Jul 2026· Journal of the American Chemical Society· Vol 148, pp. 30301-30313· 0 citations· 47 references
Medicine
TL;DR
The rational design, synthesis, and mechanistic investigation of cyclopentene-based γ-aminobutyric acid analogues bearing alkyne or nitrile warheads as potent hOAT inactivators are reported, expanding the mechanistic repertoire of PLP-dependent enzyme inactivation and providing a generalizable framework for designing highly selective mechanism-based inactivators.
Abstract
Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, plays a central role in glutamine, proline, and polyamine metabolism and is increasingly recognized as a metabolic vulnerability in multiple cancers. Previously, we established a second deprotonation strategy to achieve efficient mechanism-based inactivation of hOAT over closely related aminotransferases. Building on this concept, we report the rational design, synthesis, and mechanistic investigation of cyclopentene-based γ-aminobutyric acid analogues bearing alkyne or nitrile warheads as potent hOAT inactivators. These compounds undergo enzyme-catalyzed γ-deprotonation to form ketimine intermediates, priming for a subsequent tautomerization event that leads to irreversible inhibition. Inhibitory activity evaluation revealed pronounced stereochemical effects on binding affinity and partition ratio, with one nitrile analogue (4b) exhibiting an exceptional inactivation efficiency (kinact/KI = 111.8 mM-1·min-1) and ∼400-fold selectivity for hOAT over γ-aminobutyric acid aminotransferase. Intact protein mass spectrometry and X-ray crystallography demonstrated that alkyne-containing analogues form covalent adducts with hOAT, whereas nitrile-containing analogues generate noncovalent but tight-binding species. Kinetic isotope effect studies identified γ-deprotonation as the rate-determining step, and a complementary small-molecule mass and computational study elucidated the inactivation and turnover pathways. Collectively, these results expand the mechanistic repertoire of PLP-dependent enzyme inactivation and provide a generalizable framework for designing highly selective mechanism-based inactivators.
Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that has been implicated in several cancers due to the essential role in the synthesis of glutamine and proline. Inhibition of hOAT may provide a novel method to treat a variety of cancers. In recent years, selective inhibition of hOAT through mechanism-based inactivation has shown promise in the treatment of hepatocellular carcinoma. Inspired by the recently reported hOAT-selective δ-deprotonation and previously proposed ring-strain mediated mechanisms of inactivation, we designed, synthesized, and evaluated (1S,3R,5R)-3-amino-6,6-difluorobicyclo[3.1.0]hexane-1-carboxylic acid (5), a time-dependent inhibitor of hOAT. Structural and mechanistic studies validated the proposed mechanism of 5, which undergoes a ring-opening mechanism to form a PLP-inactivator adduct that is tightly bound in the active site of hOAT. Intact protein mass spectrometry, 19F NMR spectroscopy, transient-state kinetic studies, X-ray crystallography, and QM calculations were used to determine the final adduct and explore the mechanism of inactivation of 5. This is the first report of a ring-opening event in the active site of hOAT.
Allison N Devitt, Abigail L. Vargas, C. K. Zhang et al.· Journal of the American Chem...· 0 citations
(±)-Hypericandrone A (HCA, 1), an unprecedented pair of enantiomeric polycyclic polyprenylated acylphloroglucinols (PPAPs), and its biosynthetic congener hypericandrone B (HCB, 2) were isolated from the fruits of Hypericum androsaemum. Compound 1 features a unique 2-cyclopentane-bicyclo[3.3.0]octane scaffold, while 2 possesses a rare 2-cyclopentyltetrahydrofuran framework. Their absolute configurations were unequivocally determined by spectroscopic analysis, single-crystal X-ray diffraction, and ECD calculations. Remarkably, (+)-1 displayed potent antitubercular activity by selectively inhibiting the acetyltransferase domain of Mycobacterium tuberculosis GlmU through a dual mechanism, acting competitively against acetyl-CoA (Ki = 0.07 μM) and noncompetitively against glucosamine-1-phosphate (GlcN-1-P, Ki = 0.20 μM).
Three phenylene-linked bis(acetamide) compounds were synthesized and evaluated to determine how pyrazolone or benzothiazole terminal groups alter chemical radical-scavenging, cellular metabolic viability, and predicted accommodation within human thymidylate synthase (hTS). Spectroscopic characterization supported the proposed structures, while RP-HPLC indicated purities of 94.80% for R1, 96.72% for R2, and 90.16% for R3. Redocking of co-crystallized dUMP reproduced its experimental pose with an RMSD of 1.3 Å, supporting pose recovery by the docking setup. R2 and R3 produced AutoDock Vina scores of -9.2 and - 9.4 kcal/Mol, respectively, compared with -7.45 kcal/Mol for FdUMP; these values were interpreted as exploratory scoring outputs rather than evidence of hTS inhibition. In the cell-free DPPH assay, R3 showed the strongest response among the synthesized compounds, with an IC₅₀ of 28.95 μg/mL, whereas R1 and R2 gave 46.29 and 47.64 μg/mL, respectively, and ascorbic acid gave 19.79 μg/mL. In Caco-2 cells, R2 and R3 yielded comparable MTT-derived IC₅₀ values of 59.94 and 60.43 μg/mL, both lower than R1 at 148.7 μg/mL. Their corresponding HDF IC₅₀ values were 141.2, 109.5, and 161.7 μg/mL, giving HDF/Caco-2 indices of 2.36, 1.81, and 1.09. 5-fluorouracil showed an index of 3.16. Comparisons between Caco-2 and HDF fitted logIC₅₀ values were significant for R2 and R3 but not R1, and no compound reached the stringent SI threshold of 10. Thus, DPPH reactivity and cellular response followed different trends, and the modest differential sensitivity, limited compound series, and suboptimal purity of R3 support cautious interpretation and further mechanistic validation using adequately purified compound batches.
Ihab Abbas Taher, F. W. Askar, Fatin Fadhel Al-Kazzaz· Bioorganic chemistry (Print)· 0 citations
Diaminopimelate desuccinylase (DapE) is a promising antibacterial target involved in lysine biosynthesis and bacterial cell wall formation. Previously, N-aryl sulfonamide hit 1 (IC₅₀ = 72.9 μM) was optimized to 2 with enhanced potency (IC₅₀ = 8.9 ± 5.0 μM, Kᵢ = 1.36 ± 0.61 μM). In this paper we describe the synthesis of N-heterocyclic sulfonamides and N-aryl sultams to explore the effects of heterocycles and conformational constraints on DapE inhibition. Aminopyridine substitutions generally reduced activity, although N-pyridyl sulfonamide 6e retained moderate inhibition (68.5% at 100 μM). Five- and seven-membered sultams showed modest activity, with p-methoxybenzene 7b displaying the highest inhibition (60.4% at 100 μM). Docking studies predicted favorable zinc-binding for several analogues, particularly those with carboxylic acids, but these interactions generally did not improve experimental potency.
Sebastian Flieger, Emma H. Kelley, Megan Beulke et al.· Bioorganic & Medicinal Chemi...· 0 citations
Non-canonical amino acids (ncAAs) are privileged building blocks for the synthesis of natural products, biocatalysts, and drug molecules. Among them, γ-tertiary-nitro-α-amino acids are highly valuable yet remain scarcely explored due to challenges in synthetic accessibility. Although pyridoxal-5′-phosphate (PLP)-dependent enzymes are powerful biocatalysts for ncAA production, no enzymatic platform has, to the best of our knowledge, enabled efficient access to γ-tertiary-nitro-α-amino acids. Here, we report a PLP-dependent enzymatic platform that selectively couples
O
-acetyl-serine with secondary nitroalkanes to yield γ-tertiary-nitro-α-amino acids. Through reaction design, enzyme screening, and directed evolution, we repurpose SidM, an enzyme that catalyzes pyrrole ring formation in the siderochelin biosynthetic pathway, into a highly active and stereoselective biocatalyst, affording γ-tertiary-nitro-α-amino acids in up to 99% yield and 99% diastereomeric excess. This enzyme exhibits broad substrate scope and good evolvability, allowing enhancement of catalytic efficiency or inversion of diastereoselectivity at the Cγ nitro center, and is readily scalable to gram synthesis. Mechanistic analyses combining computation and mutagenesis elucidate the structural determinants of activity and stereocontrol. This work establishes a general, tunable and scalable biocatalytic platform for the synthesis of γ-tertiary-nitro-α-amino acids, expanding the synthetic repertoire of PLP-dependent enzymes for asymmetric C–C bond formation.
Wenping Ding, Shenggan Luo, Yike Zou et al.· Nature Communications· 0 citations
Although flavan-3-ols are extensively utilized as acrylamide inhibitors in thermal food processing, their variable efficacies suggest a more complex, unresolved mechanism. Overcoming the traditional perspective of simple inhibition, this study reveals a paradoxical, structure-dependent dual effect of four typical flavan-3-ols (EGCG, EGC, ECG, and EC) on acrylamide formation. While the net inhibitory efficacy in an asparagine-glucose system followed EGCG > EGC ≈ ECG > EC, investigations using a 3-aminopropionamide (3-APA) intermediate model demonstrated that the oxidation of the flavan-3-ol B-ring generated highly reactive quinone intermediates that actively facilitated acrylamide formation. This promotional capacity (EC > ECG > EGC > EGCG) inversely mirrored the net inhibitory trend. Crucially, UHPLC-MS/MS analysis tentatively identified nine novel quinone-amine adducts, providing structural evidence for this B-ring-mediated promotion. Consequently, the net impact of flavan-3-ols on acrylamide formation is not a simple linear inhibition, but a dynamic competition between A-ring-mediated carbonyl trapping and B-ring-mediated quinone generation. EGCG exhibits the most potent net inhibition because its highly substituted structure is exceptionally susceptible to competing side reactions, such as autoxidation and ester hydrolysis, which effectively quench the B-ring-driven promotion. These findings provide direct mechanistic and practical guidance for selecting galloylated tea polyphenols to mitigate acrylamide risk in thermally processed starchy foods.
Yajing Qi, Wen-Jun Wang, Jiahao Cheng et al.· Food Research International· 0 citations