC ĐIỂM SINH HỌC CỦA ENZYME XYLANASE TỪ LÚA MÌ NẨY MẦM

Enzyme nội sinh 1?4-??D-xylanase, gọi tắt là xylanase, là enzyme đóng vai trò quan trọng nhất trong quá trình thủy phân arabinoxylan. Để hiểu sâu hơn về tính chất của xylanase, xylanase được phân lập và xác định một số đặc điểm sinh học từ hạt lúa mì nảy mầm 24 ngày. Các dạng đồng đẳng xylanase khác nhau được tinh chế từ dịch chiết. Protein hòa tan được tủa phân đoạn với ammonium sulfate và sắc ký trao đổi ion. Sau đó xylanase được phân lập có lựa chọn bởi sắc ký ái lực với kháng thể của xylanase từ lúa mạch. Trình tự sắp xếp của các acid amin trong chuỗi polypeptide ở đầu N tận cùng của các dạng đồng đẳng xylanase được quyết định bởi phương pháp Edman. Dựa vào trình tự sắp xếp acid amin ở đầu N của xylanase, trọng lượng phân tử lý thuyết nằm trong khoảng 60 kDa đến 20 kDa và điểm đẳng điện pI từ 5,75 đến 4,69 được xác định cho các dạng đồng đẳng xylanase từ lúa mì

Post-translational processing of β-D-xylanases and changes in extractability of arabinoxylans during wheat germination

Endo-1,4-b-D-xylanase (EC 3.2.1.8, b-D-xylanase) activity, and arabinoxylan (AX) level and extractability were monitored for the first time simultaneously in wheat kernels (Triticum aestivum cv. Glasgow) up to 24 days post-imbibition (DPI), both in the absence and presence of added gibberellic acid (GA). Roughly three different stages (early, intermediate and late) can be discriminated. Addition of GA resulted in a faster increase of water extractable arabinoxylan (WEAX) level in the early stage (up to 3e4 DPI). This increase was not accompanied by the discernible presence of homologues of the barley X-I b-D-xylanase as established by immunodetection. This suggests that other, yet unidentified b-D-xylanases operate in this early time window. The intermediate stage (up to 13 DPI) was characterized by the presence of unprocessed 67 kDa X-I like b-D-xylanase, which was much more abundant in the presence of GA. The occurrence of higher levels of the unprocessed enzyme was related with higher b-D-xylanase activities and a further increase in WEAX level, pointing to in vivo activity of the unprocessed 67 kDa b-D-xylanase. During the late stage (up to 24 DPI) gradual processing of the 67 kDa b-D-xylanase occurred and was associated with a drastic increase in b-D-xylanase activity. Up to 120-fold higher activity was recorded at 24 DPI, with approx. 85% thereof originating from the kernel remnants. The WEAX level decreased during the late stage, suggesting that the b-D-xylanase is processed into more active forms to achieve extensive AX breakdown.status: publishe

A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical Wnt/PCP signaling

Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators of the BMP and Wnt signaling pathways. One of the bioactive plant extracts identified in this screen - Jasminum gilgianum, an Oleaceae species native to Papua New Guinea - induced ectopic tails during zebrafish embryonic development. As ectopic tail formation occurs when BMP or non-canonical Wnt signaling is inhibited during the tail protrusion process, we suspected a constituent of this extract to act as a modulator of these pathways. A bioassay-guided isolation was carried out on the basis of this zebrafish phenotype, identifying para-coumaric acid methyl ester (pCAME) as the active compound. We then performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos, including a tissue-specific marker analysis of the secondary tails. We found pCAME to synergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails, and causing convergence-extension defects in compound-treated embryos. These results indicate that pCAME may interfere with non-canonical Wnt signaling. Inhibition of Jnk, a downstream target of Wnt/PCP signaling (via morpholino antisense knockdown and pharmacological inhibition with the kinase inhibitor SP600125) phenocopied pCAME-treated embryos. However, immunoblotting experiments revealed pCAME to not directly inhibit Jnk-mediated phosphorylation of c-Jun, suggesting additional targets of SP600125, and/or other pathways, as possibly being involved in the ectopic tail formation activity of pCAME. Further investigation of pCAME's mechanism of action will help determine this compound's pharmacological utility

Tissue marker analysis in SP600125-induced ectopic tails.

<p>(A) <i>eve1</i>; (B) <i>ntl</i>; (C) <i>myoD</i>; (D) c<i>ol2a</i>; (E) <i>shh</i>; and (F) c<i>dx4</i> expression. All embryos were pulsed with 140 μM pCAME for 1h at tailbud stage and fixed at 30 hpf. Lateral views.</p

pCAME induces convergence and extension defects.

<p>(A) and (B) <i>flh</i> expression; (C) and (D) <i>ntl</i>; (E) and (F) <i>papc</i>; (G) and (H) <i>myoD</i>. All embryos were treated at 2-4 cell stages with 14 or 28 μM pCAME and fixed at 1 somite stage. Dorsal view. Expression domains of compound-treated fish are altered as would be predicted for CE defects. Embryos have a broader and shorter notochord and impaired convergence and extension of the para-axial mesoderm and the somites.</p

<i>jnk2</i> morpholino knockdown induces convergence extension defects.

<p>(A) and (B) 2 mM <i>jnk2</i> MO-injected fish A: 38 % B: 9 %; (C) Wild type; (D) 150 μM SP600125 pulsed for 1 h at tailbud stage. All embryos are at 48 hpf. Black arrow denotes duplicated tail. (E-H) Control MO injected fish; (E’-H’) <i>jnk2</i> MO injected fish; (E) <i>flh</i>; (F) <i>ntl</i>; (G) <i>myoD</i>; (H) <i>papc</i>.</p

Analysis of tissue-specific marker expression in pCAME-induced ectopic tails.

<p>(A) <i>eve1</i>; (B) <i>ntl</i>; (C) <i>myoD</i>; (D) c<i>ol2a</i>; (E) <i>shh</i>; and (F) <i>cdx4</i> expression. All embryos were pulsed with 140 μM pCAME for 1h at tailbud stage and fixed at 30 hpf. Lateral views.</p

Isolation of pCAME.

<p>(A) Typical normal phase TLC separation of the crude methanolic extract of <i>Jasminum gilgianum</i>, daylight view (left), plate sprayed with 1% vanillin/H₂SO4 (right). Black star denotes band of interest. (B) Typical HPLC chromatograms of preparative fraction 4 at 200 and 300 nm, black arrows denote signal due to pCAME. (C) Molecular structure and molecular weight of pCAME. (D) NMR spectral data.</p