Snyder–Robinson syndrome (SRS) results from mutations in spermine synthase (SMS), which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonia, and seizures. Symptom management is the only treatment. Reduced SMS activity causes spermidine accumulation while spermine levels are reduced. The resulting exaggerated spermidine:spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity. Our studies aim to pharmacologically manipulate polyamine metabolism to correct this imbalance as a therapeutic strategy for SRS. Here we report the repurposing of 2‐difluoromethylornithine (DFMO), an FDA‐approved inhibitor of polyamine biosynthesis, in rebalancing spermidine:spermine ratios in SRS patient cells. Mechanistic in vitro studies demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of spermidine into spermine in hypomorphic SMS cells and induces uptake of exogenous spermine, altogether reducing the aberrant ratios. In a Drosophila SRS model characterized by reduced lifespan, DFMO improves longevity. As nearly all SRS patient mutations are hypomorphic, these studies form a strong foundation for translational studies with significant therapeutic potential.

The fast spread of bacteria that are resistant to many classes of antibiotics (multidrug resistant) is a global threat to human and animal health with a worrisome scenario ahead. Novel therapeutical strategies are of crucial importance to combat this phenomenon. For this purpose, we investigated the antimicrobial properties of the naturally occurring tripeptide Bialaphos and a dipeptide L-leucyl-L-phosphinoithricin, the synthesis and diastereomers separation of which are herein described. We demonstrate that these compounds are effective on clinical isolates of the human pathogen Klebsiella pneumoniae, causing hospital-acquired and community-acquired infections. The tested isolates were remarkable for their resistance to more than 20 commercial antibiotics of different classes. Based on previous literature data and our experiments consisting of glutamine supplementation, we suggest that both compounds release phosphinothricin—a well-known nanomolar inhibitor of glutamine synthetase—after their penetration in the bacterial cells; and, in this way, exert their antibacterial effect by negatively affecting nitrogen assimilation in this pathogen.

S-Adenosyl-L-methionine (SAM) is a central cofactor in cellular methylation, donating methyl groups to a wide range of biological substrates. SAM analogues are promising tools for selective modulation of methyltransferase activity. Here, we investigated phosphorus-containing analogues of SAM and S-adenosyl-L-homocysteine (SAH), focusing on the H-phosphinic SAM analogue ((R,S)-SAM-P_H) with the HO(H)(O)P group replacing the carboxyl group of SAM. We examined the interaction of (R,S)-SAM-P_H with three representative methyltransferases: Dnmt1, responsible for maintenance of DNA methylation; Dnmt3a, which establishes de novo DNA methylation; and catechol-O-methyltransferase (COMT), which methylates protocatechuic aldehyde to yield vanillin and isovanillin. (R,S)-SAM-P_H is a methyl group donor for Dnmt3a and COMT, but not for Dnmt1, despite the high structural similarity of the Dnmt1 and Dnmt3a catalytic domains. These results demonstrate that targeted modification of the carboxyl group of SAM can yield analogues with specific activity towards various methyltransferases. The different recognition of (R,S)-SAM-P_H by Dnmt3a and Dnmt1 highlights its potential as a molecular probe for distinguishing de novo from maintenance DNA methylation. This work enriches our understanding of methyltransferase substrate specificity and provides a new tool for selective modulation of epigenetic processes.

Homeostasis of the biogenic polyamines spermine (Spm) and spermidine (Spd), present in μM-mM concentrations in all eukaryotic cells, is precisely regulated by coordinated activities of the enzymes of polyamine synthesis, degradation, and transport, in order to sustain normal cell growth and viability. Spermine oxidase (SMOX) is the key and most recently discovered enzyme of polyamine metabolism that plays an essential role in regulating polyamine homeostasis by catalyzing the back-conversion of Spm to Spd. The development of many types of epithelial cancer is associated with inflammation, and disease-related inflammatory stimuli induce SMOX. MDL72527 is widely used in vitro and in vivo as an irreversible inhibitor of SMOX, but it is also potent towards N¹-acetylpolyamine oxidase. Although SMOX has high substrate specificity, Spm analogues have not been systematically studied as enzyme inhibitors. Here we demonstrate that 1,12-diamino-2,11-bis(methylidene)-4,9-diazadodecane (2,11-Met₂-Spm) has, under standard assay conditions, an IC₅₀ value of 169 μM towards SMOX and is an interesting instrument and lead compound for studying polyamine catabolism.

The polyamines, spermine (Spm) and spermidine (Spd), are important for cell growth and function. Their homeostasis is strictly controlled, and a key downregulator of the polyamine pool is the polyamine-inducible protein, antizyme 1 (OAZ1). OAZ1 inhibits polyamine uptake and targets ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, for proteasomal degradation. Here we report, for the first time, that polyamines induce dimerization of mouse recombinant full-length OAZ1, forming an (OAZ1)₂-Polyamine complex. Dimerization could be modulated by functionally active C-methylated spermidine mimetics (MeSpds) by changing the position of the methyl group along the Spd backbone—2-MeSpd was a poor inducer as opposed to 1-MeSpd, 3-MeSpd, and Spd, which were good inducers. Importantly, the ability of compounds to inhibit polyamine uptake correlated with the efficiency of the (OAZ1)₂-Polyamine complex formation. Thus, the (OAZ1)₂-Polyamine complex may be needed to inhibit polyamine uptake. The efficiency of polyamine-induced ribosomal +1 frameshifting of OAZ1 mRNA could also be differentially modulated by MeSpds—2-MeSpd was a poor inducer of OAZ1 biosynthesis and hence a poor downregulator of ODC activity unlike the other MeSpds. These findings offer new insight into the OAZ1-mediated regulation of polyamine homeostasis and provide the chemical tools to study it.

Amino acid analogues with a phosphorus-containing moiety replacing the carboxylic group are promising sources of biologically active compounds. The H-phosphinic group, with hydrogen–phosphorus–carbon (H-P-C) bonds and a flattened tetrahedral configuration, is a bioisostere of the carboxylic group. Consequently, amino-H-phosphinic acids undergo substrate-like enzymatic transformations, leading to new biologically active metabolites. Previous studies employing NMR-based metabolomic and proteomic analyses show that in Escherichia coli, α-KG-γ-P_H (the distal H-phosphinic analogue of α-ketoglutarate) can be converted into L-Glu-γ-P_H. Notably, α-KG-γ-P_H and L-Glu-γ-P_H are antibacterial compounds, but their intracellular targets only partially overlap. L-Glu-γ-P_H is known to be a substrate of aspartate transaminase and glutamate decarboxylase, but its substrate properties with NAD⁺-dependent glutamate dehydrogenase (GDH) have never been investigated. Compounds containing P-H bonds are strong reducing agents; therefore, enzymatic NAD⁺-dependent oxidation is not self-evident. Herein, we demonstrate that L-Glu-γ-P_H is a substrate of eukaryotic GDH and that the pH optimum of L-Glu-γ-P_H NAD⁺-dependent oxidative deamination is shifted to a slightly alkaline pH range compared to L-glutamate. By ³¹P NMR, we observe that α-KG-γ-P_H exists in a pH-dependent equilibrium of keto and germinal diol forms. Furthermore, the stereospecific enzymatic synthesis of α-KG-γ-P_H from L-Glu-γ-P_H using GDH is a possible route for its bio-based synthesis.