Jeffrey Aubé

The Internal Quaternary Ammonium Receptor Site of Shaker Potassium Channels

Quaternary ammonium (QA) compounds inhibit K+ conductance by entering and occluding the open pore of voltage-activated K+ channels. We characterized the effects of a series of alkyl-triethylammonium blockers on the Shaker K+ channel and tested them on a series of site-directed mutants of the channel protein in order to define the structural features of the binding sites. We found that mutations in two regions of the channel protein, the pore (P) region and the last transmembrane sequence (S6), appear to alter QA binding, not through their effects on gating but perhaps through direct effects on the binding site. Several mutations in the P region affect tetraethylammonium binding but have minimal effects on longer blockers, suggesting that the hydrophobic tail contributes to binding in a nonadditive fashion. Binding of the longer blockers can be affected by varying the hydrophobicity of 1 residue within S6 by site-specific substitution, in a manner consistent with a direct hydrophobic interaction between the side chain at this site and the alkyl chains of the blocker.

Improvement of oral peptide bioavailability : Peptidomimetics and prodrug strategies

Clinical development of orally active peptide drugs has been restricted by their unfavorable physicochemical properties, which limit their intestinal mucosal permeation and their lack of stability against enzymatic degradation. Successful oral delivery of peptides will depend, therefore, on strategies designed to alter the physicochemical characteristics of these potential drugs, without changing their biological activity, in order to overcome the physical and biochemical barrier properties of the intestinal cells. This manuscript will focus on the physiological limitations for oral peptide delivery and on various strategies using chemical modifications to improve oral bioavailability of peptide-based drugs.

Development of functionally selective, small molecule agonists at kappa opioid receptors

Background: Kappa opioid receptor (KOR) signaling may produce antinociception through G protein or dysphoria through βarrestin pathways. Results: Two highly selective, brain penetrant agonist scaffolds bias KOR signaling toward G protein coupling and produce antinociception in mice. Conclusion: Described are first-in-class small molecule agonists that bias KOR signaling through G proteins. Significance: Functionally selective KOR agonists can now be used in vivo. The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from βarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit βarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.

A Functional Assay for Quantitation of the Apparent Affinities of Ligands of P-Glycoprotein in Caco-2 Cells

AbstractPurpose. To develop a facile functional assay for quantitative determination of the apparent affinities of compounds that interact with the taxol binding site of P-glcoprotein (P-gp) in Caco-2 cell monolayers. Methods. A transport inhibition approach was taken to determine the inhibitory effects of compounds on the active transport of [3H]-taxol, a known substrate of P-gp. The apparent affinities (KI values) of the compounds were quantitatively determined based on the inhibitory effects of the compounds on the active transport of [3H]-taxol. Intact Caco-2 cell monolayers were utilized for transport inhibition studies. Samples were analyzed by liquid scintillation counting. Results. [3H]-Taxol (0.04 μM) showed polarized transport with the basolateral (BL) to apical (AP) flux rate being about 10-20 times faster than the flux rate in the AP-to-BL direction. This difference in [3H]-taxol flux could be totally abolished by inclusion of (±)-verapamil (0.2 mM), a known inhibitor of P-gp, in the incubation medium. However, inclusion of probenecid (1.0 mM), a known inhibitor for the multidrug resistance associated protein (MRP), did not significantly affect the transport of [3H]-taxol under the same conditions. These results suggest that P-gp, not MRP, was involved in taxol transport. Quinidine, daunorubicin, verapamil, taxol, doxorubicin, vinblastine, etoposide, and celiprolol were examined as inhibitors of the BL-to-AP transport of [3H]-taxol with resulting KI values of 1.5 ± 0.8, 2.5 ± 1.0, 3.0 ± 0.3, 7.3 ± 0.7, 8.5 ± 2.8, 36.5 ± 1.5, 276 ± 69, and 313 ± 112 μM, respectively. With the exception of that of quinidine, these KI values were comparable with literature values. Conclusions. This assay allows a facile quantitation of the apparent affinities of compounds to the taxol-binding site in P-gp; however, this assay does not permit the differentiation of substrates and inhibitors. The potential of drug-drug interactions involving the taxol binding site of P-gp can be conveniently estimated using the protocol described in this paper.

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Characterization of a Cdc42 Protein Inhibitor and Its Use as a Molecular Probe

Background: By integrating extracellular signals with actin cytoskeletal changes, Cdc42 plays important roles in cell physiology and has been implicated in human diseases. Results: A small molecule was found to selectively inhibit Cdc42 in biochemical and cellular assays. Conclusion: The identified compound is a highly Cdc42-selective inhibitor. Significance: The described first-in-class Cdc42 GTPase-selective inhibitor will have applications in drug discovery and fundamental research. Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.

Syntheses of the Stemona Alkaloids (±)-Stenine, (±)-Neostenine, and (±)-13-Epineostenine Using a Stereodivergent Diels-Alder/Azido-Schmidt Reaction

A tandem Diels-Alder/azido-Schmidt reaction sequence provides rapid access to the core skeleton shared by several Stemona alkaloids including stenine, neostenine, tuberostemonine, and neotuberostemonine. The discovery and evolution of inter- and intramolecular variations of this process and their applications to total syntheses of (+/-)-stenine and (+/-)-neostenine are described. The stereochemical outcome of the reaction depends on both substrate type and reaction conditions, enabling the preparation of both (+/-)-stenine and (+/-)-neostenine from the same diene/dienophile combination.

Optimization of Potent Hepatitis C Virus NS3 Helicase Inhibitors Isolated from the Yellow Dyes Thioflavine S and Primuline

A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM.

A tandem prins/schmidt reaction approach to marine alkaloids: Formal and total syntheses of lepadiformines A and C

The tricyclic core of the cylindricine or lepadiformine families of alkaloid natural products was assembled via a Prins addition/intramolecular Schmidt rearrangement under Lewis acid conditions. Both single-pot and two-stage variations of this process were examined, with particular attention to the stereochemical outcome of the processes. This technology has been applied to a formal total synthesis of lepadiformine A and a total synthesis of lepadiformine C.

Highly Stereoselective Ring Expansion Reactions Mediated by Attractive Cation–n Interactions

Most stereoselective reactions are ruled by steric effects. In particular, kinetically controlled asymmetric transformations utilizing chiral reagent, auxiliaries, or catalysts succeed due to energy differences in transition states that most often arise by the minimization of repulsive, non-bonded interactions. Stereoelectronic considerations, which arise when the alignment of particular orbitals are necessary for a successful reaction, can also play a role.[1] An iconic stereoelectronic effect in organic chemistry is the anomeric effect.[2] Reactions controlled by the anomeric effect, such as glycosidations, largely depend on the relative orientation of the non-bonding or n electrons of a nearby alkoxy group. In recent years, alkoxy group control of stereoselective reactions via electrostatic interactions has received renewed scrutiny, led by the Woerpel group.[3] In this communication, we report an alternative and highly effective approach to stereocontrol through the maximization of attractive non-bonded interactions between an alkoxy or alkylthio group and a positively charged leaving group. The Lewis acid-promoted reaction of a symmetrically substituted cyclic ketone with a chiral hydroxyalkyl azide provides a stereoselective route to lactams (Scheme 1).[4] In this reaction, initial formation of a spirocyclic intermediate sets up the selective migration of one of the alkyl groups originally adjacent to the ketone carbonyl. Migration of a C–C bond antiperiplanar to the N2+ leaving group (only possible when the latter is in an axial position as shown) affords an iminium ether that is converted into lactam by workup with aqueous base. For 1- or 3-substituted azidopropanols (not shown), 10:1 selectivities are obtained, corresponding to preferential reaction through the most stable chairlike heterocyclic ring (A or B) resulting from equatorial addition of azide relative to the tert-butyl group. Intermediates A and B can interconvert through conformational reorganization or by reversion to the initially formed oxonium ion followed by reclosure. In this scenario, selectivity is attained by stabilization of A over B due to traditional minimization of 1,3-diaxial interactions by placement of the R1 or R3 into equatorial positions in the former. Scheme 1 Origin of selectivity in asymmetric Schmidt reactions. 2-Substituted 1,3-azidopropanols present a special case that is unusually susceptible to stereoelectronic control due to three factors: (1) the methylene groups near the spiro linkage are locally isoelectronic, so the reaction cannot be controlled by “migratory aptitude”, (2) the presence of either an oxygen ether or an N–N2+ group in a 1,3 relationship to the R group means that 1,3-diaxial steric interactions will be minimized, and (3) the 1,3 relationship between axial R and N2+ groups provides a strong opportunity for attractive electrostatic interactions to occur between these groups in intermediate B. In previous work, it was demonstrated that unusually low selectivities obtained in this system when R = aryl could be ascribed to preferential stabilization of intermediate B by attractive, non-bonded cation–π interactions between the aromatic group and the N2+ leaving group (Table 1).[5] Although such interactions are commonly proposed in biological systems,[6] they are rarely invoked as stereocontrolling features of small-molecule stereoselective reactions.[7] Table 1 Selectivity of reactions of substituted 1,3-hydroxyalkyl azides. A computational study[8] and analogy to the well-known ability of ether groups to bind cations suggested that intermediates like B should be even more enhanced in compounds where R = alkoxy. As shown in Figure 1, isomer B containing a diaxial relationship between methoxy group and leaving group was calculated to be ca. 3.8 kcal/mol more stable than the equatorial isomer for which no interaction between methoxy and N2+ groups are possible. To test this, 1-azido-2-methoxypropanol 3 was prepared and reacted with 4-tert-butylcyclohexanone using BF3•OEt2 as Lewis acid promoter. A striking 24:1 selectivity in favor of the isomer emanating from an axially disposed methoxy group was obtained in high yield (Table 1, entry 3). Figure 1 Calculations for proposed intermediates A and B performed at the MP2/6-311+G**//MP2/6-31G* level of theory.[8] This result suggests that the methoxy cation–n interaction is considerably stronger than the previously reported cation–π effect, due to the fact that the highest 3:2 ratio observed to date was 57:43 for the electron-rich 3,4,5-trimethoxyphenyl group (not shown).[5] The fact that the small MeO group (A value = 0.6[9]) pays a relatively small steric penalty in the axial orientation is a likely contributor to the high selectivity of this reaction as well. However, the much higher selectivity and opposite direction of stereocontrol obtained for the smaller MeO group as compared to alkyl or aryl substituents (Table 1, entries 1 and 2) is strong evidence for the proposed role of electrostatics in this reaction. We proposed that a similar effect might be observed with a more polarizable heteroatom.[3m] Accordingly, 1d where R = SMe, was prepared and submitted to the asymmetric Schmidt reaction protocol. Remarkably, a >98:2 dr was obtained for this system, favoring 3d. The selectivities obtained with both methoxy and methylthio, which depend mainly on electrostatics and feature axially disposed substituents, are higher than any previously reported, sterically-based, example of this ring-expansion reaction.[4,5] Although the first example of an asymmetric azido-Schmidt reaction reported utilized an azidoethanol reagent, that series has typically provided lower selectivities relative to the three-carbon-containing reagents like 1 and has more recently been shown to occur via predominant steric control, even when a phenyl group is in a position to participate in a cation–π interaction.[5] In sharp contrast to these previous results, the reaction using reagent 4 afforded a high 97:3 ratio of 5 over 6, in which the major product goes through an intermediate in which a syn relationship between the methoxy group and the leaving N2+ substituent is possible (Scheme 2). A computational investigation showed that the cation–n intermediate C is stabilized by 3.9 kcal/mol. Notably, the O–N2+ distances, energy differences, and ratios are similar between systems B and C. Previous work in the reactions of substituted 1,2-azidoethanols has shown the predominant steric feature affecting stereochemistry to exist between the migrating carbon and substituents on the five-membered heterocyclic ring.[4,5b] In cases where the alkyl group is adjacent to oxygen (i.e., across the ring from the migrating methylene group and the N2+ leaving group), steric effects do not play an important role in determining reaction stereochemistry, as clearly demonstrated by the non-selective cyclohexyl case shown in Scheme 2b. Scheme 2 (a) Electrostatically controlled reaction of 1-azidoethanol derivative 4 with 4-tert-butylcyclohexanone (including calculated energies of proposed, minimized intermediates C and D[8]) and (b) a cyclohexyl-containing control.[5b] The model systems used ... The opposite situation occurs when the methoxymethyl group is placed adjacent to the azido group. In this case, there is no substantial difference in distance between the methoxy group and either isomeric intermediate, so electrostatic considerations cannot play a role and the preference for syn E over anti F drops to 0.6 kcal/mol computationally. Instead, the usual steric course of the reaction leads to the same product observed for the analogous example to 14 (Scheme 3). Scheme 3 Sterically controlled reactions of (a) 10 and (b) a previously reported cyclohexanyl example.[5] The model systems used for the calculations are given in the Supporting Information (Figure S1). The most interesting elements of this approach are that: (1) intermediates are subject to non-bonded, attractive interactions that are able to strongly favor one stereoisomeric form over the other, (2) these intermediates lead to the corresponding products in a process entirely controlled by stereoelectronic considerations, and (3) the overall stereoselectivity ultimately depends on the control of leaving group stereoslectivity at an epimerizable nitrogen atom. The high yields of these reactions combined with the utility of the lactam products suggests a high level of utility of the present reaction. Of perhaps greater long-term interest will be the attempted utilization of cation nonbonding electron stabilization in other stereoselective processes.[10]