Adaptive Focused Acoustics™ for Nanosuspensions to Enable Pharmacology Assessment of Poorly Soluble Molecules in Lead Optimization.

In Drug Discovery, pharmacology studies often require benign formulation compositions for safe administration in animal models. Here, we applied Adaptive Focused Acoustics™ (AFA) to a molecular scaffold with challenging physicochemical properties for intraperitoneal administration. Nanosuspensions can be prepared at small scales and provide broad applicability. Our results show that nanosuspension formulations prepared by AFA have improved PK performance relative to a DMSO solution formulation that is prone to precipitation in-vivo.

Discovery of Highly Isoform Selective Thiazolopiperidine Inhibitors of Phosphoinositide 3-Kinase γ.

A series of high affinity second-generation thiazolopiperidine inhibitors of PI3Kγ were designed based on some general observations around lipid kinase structure. Optimization of the alkylimidazole group led to inhibitors with higher levels of PI3Kγ selectivity. Additional insights into PI3K isoform selectivity related to sequence differences in a known distal hydrophobic pocket are also described.

Detection of an abasic site in RNA with stem-loop DNA beacons: application to an activity assay for Ricin Toxin A-Chain.

The catalytic ability of Ricin Toxin A-Chain (RTA) to create an abasic site in a 14-mer stem-tetraloop RNA is exploited for its detection. RTA catalyzes the hydrolysis of the N-glycosidic bond of a specific adenosine in the GAGA tetraloop of stem-loop RNA. Thus, a 14-mer stem-loop RNA substrate containing an intact "GAGA" sequence can be discriminated from the product containing an abasic "GabGA" sequence by hybridization with a 14-mer DNA stem-loop probe sequence and following the fluorescent response of the heteroduplexes. Three DNA beacon probe designs are described. Beacon 1 probe is a stem-loop structure and has a fluorophore and a quencher covalently linked to the 5'- and 3'-ends. In this format the probe-substrate heteroduplex gives a fluorescent signal while the probe-product one remains quenched. Beacon 2 is a modified version of 1 and incorporates a pyrene deoxynucleoside for recognition of the abasic site. In this format both the substrate and product heteroduplexes give a fluorescent response. Beacon 3 utilizes a design where the fluorophore is on the substrate RNA sequence at its 5'-end while the quencher is on the probe DNA sequence at its 3'-end. In this format the fluorescence of the substrate-probe heteroduplex is quenched while that of the product-probe one is enhanced. The lower limit of detection with beacons is 14 ng/mL of RTA.

Vinyldeoxyadenosine in a sarcin-ricin RNA loop and its binding to ricin toxin a-chain.

8-Vinyl-2'-deoxyadenosine (8vdA) is a fluorophore with a quantum yield comparable to that of 2-aminopurine nucleoside. 8vdA was incorporated into a 10-mer stem-tetraloop RNA (8vdA-10) structure for characterization of the properties of the base, 8-vinyladenine (8-vA), with respect to adenine as a substrate or inhibitor for ribosome-inactivating proteins. Ricin toxin A-chain (RTA) and pokeweed antiviral protein (PAP) catalyze the release of adenine from a specific adenosine on a stem-tetraloop (GAGA) sequence at the elongation factor (eEF2) binding site of the 28S subunit of eukaryotic ribosomes, thereby arresting translation. RTA does not catalyze the release of 8-vinyladenine from 8vdA-10. Molecular dynamics simulations implicate a role for Arg180 in oxacarbenium ion destabilization and the lack of catalysis. However, 8vdA-10 is an active site analogue and inhibits RTA with a Ki value of 2.4 microM. Adenine is also released from the second adenosine in the modified tetraloop, demonstrating an alternative mode for the binding of this motif in the RTA active site. The 8vdA analogue defines the specificities of RTA for the two adenylate depurination sites in a RNA substrate with a GAGA tetraloop. The rate of nonenzymatic acid-catalyzed solvolysis of 8-vinyladenine from the stem-loop RNA is described. Unlike RTA, PAP catalyzes the slow release of 8-vinyladenine from 8vdA-10. The isolation of 8-vA and its physicochemical characterization is described.

Circular DNA and DNA/RNA hybrid molecules as scaffolds for ricin inhibitor design.

Ricin Toxin A-chain (RTA) catalyzes the hydrolytic depurination of A4324, the first adenosine of the GAGA tetra-loop portion of 28S eukaryotic ribosomal RNA. Truncated stem-loop versions of the 28S rRNA are RTA substrates. Here, we investigate circular DNA and DNA/RNA hybrid GAGA sequence oligonucleotides as minimal substrates and inhibitor scaffolds for RTA catalysis. Closing the 5'- and 3'-ends of a d(GAGA) tetraloop creates a substrate with 92-fold more activity with RTA (kcat/Km) than that for the d(GAGA) linear form. Circular substrates have catalytic rates (kcat) comparable to and exceeding those of RNA and DNA stem-loop substrates, respectively. RTA inhibition into the nanomolar range has been achieved by introducing an N-benzyl-hydroxypyrrolidine (N-Bn) transition state analogue at the RTA depurination site in a circular GAGA motif. The RNA/DNA hybrid oligonucleotide cyclic GdAGA provides a new scaffold for RTA inhibitor design, and cyclic G(N-Bn)GA is the smallest tight-binding RTA inhibitor (Ki = 70 nM). The design of such molecules that lack the base-paired stem-loop architecture opens new chemical synthetic approaches to RTA inhibition.

Structural and kinetic characterization of Escherichia coli TadA, the wobble-specific tRNA deaminase.

The essential tRNA-specific adenosine deaminase catalyzes the deamination of adenosine to inosine at the wobble position of tRNAs. This modification allows for a single tRNA species to recognize multiple synonymous codons containing A, C, or U in the last (3'-most) position and ensures that all sense codons are appropriately decoded. We report the first combined structural and kinetic characterization of a wobble-specific deaminase. The structure of the Escherichia coli enzyme clearly defines the dimer interface and the coordination of the catalytically essential zinc ion. The structure also identifies the nucleophilic water and highlights residues near the catalytic zinc likely to be involved in recognition and catalysis of polymeric RNA substrates. A minimal 19 nucleotide RNA stem substrate has permitted the first steady-state kinetic characterization of this enzyme (k(cat) = 13 +/- 1 min(-)(1) and K(M) = 0.83 +/- 0.22 microM). A continuous coupled assay was developed to follow the reaction at high concentrations of polynucleotide substrates (>10 microM). This work begins to define the chemical and structural determinants responsible for catalysis and substrate recognition and lays the foundation for detailed mechanistic analysis of this essential enzyme.

Ricin A-chain activity on stem-loop and unstructured DNA substrates.

Ricin toxin A-chain (RTA) depurinates a single adenylate on a GAGA stem-loop region of eukaryotic 28S RNA, making it a potent toxin. Steady state rate analysis is used to establish the kinetic parameters for depurination of short RNA, DNA, and RNA-DNA hybrids of GAGA linear segments and stem-loop regions as substrates for RTA. Both stem and tetraloop structures are essential for action on RNA. For DNA stem-loop substrates, stem stability plays a small role in enhancing catalytic turnover but can enhance binding by up to 3 orders of magnitude. DNA sequences of d[GAGA] without stem-loop structures are found to be slow substrates for RTA. In contrast, equivalent RNA sequences exhibit no activity with RTA. Introduction of a deoxyadenosine at the depurination site of short RNA oligonucleotides restores catalytic function. NMR analysis indicates that the short, nonsubstrate GAGA is converted to substrate in GdAGA by the presence of a more flexible ribosyl group at the deoxyadenosine site. Conversion between C2'-endo and C2'-exo conformations at the deoxyadenosine site moves the 3'- and 5'-phosphorus atoms by 1.1 A, and the former is proposed to place them in a catalytically favorable configuration. The ability to use short RNA-DNA hybrids as substrates for RTA permits exploration of related structures to function as substrates and inhibitors.

Inhibition of ricin A-chain with pyrrolidine mimics of the oxacarbenium ion transition state.

Ricin A-chain (RTA) catalyzes the hydrolytic depurination of a specific adenosine at position 4324 of 28S rRNA. Kinetic isotope effects on the hydrolysis of a small 10mer stem-tetraloop oligonucleotide substrate established the mechanism of the reaction as D(N)*A(N), involving an oxacarbenium ion intermediate in a highly dissociative transition state. An inhibitor with a protonated 1,4-dideoxy-1,4-imino-D-ribitol moiety, a 4-azasugar mimic, at the depurination site in the tetraloop of a 14mer oligonucleotide with a 5 bp duplex stem structure had previously been shown to bind to RTA with a K(d) of 480 nM, which improved to 12 nM upon addition of adenine. Second-generation stem-tetraloop inhibitors have been synthesized that incorporate a methylene bridge between the nitrogen of a 1-azasugar mimic, namely, (3S,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidine, and substituents, including phenyl, 8-aza-9-deazaadenyl, and 9-deazaadenyl groups, that mimic the activated leaving group at the transition state. The values for the dissociation constants (K(i)) for these were 99 nM for the phenyl 10mer, 163 and 94 nM for the 8-aza-9-deazaadenyl 10- and 14mers, respectively, and 280 nM for the 9-deazaadenyl 14mer. All of these compounds are among the tightest binding molecules known for RTA. A related phenyl-substituted inhibitor with a deoxyguanosine on the 5'-side of the depurination site was also synthesized on the basis of stem-loop substrate specificity studies. This molecule binds with a K(i) of 26 nM and is the tightest binding "one-piece" inhibitor. 8-Aza-9-deaza- and 9-deazaadenyl substituents provide an increased pK(a) at N7, a protonation site en route to the transition state. The binding of these inhibitors is not improved relative to the binding of their phenyl counterpart, however, suggesting that RTA might also employ protonation at N1 and N3 of the adenine moiety to activate the substrate during catalysis. Studies with methylated adenines support this argument. That the various stem-loop inhibitors have similar potencies suggests that an optimal one-piece inhibitor remains to be identified. The second-generation inhibitors described here incorporate ribose mimics missing the 2-hydroxy group. On the basis of inhibition data and substrate specificity studies, the 2'-hydroxyl group at the depurination site seems to be critical for recruitment as well as catalysis by RTA.