Two-dimensional combinatorial screening and the RNA Privileged Space Predictor program efficiently identify aminoglycoside-RNA hairpin loop interactions.

Herein, we report the identification of RNA hairpin loops that bind derivatives of kanamycin A, tobramycin, neamine, and neomycin B via two-dimensional combinatorial screening, a method that screens chemical and RNA spaces simultaneously. An arrayed aminoglycoside library was probed for binding to a 6-nucleotide RNA hairpin loop library (4096 members). Members of the loop library that bound each aminoglycoside were excised from the array, amplified and sequenced. Sequences were analyzed with our newly developed RNA Privileged Space Predictor (RNA-PSP) program, which analyzes selected sequences to identify statistically significant trends. RNA-PSP identified the following unique trends: 5'UNNNC3' loops for the kanamycin A derivative (where N is any nucleotide); 5'UNNC3' loops for the tobramycin derivative; 5'UNC3' loops for the neamine derivative; and 5'UNNG3' loops for the neomycin B derivative. The affinities and selectivities of a subset of the ligand-hairpin loop interactions were determined. The selected interactions have K(d) values ranging from 10 nM to 605 nM. Selectivities ranged from 0.4 to >200-fold. Interestingly, the results from RNA-PSP are able to qualitatively predict specificity based on overlap between the RNA sequences selected for the ligands. These studies expand the information available on small molecule-RNA motif interactions, which could be useful to design ligands targeting RNA.

A rapid and sensitive LC-MS/MS method for determination of fluconazole in human plasma and its application in infants with Candida infections.

A rapid and sensitive LC-MS/MS method was developed to quantify fluconazole in human plasma. Seventy microliters of plasma were treated with protein precipitation procedures. Chromatographic separation was achieved on a C18 column using a gradient mobile phase of acetonitrile and water in 0.1% formic acid. Fluconazole and its deuterium-labeled internal standard were monitored in positive mode using electrospray ionization source. The method was fully validated over the range of 0.01 to 10 microg/mL. Intraday and interday precision ranged from 2.84% to 10.8% and 5.27% to 11.5%, respectively. The process recovery efficiency for fluconazole ranged from 98.6% to 104.4%. No carryover and minimal matrix effects were observed. Acceptable stability of fluconazole in blood at room temperature for up to 72 hours guaranteed that fluconazole concentrations in scavenged blood specimens were usable for infant PK analysis and model development. This method has been utilized for a fluconazole pharmacokinetic trial with 55 preterm and term infants younger than 90 days of age. The fast sample preparation cycle and lower limit of quantitation make this method a potential tool for therapeutic drug monitoring of fluconazole to optimize pediatric antifungal therapy. Optimal dose regimen of fluconazole in neonates and young infants might be achieved with application of TDM and pharmacometric approach designed to achieve AUC/MIC >50 for most Candida species with a MIC90 less than 8 microg/mL.

Infant with protein C deficiency and stroke in the setting of iron deficiency anemia.

We report an 18-month-old infant with ischemic stroke, neurocognitive impairment, and psychomotor retardation in the setting of severe iron deficiency anemia. Although an uncommon outcome in anemic children, stroke is important to consider as a cause for developmental delay in children with iron deficiency anemia.

Two-dimensional combinatorial screening identifies specific 6'-acylated kanamycin A- and 6'-acylated neamine-RNA hairpin interactions.

Herein, we report the RNA hairpin loops from a six-nucleotide hairpin library that bind 6'-acylated kanamycin A (1) and 6'-acylated neamine (2) identified by two-dimensional combinatorial screening (2DCS). Hairpins selected to bind 1 have K(d)'s ranging from 235 to 1035 nM, with an average K(d) of 618 nM. For 2, the selected hairpins bind with K(d)'s ranging from 135 to 2300 nM, with an average K(d) of 1010 nM. The selected RNA hairpin-ligand interactions are also specific for the ligand that they were selected to bind compared with the other arrayed ligand. For example, the mixture of hairpins selected for 1 on average bind 33-fold more tightly to 1 than to 2, while the mixtures of hairpins selected for 2 on average bind 11-fold more tightly to 2 than to 1. Secondary structure prediction of the selected sequences was completed to determine the motifs that each ligand binds, and the hairpin loop preferences for 1 and 2 were computed. For 1, the preferred hairpin loops contain an adenine separated by at least two nucleotides from a cytosine, for example, ANNCNN (two-tailed p-value = 0.0010) and ANNNCN (two-tailed p-value <0.0001). For 2, the preferred hairpin loops contain both 5'GC and 5'CG steps (two-tailed p-value <0.0001). These results expand the information available on the RNA hairpin loops that bind small molecules and could prove useful for targeting RNA.

Two-dimensional combinatorial screening identifies specific aminoglycoside-RNA internal loop partners.

Herein is described the identification of RNA internal loops that bind to derivatives of neomycin B, neamine, tobramycin, and kanamycin A. RNA loop-ligand partners were identified by a two-dimensional combinatorial screening (2DCS) platform that probes RNA and chemical spaces simultaneously. In 2DCS, an aminoglycoside library immobilized onto an agarose microarray was probed for binding to a 3 x 3 nucleotide RNA internal loop library (81,920 interactions probed in duplicate in a single experiment). RNAs that bound aminoglycosides were harvested from the array via gel excision. RNA internal loop preferences for three aminoglycosides were identified from statistical analysis of selected structures. This provides consensus RNA internal loops that bind these structures and include: loops with potential GA pairs for the neomycin derivative, loops with potential GG pairs for the tobramycin derivative, and pyrimidine-rich loops for the kanamycin A derivative. Results with the neamine derivative show that it binds a variety of loops, including loops that contain potential GA pairs that also recognize the neomycin B derivative. All studied selected internal loops are specific for the aminoglycoside that they were selected to bind. Specificity was quantified for 16 selected internal loops by studying their binding to each of the arrayed aminoglycosides. Specificities ranged from 2- to 80-fold with an average specificity of 20-fold. These studies show that 2DCS is a unique platform to probe RNA and chemical space simultaneously to identify specific RNA motif-ligand interactions.

A sensitive and rapid liquid chromatography-tandem mass spectrometry method for the quantification of the novel neurokinin-1 receptor antagonist aprepitant in rhesus macaque plasma, and cerebral spinal fluid, and human plasma with application in translational NeuroAIDs research.

A sensitive and rapid liquid chromatography-tandem mass spectrometry method has been developed for to assess therapeutic exposures of aprepitant in HIV-infected patients and rhesus macaques. The method utilized a simple sample-preparation procedure of protein precipitation with methanol. Chromatographic separation was performed on a reversed phase C(8) column (Hypersil Gold, 50 mm x 2.1 mm, 3 microm) using a mobile phase composed of acetonitrile and water in 0.5% formic acid through gradient elution. Electro-spray ionization in positive mode was incorporated in the tandem mass spectrometric detection. The lower limit of quantitation of aprepitant in plasma of rhesus macaques and human and cerebral spinal fluid of rhesus macaques were 1, 1, and 0.1 ng/mL, respectively. The method has been successfully employed to measure aprepitant in preclinical and clinical samples collected from three SIV-infected rhesus macaques and ten patients with HIV infection. In conclusion, this liquid chromatography-tandem mass spectrometry method is suitable for preclinical-clinical translational research exploring exposure-response relationships with aprepitant as well as therapeutic drug monitoring of aprepitant.