Solvent-controlled synthesis of bulky and polar-bulky galactonoamidines.

The goal of this study was the design and synthesis of bulky and polar-bulky galactonoamidines that have a potential to interact with both catalytic amino acids in the active site of human α-galactosidase. While a library of more than 25 compounds was previously synthesized following established protocols, the coupling of the selected amines with activated perbenzylated galactothionolactam yielded only small amounts for some of the perbenzylated targets. A computational approach disclosed relative energy differences of selected adducts and suggested a solvent change that then allowed a successful synthesis of the precursor compounds in 20-75%. Subsequent attempts to globally deprotect perbenzylated galactonoamidines by Pd catalyzed hydrogenation resulted in unwanted Pd coordination, incomplete debenzylation reactions, partial compound hydrolysis, and even complete decomposition. A lengthy protocol was elaborated to purify the targeted carbohydrate derivatives after modified debenzylation conditions.

Copper(II)-N-hydroxy-N,N'-diarylformamidine complexes: Synthesis, crystal structures, antibacterial and molecular docking studies.

A series of Cu(II) complexes were synthesized by using N-hydroxy-N,N'-diarylformamidine ligands: N-hydroxy-N,N'-(phenyl)formamidine (L1), N-hydroxy-N'-(4-methylphenyl)formamidine (L2), N-hydroxy-N,N'-(2,6-dimethylphenyl)formamidine (L3), N-hydroxy-N,N'-(2,6-diisopropylphenyl)formamidine (L4). Reaction of ligands L1-L4 with hydrated copper acetate furnished mononuclear Cu(II) complexes 1-4 with general formula [Cu-(L)2]. The molecular structures of complexes 3 and 4, as determined by single crystal X-ray diffraction, showed both to have square planar geometry with a near C2 symmetry. The antimicrobial potency of all four complexes was evaluated against three gram-(-) bacteria (S. typhimurium, P. aeruginosa, and E. coli) and two gram-(+) bacteria (Methicillin-resistant S. aureus (MRSA) and S. aureus), with ciprofloxacin as the reference drug. All tested complexes were inactive against gram-(+) bacteria strains except for complex 1, which displayed excellent activity when compared to the reference. Molecular docking studies showed that hydrogen bonding, pi-sigma and van der Waals interactions are prominent complex-protein connections, with complex 2 displaying good binding affinities with the studied biological targets.

A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures.

G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small "druglike" optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure-activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.

Discovery of 5-(N-hydroxycarbamimidoyl) benzofuran derivatives as novel indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) are rate-limiting enzymes in the kynurenine pathway (KP) of l-tryptophan (l-Trp) metabolism and are becoming key drug targets in the combination therapy of checkpoint inhibitors in immunoncology. To discover a selective and potent IDO1 inhibitor, a structure-activity relationship (SAR) study of N-hydroxybenzofuran-5-carboximidamide as a novel scaffold was investigated in a systematic manner. Among the synthesized compounds, the N-3-bromophenyl derivative 19 showed the most potent inhibition, with an IC50 value of 0.44 µM for the enzyme and 1.1 µM in HeLa cells. The molecular modeling of 19 with the X-ray crystal structure of IDO1 indicated that dipole-ionic interactions with heme iron, halogen bonding with Cys129 and the two hydrophobic interactions were important for the high potency of 19.

AAPH or Peroxynitrite-Induced Biorelevant Oxidation of Methyl Caffeate Yields a Potent Antitumor Metabolite.

Hydroxycinnamic acids represent a versatile group of dietary plant antioxidants. Oxidation of methyl-p-coumarate (pcm) and methyl caffeate (cm) was previously found to yield potent antitumor metabolites. Here, we report the formation of potentially bioactive products of pcm and cm oxidized with peroxynitrite (ONOO¯), a biologically relevant reactive nitrogen species (RNS), or with α,α'-azodiisobutyramidine dihydrochloride (AAPH) as a chemical model for reactive oxygen species (ROS). A continuous flow system was developed to achieve reproducible in situ ONOO¯ formation. Reaction mixtures were tested for their cytotoxic effect on HeLa, SiHa, MCF-7 and MDA-MB-231 cells. The reaction of pcm with ONOO¯ produced two fragments, an o-nitrophenol derivative, and a new chlorinated compound. Bioactivity-guided isolation from the reaction mixture of cm with AAPH produced two dimerization products, including a dihydrobenzofuran lignan that exerted strong antitumor activity in vitro, and has potent in vivo antimetastatic activity which was previously reported. This compound was also detected from the reaction between cm and ONOO¯. Our results demonstrate the ROS/RNS dependent formation of chemically stable metabolites, including a potent antitumor agent (5), from hydroxycinnamic acids. This suggests that diversity-oriented synthesis using ROS/RNS to obtain oxidized antioxidant metabolite mixtures may serve as a valid natural product-based drug discovery strategy.

Mechanism of the effect of 2, 2'-azobis (2-amidinopropane) dihydrochloride simulated lipid oxidation on the IgG/IgE binding ability of ovalbumin.

In this work, the mechanism of the effect of lipid oxidation on the IgG/IgE binding ability of ovalbumin (OVA) was investigated via the peroxyl radicals produced by 2, 2'-azobis (2-amidinopropane) dihydrochloride to simulate lipid oxidation. Results showed that the structure of OVA unfolded partially with an increase in oxidation degree, leading to the exposure of the allergenic epitopes and increasing the IgG/IgE binding ability of OVA. Nine oxidation sites were found on the α-helix, and these sites may unwind the α-helix and expose the allergenic epitopes on the OVA surface, leading to antibody recognition and combination. Consequently, the IgG/IgE binding ability of OVA was increased. In conclusion, the allergenic capacity of OVA can be promoted by modifying peroxyl radical oxidation in processing egg products.

Synthesis, docking studies, and pharmacological evaluation of 5HT2C ligands containing the N'-cyanoisonicotinamidine or N'-cyanopicolinamidine nucleus.

N'-Cyanoisonicotinamidine and N'-cyanopicolinamidine derivatives, linked to an arylpiperazine moiety, were prepared and their affinities to the 5-HT1A , 5-HT2A , and 5-HT2C receptors were evaluated. Several of the newly synthesized compounds, tested by binding studies, showed nanomolar affinity at the 5-HT1A and 5-HT2C receptors and moderate or no affinity for other relevant receptors (D1 , D2 , α1 , and α2 ). Compound 8e (Ki = 21.4 nM) was the most affine for the 5-HT2C receptor, showing, at the same time, a high selectivity with respect to the other receptors analyzed. Compounds 4a and 4c, instead, showed an interesting mixed 5-HT1A /5-HT2C activity with Ki values of 21.3/11.5 and 23.2/6.48 nM, respectively. The compounds with better affinity and selectivity binding profiles toward 5-HT2C (4a, 4c, 8b, and 8e) were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that compounds 4a, 8b, and 8e exerted antidepressant-like effects and 4a and 8e revealed also significant anxiolytic properties. Among the developed derivatives, the most promising compound seems to be 4a, which displayed antipsychotic-, antidepressant- and anxiolytic-like properties. No side effects, like catalepsy, motor-impairment or ethanol-potentiating effects, were observed after the injection of the tested compounds.

Fragment-Based Discovery of an Apolipoprotein E4 (apoE4) Stabilizer.

Apolipoprotein E is a 299-residue lipid carrier protein produced in both the liver and the brain. The protein has three major isoforms denoted apoE2, apoE3, and apoE4 which differ at positions 112 and 158 and which occur at different frequencies in the human population. Genome-wide association studies indicate that the possession of two apoE4 alleles is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). In an attempt to identify a small molecule stabilizer of apoE4 function that may have utility as a therapy for Alzheimer's disease, we carried out an NMR-based fragment screen on the N-terminal domain of apoE4 and identified a benzyl amidine based fragment binder. In addition to NMR, binding was characterized using various other biophysical techniques, and a crystal structure of the bound core was obtained. Core elaboration ultimately yielded a compound that showed activity in an IL-6 and IL-8 cytokine release assay.

Charged phospholipid effects on AAPH oxidation assay as determined using liposomes.

The capacity of molecules to inhibit oxidation is widely tested using liposomes as host matrices of the antioxidant molecule of interest. Spectroscopic assays are readily used for this purpose, specifically assays using 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). In this work the effect that charged lipids have on an AAPH antioxidation assay using 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY® 581/591) as the reporter molecule was investigated. We measured the diameter, zeta potential and spectroscopic rate of decay and area-under-the-curve (AUC) associated with liposomes containing C11-BODIPY® 581/591 at varying molar percentages (0-10 mol%) of charged (cationic or anionic) lipids and compared the results. We showed that although increasing amounts of cationic or anionic lipids did change the diameter of the liposomes, size had little to no effect on the area-under-the-curve or decay rate of fluorescence. Increased (more positive) or decreased (more negative) zeta potentials did, on the other hand, affect the spectroscopic decay rates and area-under-the-curve. The results demonstrate the importance of considering the presence of charged lipids in the AAPH antioxidation assay.

Development and validation of GC-MS methods for the comprehensive analysis of amino acids in plasma and urine and applications to the HELLP syndrome and pediatric kidney transplantation: evidence of altered methylation, transamidination, and arginase activity.

We developed and validated gas chromatography-mass spectrometry (GC-MS) methods for the simultaneous measurement of amino acids and their metabolites in 10-µL aliquots of human plasma and urine. De novo synthesized trideutero-methyl esters were used as internal standards. Plasma proteins were precipitated by acidified methanol and removed by centrifugation. Supernatants and native urine were evaporated to dryness. Amino acids were first esterified using 2 M HCl in methanol and then amidated using pentafluoropropionic anhydride for electron-capture negative-ion chemical ionization. Time programmes were used for the gas chromatograph oven and the selected-ion monitoring of specific anions. The GC-MS methods were applied in clinical studies on the HELLP syndrome and pediatric kidney transplantation (KTx) focusing on L-arginine-related pathways. We found lower sarcosine (N-methylglycine) and higher asymmetric dimethylarginine (ADMA) plasma concentrations in HELLP syndrome women (n = 7) compared to healthy pregnant women (n = 5) indicating altered methylation. In plasma of pediatric KTx patients, lower guanidinoacetate and homoarginine concentrations were found in plasma but not in urine samples of patients treated with standard mycophenolate mofetil-based immunosuppression (MMF; n = 22) in comparison to matched patients treated with MMF-free immunosuppression (n = 22). On average, the global arginine bioavailability ratio was by about 40% lower in the MMF group compared to the EVR group (P = 0.004). Mycophenolate, the major pharmacologically active metabolite of MMF, is likely to inhibit the arginine:glycine amidinotransferase (AGAT), and to enhance arginase activity in leukocytes and other types of cell of MMF-treated children.

Pharmacokinetics and brain distribution of amitraz and its metabolites in rats.

Amitraz is an acaricide and insecticide widely used in agriculture and veterinary medicine. Although central nervous system (CNS) toxicity is one of major toxicities following oral ingestion of amitraz, the understanding of the cause of the toxicity is limited. This study evaluated the systemic and brain exposure of amitraz and its major metabolites, BTS27271, 2',4'-formoxylidide, and 2,4-dimethylaniline following administration of amitraz in male Sprague-Dawley rats. Significant metabolism of amitraz was observed following the intravenous and oral administration. Amitraz related metabolites were majority of the total exposure observed, especially following oral administration. BTS27271 had higher brain exposure than amitraz and its other metabolites, which was due to low plasma protein binding but high brain tissue binding of BTS27271. Since BTS27271 has similar or higher toxicity and α2-adrenoreceptor agonist potency than amitraz, its exposure in brain tissues may be the major cause of CNS toxicity of amitraz in animals and humans.

Synthesis and characterization of 11 C-labeled benzyl amidine derivatives as PET radioligands for GluN2B subunit of the NMDA receptors.

GluN2B-containing NMDA receptors (NMDARs) play fundamental roles in learning and memory, although they are also associated with various brain disorders. In this study, we synthesized and evaluated three 11 C-labeled N-benzyl amidine derivatives 2-[11 C]methoxybenzyl) cinnamamidine ([11 C]CBA), N-(2-[11 C]methoxybenzyl)-2-naphthamidine ([11 C]NBA), and N-(2-[11 C]methoxybenzyl)quinoline-3-carboxamidine ([11 C]QBA) as PET radioligands for these receptors. The 11 C-benzyl amidines were synthesized via conventional methylation of corresponding des-methyl precursors with [11 C]CH3 I. In vitro binding characteristics were examined in brain sagittal sections using various GluN2B modulators and off-target ligands. Further, in vivo brain distribution studies were performed in normal mice. The 11 C-labeled benzyl amidines showed high-specific binding to the GluN2B subunit at in vitro. In particular, the quinoline derivative [11 C]QBA had the best binding properties in terms of high-brain localization to GluN2B-rich regions and specificity to the GluN2B subunit. Conversely, these 11 C-radioligands showed the brain distributions were inconsistent with GluN2B expression in biodistribution experiments. The majority of the radiolabeled compounds were identified as metabolized forms of which amido derivatives seemed to be the major species. Although these 11 C-ligands had high-specific binding to the GluN2B subunit, significant improvement in metabolic stability is necessary for successful positron emission tomography (PET) imaging of the GluN2B subunit of NMDARs.

Evaluating hydrophobic galactonoamidines as transition state analogs for enzymatic ß-galactoside hydrolysis.

A spectroscopic examination of six galactonoamidines with inhibition constants and efficacy in the low nanomolar concentration range (Ki = 6-11 nM, IC50 = 12-36 nM) suggested only two of them as putative transition state analogs for the hydrolysis of ß-galactosides by ß-galactosidase (A. oryzae). A rationale for the experimental results was elaborated using docking and molecular dynamics studies. An analysis of the combined observations reveals several common factors of the compounds suggested as transition state analogs (TSAs): the putative TSAs have a similar orientation in the active site; show conserved positioning of the glycon; display a large number of H-bond interactions toward the catalytically active amino acid residues via their glycon; and exhibit hydrophobic interactions at the outer rim of the active site with small changes of the position and orientation of their respective aglycons.

Uniformity and Diversity of Cortical Projections to Precuneate Areas in the Macaque Monkey: What Defines Area PGm?

We report on the corticocortical connections of areas on the mesial surface of the macaque posterior parietal cortex, based on 10 retrograde tracer injections targeting different parts of the precuneate gyrus. Analysis of afferent connections supported the existence of two areas: PGm (also known as 7 m) and area 31. Both areas received major afferents from the V6A complex and from the external subdivision of area 23, but they differed in most other aspects. Area 31 showed greater emphasis on connections with premotor and parietal sensorimotor areas, whereas PGm received a greater proportion of its afferents from visuomotor structures involved in spatial cognition (including the lateral intraparietal cortex, inferior parietal lobule, and the putative visual areas in the ventral part of the precuneus). Medially, the anterior cingulate cortex (area 24) preferentially targeted area 31, whereas retrosplenial areas preferentially targeted PGm. These results indicate that earlier views on the connections of PGm were based on tracer injections that included parts of adjacent areas (including area 31), and prompt a reassessment of the limits of PGm. Our findings are compatible with a primary role of PGm in visuospatial cognition (including navigation), while supporting a role for area 31 in sensorimotor planning and coordination.

Morphology of P2X3-immunoreactive nerve endings in the rat tracheal mucosa.

Nerve endings with immunoreactivity for the P2X3 purinoreceptor (P2X3) in the rat tracheal mucosa were examined by immunohistochemistry of whole-mount preparations with confocal scanning laser microscopy. P2X3 immunoreactivity was observed in ramified endings distributed in the whole length of the trachea. The myelinated parent axons of P2X3-immunoreactive nerve endings ramified into several branches that extended two-dimensionally in every direction at the interface between the epithelial layer and lamina propria. The axonal branches of P2X3-immunoreactive endings branched off many twigs located just beneath the epithelium, and continued to intraepithelial axon terminals. The axon terminals of P2X3-immunoreactive endings were beaded, rounded, or club-like in shape and terminated between tracheal epithelial cells. Flat axon terminals sometimes partly ensheathed neuroendocrine cells with immunoreactivity for SNAP25 or CGRP. Some axons and axon terminals with P2X3 immunoreactivity were immunoreactive for P2X2, while some terminals were immunoreactive for vGLUT2. Furthermore, a retrograde tracing method using fast blue (FB) revealed that 88.4% of FB-labeled cells with P2X3 immunoreactivity originated from the nodose ganglion. In conclusion, P2X3-immunoreactive nerve endings in the rat tracheal mucosa have unique morphological characteristics, and these endings may be rapidly adapting receptors and/or irritant receptors that are activated by mucosal irritant stimuli.

Thalamic connections of the core auditory cortex and rostral supratemporal plane in the macaque monkey.

In the primate auditory cortex, information flows serially in the mediolateral dimension from core, to belt, to parabelt. In the caudorostral dimension, stepwise serial projections convey information through the primary, rostral, and rostrotemporal (AI, R, and RT) core areas on the supratemporal plane, continuing to the rostrotemporal polar area (RTp) and adjacent auditory-related areas of the rostral superior temporal gyrus (STGr) and temporal pole. In addition to this cascade of corticocortical connections, the auditory cortex receives parallel thalamocortical projections from the medial geniculate nucleus (MGN). Previous studies have examined the projections from MGN to auditory cortex, but most have focused on the caudal core areas AI and R. In this study, we investigated the full extent of connections between MGN and AI, R, RT, RTp, and STGr using retrograde and anterograde anatomical tracers. Both AI and R received nearly 90% of their thalamic inputs from the ventral subdivision of the MGN (MGv; the primary/lemniscal auditory pathway). By contrast, RT received only ∼45% from MGv, and an equal share from the dorsal subdivision (MGd). Area RTp received ∼25% of its inputs from MGv, but received additional inputs from multisensory areas outside the MGN (30% in RTp vs. 1-5% in core areas). The MGN input to RTp distinguished this rostral extension of auditory cortex from the adjacent auditory-related cortex of the STGr, which received 80% of its thalamic input from multisensory nuclei (primarily medial pulvinar). Anterograde tracers identified complementary descending connections by which highly processed auditory information may modulate thalamocortical inputs.

Tyrosine oxidation and nitration in transmembrane peptides is connected to lipid peroxidation.

Tyrosine nitration is an oxidative post-translational modification that can occur in proteins associated to hydrophobic bio-structures such as membranes and lipoproteins. In this work, we have studied tyrosine nitration in membranes using a model system consisting of phosphatidylcholine liposomes with pre-incorporated tyrosine-containing 23 amino acid transmembrane peptides. Tyrosine residues were located at positions 4, 8 or 12 of the amino terminal, resulting in different depths in the bilayer. Tyrosine nitration was accomplished by exposure to peroxynitrite and a peroxyl radical donor or hemin in the presence of nitrite. In egg yolk phosphatidylcholine liposomes, nitration was highest for the peptide with tyrosine at position 8 and dramatically increased as a function of oxygen levels. Molecular dynamics studies support that the proximity of the tyrosine phenolic ring to the linoleic acid peroxyl radicals contributes to the efficiency of tyrosine oxidation. In turn, α-tocopherol inhibited both lipid peroxidation and tyrosine nitration. The mechanism of tyrosine nitration involves a "connecting reaction" by which lipid peroxyl radicals oxidize tyrosine to tyrosyl radical and was fully recapitulated by computer-assisted kinetic simulations. Altogether, this work underscores unique characteristics of the tyrosine oxidation and nitration process in lipid-rich milieu that is fueled via the lipid peroxidation process.

Altered intracortical myelin staining in the dorsolateral prefrontal cortex in severe mental illness.

Imaging and postmortem studies into the severe mental illnesses of major depressive disorder (MDD), bipolar disorder (BD), and schizophrenia (SZ) have revealed deficiencies in the myelination of deep white matter tracts of the brain. Recent studies have further suggested that deficits could extend to myelinated fibers running through the cortex in those illnesses. Disruptions in this intracortical myelin may underlie functional symptomology in MDD, BD, and SZ; thus, in this study, we hypothesized that individuals with these illnesses may have reduced myelin staining relative to controls in the cerebral cortex. We stained 60 sections of dorsolateral prefrontal cortex for myelin with Luxol® fast blue in four groups: 15 BD, 15 MDD, 15 SZ, and 15 controls with no psychiatric illness. We digitally measured optical tissue attenuation reflecting the amount of myelin staining across six cortical depths in the middle frontal gyrus (MFG), in superficial white matter in the crown of the MFG, and in deep white matter. We found that a diagnosis of MDD or SZ meant that optical tissue attenuation was significantly reduced in the shallowest depths of the cortex. Furthermore, there was a trend toward reduced optical tissue attenuation in all illnesses across all myelinated regions we studied. These results encourage future studies into potential reductions in intracortical myelin in severe mental illness.

Modulation of Specific Sensory Cortical Areas by Segregated Basal Forebrain Cholinergic Neurons Demonstrated by Neuronal Tracing and Optogenetic Stimulation in Mice.

Neocortical cholinergic activity plays a fundamental role in sensory processing and cognitive functions. Previous results have suggested a refined anatomical and functional topographical organization of basal forebrain (BF) projections that may control cortical sensory processing in a specific manner. We have used retrograde anatomical procedures to demonstrate the existence of specific neuronal groups in the BF involved in the control of specific sensory cortices. Fluoro-Gold (FlGo) and Fast Blue (FB) fluorescent retrograde tracers were deposited into the primary somatosensory (S1) and primary auditory (A1) cortices in mice. Our results revealed that the BF is a heterogeneous area in which neurons projecting to different cortical areas are segregated into different neuronal groups. Most of the neurons located in the horizontal limb of the diagonal band of Broca (HDB) projected to the S1 cortex, indicating that this area is specialized in the sensory processing of tactile stimuli. However, the nucleus basalis magnocellularis (B) nucleus shows a similar number of cells projecting to the S1 as to the A1 cortices. In addition, we analyzed the cholinergic effects on the S1 and A1 cortical sensory responses by optogenetic stimulation of the BF neurons in urethane-anesthetized transgenic mice. We used transgenic mice expressing the light-activated cation channel, channelrhodopsin-2, tagged with a fluorescent protein (ChR2-YFP) under the control of the choline-acetyl transferase promoter (ChAT). Cortical evoked potentials were induced by whisker deflections or by auditory clicks. According to the anatomical results, optogenetic HDB stimulation induced more extensive facilitation of tactile evoked potentials in S1 than auditory evoked potentials in A1, while optogenetic stimulation of the B nucleus facilitated either tactile or auditory evoked potentials equally. Consequently, our results suggest that cholinergic projections to the cortex are organized into segregated pools of neurons that may modulate specific cortical areas.

T-2307, a novel arylamidine, is transported into Candida albicans by a high-affinity spermine and spermidine carrier regulated by Agp2.

OBJECTIVES: T-2307, a novel arylamidine, exhibits potent broad-spectrum activities against pathogenic fungi, particularly Candida albicans. We previously reported that T-2307 uptake was mainly mediated by a saturable high-affinity carrier at the MIC for C. albicans. Since we hypothesized that the potent anticandidal activity arose from accumulation via the high-affinity carrier, we characterized the specificity and kinetic features of the carrier. METHODS: The MICs of T-2307 for C. albicans strains were evaluated in the presence and absence of potential competitive substrates. The cells were exposed to [(14)C]T-2307, [(14)C]spermine or [(14)C]spermidine in the presence of unlabelled T-2307, pentamidine, propamidine, or competitive substrates if necessary, and the radioactivity in the cells was measured. C. albicans gene deletion was performed using a one-step PCR-based technique. RESULTS: Coapplication with exogenous spermine or spermidine decreased the antifungal activity and uptake of T-2307 in C. albicans strains. T-2307 competitively inhibited spermine and spermidine uptake with inhibition constants similar to its Km for the high-affinity carrier. The comparison of MICs and kinetic values between T-2307 and other diamidine compounds suggested that the different antifungal properties could be partially attributable to the variations in their affinity with the carrier. Studies of gene deletion mutants revealed that T-2307 was transported into C. albicans by a high-affinity spermine and spermidine carrier regulated by Agp2. CONCLUSIONS: Uptake of T-2307 via the high-affinity spermine and spermidine carrier regulated by Agp2 could contribute to its potent antifungal activity. Further investigation is required to identify the high-affinity carrier for potential targeting with novel therapies.