WU g, XU g, zhao y, Gao T, NI y, LV X, chen h, Li m, Du J, Yu c.
PMID:PPR1178249
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Abstract In antibody–drug conjugate research, the small molecule impurities requiring evaluation include not only free payloads, but also potential payload-related small molecule impurities. In this study, high-resolution mass spectrometry and triple quadrupole mass spectrometry were employed for systematically and comprehensively investigate small molecule related impurities in antibody–drug conjugate product. First, the quantitation of free payload was analyzed. High-resolution mass spectrometry enabled quantification at both precursor ion and product ion levels, the free payload exhibited good linearity over the concentration range of 0.05–100 ng/mL (R² ≥ 0.991), with a lower limit of quantification of 0.05 ng/mL. The accuracy ranged from 80% to 120%, and the precision was below 5%. The high-resolution mass spectrometry results were confirmed using triple quadrupole mass spectrometry. Second, other potential payload related small molecule impurities in the antibody–drug conjugate was investigated in depth. Multiple possible hydrolysis pathways of the payload were predicted based on molecular structure analysis, and the corresponding payload related impurities were subsequently identified using triple quadrupole mass spectrometry in accordance with the predicted precursor–product ion information. Third, stability studies of the antibody–drug conjugate were conducted under forced degradation conditions. Qualitative analyses were performed to characterize unknown payload related degradation products generated under stress conditions, including light exposure, heat, acidic, and alkaline environments. The major degradation products and their relative abundances were identified, with alkaline conditions exerting the greatest impact on sample stability. Clinical trial number: not applicable.
Alberga D, Nanna V, Papadopoulos MGE, Ancona A, Lomuscio MC, Mangiatordi GF.
J Chem Inf Model
PMID:41987658
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Achieving optimal target activity while maintaining synthetic accessibility and drug-likeness represents a major challenge in computational drug discovery. Existing de novo generative models often yield chemically invalid or synthetically intractable structures and struggle to optimize multiple objectives simultaneously. Here, we introduce ALCHIMIA, an interpretable hybrid framework combining reinforcement learning (RL) and a genetic algorithm (GA), built based on a vocabulary of 33 medicinal chemistry-inspired molecular transformations. The RL component trains a policy network to prioritize transformation sequences that improve synthetic accessibility (SA) and the quantitative estimate of drug-likeness (QED) scores, embedding these constraints directly into molecular generation. The GA component applies the learned policy as a mutational operator within population-based optimization guided by molecular docking, enabling the exploration of diverse chemical lineages while converging toward high-affinity ligands. ALCHIMIA was applied to two different pharmacologically relevant targets: human Cannabinoid Receptor 2 (CB2R) and human Sigma nonopioid intracellular Receptor 1 (S1R). We considered three different scenarios: (i) unconstrained hit identification; (ii) scaffold-constrained lead optimization; and (iii) design of dual modulators. The framework generated chemically valid molecules with QED and SA scores comparable to or better than those obtained with random baselines and selected de novo design methods. By codifying typical medicinal chemistry actions as learnable transformations and coupling multiobjective optimization with GA-based diversity maintenance, ALCHIMIA, freely available as a GitHub repository (https://github.com/alberdom88/ALCHIMIA), provides a practical, interpretable, and scalable framework for molecular de novo design.
Cai J, Wang H, He S, Li M, Peng D, Yao B.
Appl Microbiol Biotechnol
PMID:41991737
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Rainbow trout β-defensin 3 (rtDefb3) is identified as a small cationic antimicrobial peptide, but its recombinant expression and functional characterization have not been reported. In this study, the mature rtDefb3 Komagataella phaffii coding sequence was cloned from trout liver cDNA and heterologously expressed in GS115 using the pPIC9K with an α-factor secretion signal. Expression condition was optimized by adjusting methanol concentration (0.5-1.25%), induction temperature (26-30 °C), and induction duration (24-168 h). Optimized induction with 1.0% methanol at 30 ℃ for 96 h produced ~ 7 mg/mL of secreted peptide, which was purified to > 90% purity using one-step Ni-IDA affinity chromatography. The identity and purity of the recombinant rtDefb3 were confirmed by Tricine-SDS-PAGE, HPLC (~ 97% purity), and MALDI-TOF mass spectrometry. Functional assays revealed potent broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. These findings demonstrate, for the first time, the successful production of active rtDefb3 in Komagataella phaffii, establishing a scalable expression platform for fish β-defensins. The recombinant rtDefb3 peptide holds promise as a natural antimicrobial agent for aquaculture and potential therapeutic applications, addressing the urgent demand for alternatives to conventional antibiotics. KEY POINTS: • First successful recombinant production of rainbow trout β-defensin 3 in Komagataella phaffii • via optimized induction strategy. • High-yield secretory expression (~ 7 mg/mL) with > 90% purity confirmed by HPLC and biochemical analysis. • Recombinant rtDefb3 displayed potent broad-spectrum antibacterial activity, offering a repeatable and scalable system and functional validation.
Ramanathan M, Moussa Z, Peddinti RK.
Org Biomol Chem
PMID:41859767
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Owing to the valuable contribution of N -fused heterocycles to organic synthesis, medicinal chemistry, and materials research, tremendous efforts have been devoted towards the construction of N-heterocyclic scaffolds. Aryldiazonium salts are a ubiquitous class of building blocks/reagents widely used in organic synthesis owing to their distinct electrophilic nature to undergo substitution reactions with concurrent loss of molecular nitrogen (C-C, C-N bond formations). Conversely, a distinctive category of reactions known as "nitrogen retention reactions" preserve the diazonium-derived "N 2 " atoms in the end products. These classes of reactions are of great importance due to their uniqueness in constructing nitrogen-rich heterocycles and azo compounds without sacrificing the -NN- functionality. In this review we discuss 77 synthetic methodologies reported between 2015 and 2025 in which aryldiazonium salts are used as a key precursor and the characteristic nitrogen moiety is retained in the N-heterocyclic frameworks. This report groups nine classes of N-heterocycles, which are synthesized using five catalytic systems (Cu, Au, Ru, Ag and organo-photocatalysts) via three distinct activation modes (thermal, electrochemical, and photochemical). These innovative strategies incorporate several advantages, such as gram-scale experiments, one-pot conversions, solvent/transition metal-free conditions, mechanochemical/electrochemical/continuous-flow setups, and photochemical platforms. Overall, the methods discussed herein highlight the innovative trends and key breakthroughs in aryldiazonium salt based diazo retention chemistry over the years. The scope, limitations, mechanisms, and post-synthetic transformations are specifically emphasized.
Tachiiri N, Usui K, Mimasaka S.
Anal Sci
PMID:41989647
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