Recent studies suggest that apolipoprotein A-I (ApoA-I), the major protein constituent of high-density lipoprotein particles, plays a critical role in preserving cerebrovascular integrity and reducing Alzheimer's risk. ApoA-I present in brain is thought to be primarily derived from the peripheral circulation. Although plasma-to-brain delivery of ApoA-I is claimed to be handled by the blood-cerebrospinal fluid barrier (BCSFB), a contribution by the blood-brain barrier (BBB), which serves as a major portal for protein delivery to brain, cannot be ruled out. In this study, we assessed the permeability-surface area product (PS) of radioiodinated ApoA-I (125 I-ApoA-I) in various brain regions of wild-type rats after an intravenous bolus injection. The PS value at the cortex, caudate putamen, hippocampus, thalamus, brain stem, and cerebellum was found to be 0.39, 0.28, 0.28, 0.36, 0.69, and 0.76 (ml/g per second  10 26), respectively. Solutes delivered into brain via the BCSFB are expected to show greater accumulation in the thalamus due to its periventricular location. The modest permeability for 125 I-ApoA-I into the thalamus relative to other regions suggests that BCSFB transport accounts for only a portion of total brain uptake and thus BBB transport cannot be ruled out. In addition, we show that Alexa Flour 647-labeled ApoA-I (AF647-ApoA-I) undergoes clathrin-independent and cholesterol-mediated endocytosis in transformed human cerebral microvascular endothelial cells (hCMEC/D3). Further, Z-series confocal images of the hCMEC/D3 monolayers and Western blot detection of intact ApoA-I on the abluminal side demonstrated AF647-ApoA-I transcytosis across the endothelium. These findings implicate the BBB as a significant portal for ApoA-I delivery into brain.

Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by these results may be exploited to tune the rate of substrate hydrolysis to strategically alter the intracellular release of the product nucleoside monophosphate from the ProTide.

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Hint1 has recently emerged to be an important target of interest due to its involvement in the regulation of a broad range of CNS functions including opioid signaling, tolerance, neuropathic pain, and nicotine dependence. A series of inhibitors were rationally designed, synthesized, and tested for their inhibitory activity against hHint1 using isothermal titration calorimetry (ITC). The studies resulted in the development of the first small-molecule inhibitors of hHint1 with submicromolar binding affinities. A combination of thermodynamic and high-resolution X-ray crystallographic studies provides an insight into the biomolecular recognition of ligands by hHint1. These novel inhibitors have potential utility as molecular probes to better understand the role and function of hHint1 in the CNS.