Currently, most maytansine-containing antibody-drug conjugates (ADCs) in clinical trials are prepared with DM1 or DM4, which in turn is synthesized mainly from ansamitocin P-3 (AP-3), a bacterial maytansinoid, isolated from Actinosynnema pretiosum. However, due to the high self-toxicity of AP-3 to A. pretiosum, the yield of AP-3 has been difficult to improve. Herein, a new maytansinoid with much lower self-toxicity to A. pretiosum, 3-O-carbamoylmaytansinol (CAM, 3), was designed and generated by introducing the 3-O-carbamoyltransferase gene asc21b together with the N-methyltransferase genes from exogenous maytansinoid gene clusters into the 3-O-acyltransferase gene (asm19) deleted mutant HGF052. Meanwhile, two new shunt products, 20-O-demethyl-19-dechloro-N-demethyl-4,5-desepoxy-CAM (4) and 20-O-demethyl-N-demethyl-4,5-desepoxy-CAM (5) were identified from the recombinant strain. Furthermore, by screening of liquid fermentation media, overexpression of bottleneck tailoring enzymes and the pathway-specific activator, the titer of CAM reached 498 mg/L in the engineered strain. Since the 3-O-carbamoyl group of CAM can be removed by chemical cleavage as AP-3 to produce maytansinol, our work suggests that CAM may be a promising alternative to AP-3 in the future development of ADCs.

2021-01-01·Applied Microbiology and Biotechnology3区 · 工程技术

Efflux identification and engineering for ansamitocin P-3 production in Actinosynnema pretiosum

3区 · 工程技术

Article

作者: Xiao, Yifan ; Wang, Xinran ; Ning, Xinjuan ; Wei, Jianhua ; Bai, Linquan ; Luan, Shuhui

Ansamitocin P-3 (AP-3) exhibits potent biological activities against various tumor cells. As an important drug precursor, reliable supply of AP-3 is limited by low fermentation yield. Although different strategies have been implemented to improve AP-3 yield, few have investigated the impact of efflux on AP-3 production. In this study, AP-3 efflux genes were identified through combined analysis of two sets of transcriptomes. The production-based transcriptome was implemented to search for efflux genes highly expressed in response to AP-3 accumulation during the fermentation process, while the resistance-based transcriptome was designed to screen for genes actively expressed in response to the exogenous supplementation of AP-3. After comprehensive analysis of two transcriptomes, six efflux genes outside the ansamitocin BGC were identified. Among the six genes, individual deletion of APASM_2704, APASM_6861, APASM_3193, and APASM_2805 resulted in decreased AP-3 production, and alternative overexpression led to AP-3 yield increase from 264.6 to 302.4, 320.4, 330.6, and 320.6 mg/L, respectively. Surprisingly, APASM_2704 was found to be responsible for exportation of AP-3 and another macro-lactam antibiotic pretilactam. Furthermore, growth of APASM_2704, APASM_3193, or APASM_2805 overexpression mutants was obviously improved under 300 mg/L AP-3 supplementation. In summary, our study has identified AP-3 efflux genes outside the ansamitocin BGC by comparative transcriptomic analysis, and has shown that enhancing the transcription of transporter genes can improve AP-3 production, shedding light on strategies used for exporter screening and antibiotic production improvement. KEY POINTS: • AP-3-related efflux genes were identified by transcriptomic analysis. • Deletion of the identified efflux genes led in AP-3 yield decrease. • Overexpression of the efflux genes resulted in increased AP-3 production.

2020-04-01·Applied Microbiology and Biotechnology3区 · 工程技术

Metabolomic change and pathway profiling reveal enhanced ansamitocin P-3 production in Actinosynnema pretiosum with low organic nitrogen availability in culture medium

3区 · 工程技术

Article

作者: Hu, Fengxian ; Wei, Liu-Jing ; Hua, Qiang ; Yang, Linbing ; Liu, Ting ; Chen, Jun

Ansamitocin P-3 (AP-3), a 19-membered polyketide macrocyclic lactam, has potent antitumor activity. Our previous study showed that a relatively low organic nitrogen concentration in culture medium could significantly improve AP-3 production of Actinosynnema pretiosum. In the present study, we aimed to reveal the possible reasons for this improvement through metabolomic and gene transcriptional analytical methods. At the same time, a metabolic pathway profile based on metabolome data and pathway correlation information was performed to obtain a systematic view of the metabolic network modulations of A. pretiosum. Orthogonal partial least squares discriminant analysis showed that nine and eleven key metabolites directly associated with AP-3 production at growth phase and ansamitocin production phase, respectively. In-depth pathway analysis results highlighted that low organic nitrogen availability had significant impacts on central carbon metabolism and amino acid metabolic pathways of A. pretiosum and these metabolic responses were found to be beneficial to precursor supply and ansamitocin biosynthesis. Furthermore, real-time PCR results showed that the transcription of genes involved in precursor and ansamitocin biosynthetic pathways were remarkably upregulated under low organic nitrogen condition thus directing increased carbon flux toward ansamitocin biosynthesis. More importantly, the metabolic pathway analysis demonstrated a competitive relationship between fatty acid and AP-3 biosynthesis could significantly affect the accumulation of AP-3. Our findings provided new knowledge on the organic nitrogen metabolism and ansamitocin biosynthetic precursor in A. pretiosum and identified several important rate-limiting steps involved in ansamitocin biosynthesis thus providing a theoretical basis of further improvement in AP-3 production.

项与吡非尼酮/尼达尼布相关的新闻（医药）

2023-09-27

·Firstwordpharma

Avalyn pulls in $175 million to fund mid-stage studies of inhaled versions of IPF drugs

Avalyn Pharma announced Wednesday that it raised $175 million through a Series C financing, which it will use to advance inhaled formulations of two interstitial lung disease drugs into mid-stage development. The funding round was co-led by Perceptive Xontogeny Venture Funds, SR One and Eventide Asset Management, with new investors also joining.The company’s lead assets are AP01, an inhaled formulation of Roche’s Esbriet ( pirfenidone), and AP02, an inhaled formulation of Boehringer Ingelheim’s tyrosine kinase inhibitor Ofev (nintedanib). “There is an urgent need for effective treatments that patients can tolerate, which we believe can be addressed with inhaled delivery,” Avalyn CEO Lyn Baranowski said, adding that “our existing clinical data shows potential for both improved tolerability and efficacy.”Although the oral formulation of pirfenidone has been shown to reduce the decline of lung function and mortality in patients with idiopathic pulmonary fibrosis (IPF), Avalyn suggested that systemic exposure can lead to side effects including nausea, rash, photosensitivity, weight loss and fatigue. Meanwhile, in early-stage studies, AP01 has been well tolerated and demonstrated enhanced efficacy over oral pirfenidone, while AP02 has also shown a promising safety profile.Avalyn said that it plans to move AP01 into a Phase IIb trial for patients with pulmonary fibrosis, with AP02 progressing into a Phase IIa study. The drugmaker’s pipeline also includes AP02-DP, which is an inhaled dry powder formulation of nintedanib, AP03, a fixed-dose combination of pirfenidone and nintedanib, and AP04, an inhaled formulation of imatinib.In conjunction with the financing, Gianna Hoffman-Luca of Perceptive Xontogeny Venture Funds and Jill Carroll of SR One have joined Avalyn’s board. The company previously raised $35.5 million via a Series B financing.

临床2期

100 项与吡非尼酮/尼达尼布相关的药物交易

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