Potential applications for functional RNAs are rapidly expanding, not only to address functions based on primary nucleotide sequences, but also by RNA aptamer, which can suppress the activity of any target molecule. Aptamers are short DNA or RNA folded molecules that can be selected in vitro on the basis of their high affinity for a target molecule. Here, we demonstrate the ability of RNA aptamers to recognize-and bind to-human IgG with high specificity and affinity. An optimized 23-nucleotide aptamer, Apt8-2, was prepared, and was shown to bind to the Fc domain of human IgG, but not to other IgG's, with high affinity. Apt8-2 was observed to compete with protein A, but not with the Fcg receptor, for IgG binding. NMR chemical-shift analyses localized the aptamer-binding sites on the Fc subdomain, which partially overlaps the protein A binding site but not the Fcg receptor binding site. The tertiary structures of the predicted recognition sites on the Fc domain differ significantly between human IgG and other species of IgGs; this, in part, accounts for the high specificity of the selected aptamer. Apt8-2 can therefore be used as a protein A alternative for affinity purification of human IgG and therapeutic antibodies. Using Apt8-2 would have several potential advantages, raising the possibility of developing new applications based on aptamer design.

Aptamers targeting reverse transcriptase (RT) from HIV-1 inhibit viral replication in vitro, presumably by competing with binding of the primer/template complex. This site is not targeted by the currently available small-molecule anti-HIV-1 RT inhibitors. We have identified SY-3E4, a small-molecule inhibitor of HIV-1 RT, by applying a screening assay that utilizes a reporter-ribozyme regulated by the anti-HIV-1 RT aptamer. SY-3E4 displaces the aptamer from the protein, selectively inhibits DNA-dependent, but not RNA-dependent, polymerase activity, and inhibits the replication of both the wild-type virus and a multidrug-resistant strain. Analysis of available structural data of HIV-1 and HIV-2 RTs rationalizes many of the observed characteristics of the inhibitory profiles of SY-3E4 and the aptamer and suggests a previously not considered region in these RTs as a target for antiviral therapy. Our study reveals unexplored ways for rapidly identifying alternative small-molecule target sites in proteins and illustrates strategies for overcoming resistance-conferring mutations with small molecules.

A one‐pot three‐component coupling involving organolithium reagents, ATPH⋅cyclopentenone complex, and dihydrofuran⋅BCl3 complex (see scheme) gives moderate to good yields of the products with selective formation of either the 2,3‐cis or 2,3‐trans isomer, depending on the nature of the lithium reagent.

We have reported that mast cell chymase, an angiotensin II-generating enzyme, is important in cardiovascular tissues. Recently, we developed a new chymase-specific inhibitory RNA aptamer, HA28, and we evaluated the effects of HA28 on cardiac function and the mortality rate after myocardial infarction. Echocardiographic parameters, such as the left ventricular ejection fraction, fractional shortening, and the ratio of early to late ventricular filling velocities, were significantly improved by treatment with HA28 after myocardial infarction. The mortality rate was significantly reduced in the HA28-treated group. Cardiac chymase activity and chymase gene expression were significantly higher in the vehicle-treated myocardial infarction group, and these were markedly suppressed in the HA28-treated myocardial infarction group. The present study provides the first evidence that a single-stranded RNA aptamer that is a chymase-specific inhibitor is very effective in the treatment of acute heart failure caused by myocardial infarction. Chymase may be a new therapeutic target in post-myocardial infarction pathophysiology.

The binding of drugs to proteins is an important determinant of their efficacy and/or side-effects in clinical use. Many basic drugs bind mainly to a 1 -acid glycoprotein (AGP), an acute phase reactant, in plasma, but the plasma level of AGP varies greatly in various diseases. It is increased by inflammatory reaction, injury, and surgery, [1][2][3] but decreased by liver cancer, hepatitis and nephritic syndrome. [4][5][6] We have shown that there is a good correlation between the unbound fraction (f u ) of ropivacaine and AGP concentration after surgery. 3)Recently, it has been reported that human AGP exists as a heterogeneous population of three genetic variants of ORM1 F 1 and ORM1 S derived from the AGP-A gene and ORM2 A derived from the AGP-B/BЈ genes in the plasma of most individuals. 7,8) The variants can be identified by isoelectric focusing assay 9) and separated by chromatography on an immobilized copper(II) affinity adsorbent. 10,11) Fitos et al. demonstrated that the two main forms (the F 1 S and A variants) have different drug binding properties by means of circular dichroism (CD) spectroscopy using dicumarol and acridine orange as probes.12) It has been clarified that some drugs bind selectively to different AGP variants: for example, dipyridamole binds selectively to the F 1 S variant, and disopyramide to the A variant, whereas lidocaine binds to both the F 1 S and A variants. [13][14][15][16] However, there are few reports regarding the effects of drug-drug interaction arising from selective binding to AGP variants on the f u values of basic drugs. In this study, we evaluated the selective competitive binding of basic drugs to the F 1 S and A variants isolated from native human AGP. MATERIALS AND METHODS Materials Anapeine injection® (ropivacaine hydrochloride) and Xylocaine injection ® (lidocaine hydrochloride) were purchased from AstraZeneca Co., Ltd. (Osaka, Japan). Persantin injection ® (dipyridamole) was purchased from Boehringer Ingelheim Co., Ltd. (Tokyo, Japan). Vasolan injection ® (verapamil hydrochloride) was purchased from Eisai Co., Ltd. (Tokyo, Japan). Rythmodan P injection ® (disopyramide phosphate) was purchased from Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). Nifejipine and amlodipine besilate were purchased from Wako Pure Chemicals Co., Ltd. (Osaka, Japan). Herbesser injection ® (diltiazem hydrochloride) was purchased from Tanabe Co., Ltd. (Tokyo, Japan). Carvedilol was purchased from LKT Laboratories, Inc. (MN, U.S.A.). Native human a 1 -acid glycoprotein (total AGP) was purchased from Sigma-Aldrich Co., Ltd. (MO, U.S.A.). 3,3Ј-Methylene-bis(4-hydroxycoumarin)(dicumarol), acridine orange hemi(zinc chloride) salt and mepivacaine were purchased from Sigma-Aldrich Co., Ltd. Other chemicals were of reagent grade.Drug Assays Concentrations of ropivacaine, lidocaine and disopyramide were determined by GC-MS (Model GC-17 system Class 5000, Shimadzu, Kyoto, Japan). The assay for these drugs was carried out by using the procedure described for ropivacaine by Yokogawa et al. 3)Aliquots...