Category E. Drug Target Identification and Drug Design
E1. Phosphate Tether-Mediated Ring-Closing Metathesis Studies to Complex 1,3-anti Diol-Containing Subunits
Rambabu Chegondi, Soma Maitra, Jana L. Markley and Paul R. Hanson*
Department of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045.
An array of diastereoselective, phosphate tether-mediated ring-closing metathesis reactions, which highlight the importance of product ring size and substrate stereochemical compatibility, as well as complexity, is reported. Studies focus primarily on the formation of bicyclo[n.3.1]phosphates, involving the coupling of C2-symmetric dienediol subunits with a variety of simple, as well as complex alcohol cross-partners. Plausible metallocyclobutane-containing intermediates for RCM reactions to bicyclo[4.3.1]- and [5.3.1]-phosphates are proposed to rationalize observed experimental outcomes. In addition, notable trans-products in the bicyclo[4.3.1]phosphate series, as well as exclusive E-selectivity in the bicyclo[7.3.1]phosphate series, were also observed.
E2. Chemical shift mapping of the cytochrome b5, CYP17A1 interaction provides evidence of substrate-driven allostery in a human P450 enzyme
D. Fernando Estrada1, Jennifer S. Laurence2, Emily E. Scott1
The Departments of Medicinal Chemistry1 and Pharmaceutical Chemistry2, The University of Kansas, Lawrence, KS
The membrane heme protein cytochrome b5 (b5) plays an important role in the catalytic cycle of human cytochrome P450 enzymes. Extensive studies using various P450 enzymes indicate that b5 either enhances, inhibits, or has no effect on the rate of catalysis, depending on the particular P450 or substrate studied. However, the structural cause underlying these variable effects remains unclear. To date, prevailing theories to explain altered activity in the presence of b5 consist of i) a role for b5 as the donor of the second electron required for catalysis, or ii) a b5 binding-induced allosteric effect on P450 enzymes that alters the rates of catalysis. Currently, there are no high resolution binding studies of a b5, mammalian P450 interaction.
Therefore, here we report the first solution NMR study investigating the binding interaction between the soluble domain of microsomal b5 and the catalytic domain of the bi-functional androgen-producing enzyme cytochrome P450 17A1 (CYP17A1). Titration of the binding partners and chemical shift mapping indicates that the interaction occurs in an intermediate exchange regime and is mediated by surface electrostatics. Specifically, site-directed mutagenesis of either of the anionic b5 residues at the interface (E48 & E49) or any of the cationic proximal CYP17A1 residues (R347, R358, or R449) was sufficient to disrupt formation of the complex. Binding was also shown to be mutually exclusive with respect to the P450 oxidoreductase (POR), CYP17A1 interaction, in that addition of unlabeled full-length rat POR was able to compete with b5 for binding in a way that restored the free b5 signal. Finally, to probe communication between the CYP17A1 active site and the superficial b5 binding site, we characterize the complex with either 17α-hydroxylase or 17,20-lyase substrates in the CYP17A1 active site.
Significantly, we found that b5 binds CYP17A1 in a substrate-specific manner that correlates to a stronger association with CYP17A1:pregnenolone than with CYP17A1:17α-hydroxypregnenolone. These findings form the basis for a better understanding of this important protein-protein interaction by providing the first evidence of allostery between the CYP17A1 active site and the superficial proximal binding site.
E3. ROMP-Derived Oligomeric Phosphates (OBP and OTP) for the Application in Facile Benzylation and Triazolation.
Saqib Faisal, Toby R. Long, Pradip K. Maity, Alan Rolfe, Paul R. Hanson.
Department of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045.
The University of Kansas Center for Chemical Methodologies and Library Development (KU-CMLD), 2034 Becker Drive
Shankel Structural Biology Center, West Campus, Lawrence, KS 66047.
The development of new ROMP-derived oligomeric phosphorus reagents for application in purification-free diversification protocols is reported. An array of oligomeric benzyl phosphates (OBP) and their corresponding oligomeric triazole phosphate (OTP) derivatives were successfully synthesized for the efficient benzylation and triazolation of nucleophilic species. The titled oligomeric reagents were readily synthesized from commercially available materials and were conveniently polymerized and purified in a one-pot process affording pure reagent on multi-gram scale. The application of both OBP and OTP for the diversification of N, O and S-nucleophilic species, including the generation of a 108-member sultam library is reported.
E4. ML291: A Selective, Small Molecule Activator of the Apoptotic Arm of the Unfolded Protein Response
Daniel P. Flaherty1, Jennifer E. Golden1, Chunjing Liu2, Michael Hedrick4, Palak Gosalia4, Yujie Li4, Monika Milewski4, Eliot Sugarman5, Eigo Suyama5, Kevin Nguyen5, Stefan Vasile5, Sumeet Salaniwal4, Derek Stonich4, Ying Su4, Arianna Mangravita-Novo5, Michael Vicchiarella5, Layton H. Smith5, Jena Diwan4, Thomas D.Y. Chung4, Anthony B. Pinkerton4, Jeffrey Aubé3, Justin R. Miller6, Danielle M. Garshott6, Michael U. Callaghan6, Andrew M. Fribley6, Randal J. Kaufman7
1University of Kansas Specialized Chemistry Center, University of Kansas, Lawrence, KS, USA;
2University of Kansas COBRE Center for Cancer Experimental Therapeutics, University of Kansas, Lawrence, KS, USA;
3Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, USA;
4Sanford-Burnham Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA;
5Sanford-Burnham Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, Orlando, FL, USA;
6Carmen and Ann Adams Department of Pediatrics Division of Hematology/Oncology, Wayne State University, Detroit, MI, USA;
7Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
Several diseases, such as diabetes, Alzheimer’s, Parkinson’s, hemophilia, lysomal storage diseases and cancer involve folding defects or impaired transport of proteins from the endoplasmic reticulum (ER). When stressed with protein defects and transport impairment cells activate the Unfolded Protein Response (UPR). The UPR attempts to restore a homeostatic state in the cell’s protein processing mechanism via the XPB1 (adaptive arm). If the stressor continues the cell will activate the CHOP (apoptotic arm) pathway leading to selective cell death. Toward the goal of developing a small molecule activator of the apoptotic arm, a CHOP pathway selective cell-based reporter high-throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (~330,000 compounds) was performed. Hits were triaged via secondary assays to yield two scaffolds for follow up structure-activity relationship (SAR) studies. As a result of this effort we report a potent, first-in-class, probe (ML291) that selectively activates the CHOP (apoptotic arm) pathway over the XPB1 (adaptive arm) pathway. ML291 is generally non-cytotoxic, is shown to activate genes associated with the CHOP pathway of the UPR and demonstrates efficacy in inducing cell death in a relevant carcinoma cell line via this pathway. ML291 will undergo further proof-of-concept studies to determine if it can selectively activate apoptotic pathways in tumors leading to selective and efficacious killing. Animal studies are also planned to determine the probes effectiveness in vivo.
E5. Discovery of Small Molecule Kappa Opioid Receptor Agonist and Antagonist Chemotypes through a HTS and Hit Refinement Strategy
Kevin J. Frankowskia, Michael P. Hedrickb, Palak Gosalia, Kelin Lia, Shenghua Shib, David Whipplea, Partha Ghosha, Thomas E. Prisinzanoa, Frank J. Schoenena, Ying Sub, S. Vasileb, Eduard Sergienkob, Wilson Grayb, Santosh Hariharanb, Loribelle Milanb, Susanne Heynen-Genelb, Arianna Mangravita-Novoc, Michael Vicchiarellic, Layton H. Smithc, John M. Streicherd, Laura M. Bohnd, Thomas D.Y. Chungb and Jeffrey Aubéa*
a University of Kansas Specialized Chemistry Center, University of Kansas, Lawrence, KS 66047; b Conrad Prebys Center for Chemical Genomics at Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; c Conrad Prebys Center for Chemical Genomics at Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, FL 32827, d Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida 33458
The high throughput screening (HTS) of the NIH compound collection using a ß-arrestin recruitment platform has identified several new chemotypes for the modulation of the kappa opioid receptor (KOR). Here we describe the successful optimization of two agonist and two antagonist chemotypes through a brief, collaborative SAR campaign. The resulting KOR probe molecules were profiled against a set of 44 GPCR and other molecular targets and characterized in a basic set of PK assays. The four chemotypes presented complement the existing arsenal of KOR ligands through their modular, easily constructed, and, in most cases achiral structures. The development of such readily-modified, KOR-selective scaffolds provides new molecular tools for the investigation of KOR modulation pathways.
E6. Reaction Pairing: A Diversity-Oriented Modular Approach to Fused- and Bridged Sultam Library
Joanna K. Loh, Thiwanka B. Samarakoon, Alan Rolfe, Sun Young Yoon and Paul R. Hanson*
Department of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045.
The University of Kansas Center for Chemical Methodologies and Library Development (KU-CMLD), 2034 Becker Drive, Shankel Structural Biology Center, West Campus, Lawrence, KS 66047.
A reaction pairing strategy focused on the utilization of a collection of reactions (sulfonylation, SNAr addition and Mitsunobu alkylation) to generate skeletally diverse benzofused sultams is reported. The pairing of sulfonylation and SNAr reactions rapidly generates tricyclic, bridged benzofused sultams. The addition of the Mitsunobu reaction in a sulfonylation–Mitsunobu–SNAr sequence allows access to benzoxathiazocine-1,1-dioxides, while a simple change in combination to sulfonylation–SNAr–Mitsunobu affords structurally distinct tricyclic, bridged and fused sultams. With these scaffolds in hand, a unique 80-member library was efficiently prepared via a microwave-assisted intermolecular SNAr diversification reaction. Chemical diversity of these sultams was explored utilizing several computational studies including QED, Z-score, PMI and overlay analyses.
E7. ROMP-derived Oligo Phosphate Reagents: Synthesis and Application in Parallel and Flow Platforms
Pradip K. Maity, Moon Y. Hur, Alan Rolfe, Quirin M. Kainz, Saqib Faisal, Toby R. Long, Oliver Reiser, and Paul R. Hanson*
Department of Chemistry, 1251 Wescoe Hall Dr., University of Kansas, Lawrence, KS 66045.
NIH Center for Chemical Methodologies and Library Development at the University of Kansas (KU-CMLD), 2034 Becker Drive, Del Shankel Structural Biology Center, Lawrence, KS 66047.
The development of new ROMP-derived oligomeric phosphorus reagents (oligomeric benzyl phosphates, oligomeric triazole phosphates), chromatography-free Mitsunobu protocols and surface-initiated polymerization from Nb-tagged silica (Nb-Si) and Nb-tagged Cobalt-graphite (Nb-Co/C) magnetic nanoparticles for use in parallel synthesis are reported. A Monomer-on-Monomer (MoM) Mitsunobu protocol utilizing norbornenyl-tagged benzylethyl azodicarboxylate (Nb-BEAD) and Nb tagged triphenylphosphine (Nb-TPP) is presented whereby purification/ sequestration of Nb-tagged reagents is rapidly achieved using ring-opening metathesis (ROM) polymerization. Additional efforts utilizing surface-initiated polymerization from Nb-tagged silica (Nb-Si) and Nb-tagged Cobalt-graphite (Nb-Co/C) magnetic nanoparticles is also discussed. Applications of Silica-supported ROMP Reagents in Flow through cartridge for the syntheses of small molecule have been carried out in purification-free processes.
E8. Discovery and development of sulfonylpyrrolidine compounds that inhibit human respiritatory synctial virus activity
Daljit S. Matharuc, James W. Noaha, William Seversonb, Donghoon H. Chungb, Blake Moorea, Fuli Jiaa, Xiaolin Xua, Clinton Maddoxa, Lynn Rasmussena, Melinda Ingrum Sosaa, Nichole A. Towera,S. Ananthana, E. Lucile Whitea, Colleen Jonssonb, Jennifer E. Goldenc, Jeffrey Aubé c,d
a Southern Research Specialized Biocontainment Screening Center; Southern Research Institute, Birmingham, AL
bCenter for Predictive Medicine, University of Louisville, Louisville, KY
c University of Kansas Specialized Chemistry Center, Lawrence, KS
dDepartment of Medicinal Chemistry, University of Kansas, Lawrence, KS
Due to the limit in therapeutic options for human respiratory syncytial virus (RSV), a high throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR) was carried out with a focus on identifying novel, small molecule inhibitors that could be optimized through a medicinal chemistry effort utilizing structure-activity relationships. Hit compounds were validated and optimized for potency and HEp-2 cellular cytotoxicity. Pursuit of a sulfonylpyrrolidine hit scaffold resulted in a compound that inhibited a virus-induced cytopathic effect in the entry stage of infection (EC50 = 2.25 ± 0.82 µM). The inhibitor demonstrated marginal cytotoxicity (CC50 = 30.91 ± 1.09 µM) and reduced viral titer by 200-fold as determined by a plaque reduction assay. Compared to Ribavirin, the only FDA-approved RSV small molecule available, the sulfonylpyrrolidine inhibitor obtained from this effort demonstrated an improved in vitro potency and therapeutic window.
E9. Exploring Chemical Space with a Family of Spirocycles
Thomas O. Painter, Paul D. Thornton, Sarvesh Kumar, Prashi Jain, Patrick Porubsky, Jon Bunn, Justin Douglas, Victor Day and Conrad Santini*
The Center for Chemical Methodologies and Library Development, University of Kansas, Lawrence, Kansas 66047
Simple saturated spirocyclic compounds derived from 3-piperidone and similar ketones are comparatively uncommon in PubChem. Our goal was to construct a family of scaffolds from these ketones that provide reproducible and incremental changes in molecular architecture. We initiated studies designed to produce a 24-member family of scaffolds and determine their utility in parallel synthesis. The synthesis of 18 members of the scaffold family was achieved and is presented. The utilization of three of the derived scaffolds in parallel synthesis to prepare 261 compounds is also presented.
E10. Novel Analogs of the Kappa Opioid Receptor Ligand CJ-15,208 with Potential for Drug Development
Sanjeewa N. Senadheera,a Shainnel O. Eans,b Nicolette C. Ross,b Jay P. McLaughlin,b Thomas F. Murray,c Jane V. Aldricha
aDepartment of Medicinal Chemistry, The University of Kansas, Lawrence, KS, USA
bTorrey Pines Institute for Molecular Studies, Port St. Lucie, FL, USA
cDepartment of Pharmacology, Creighton University School of Medicine, Omaha, NE, USA
Recently kappa opioid receptor (KOR) antagonists have demonstrated therapeutic potential for the treatment of drug abuse. Macrocyclic tetrapeptides are promising candidates for development because of their low molecular weight, expected metabolic stability in vivo and potential oral bioavailability. The macrocyclic tetrapeptide CJ-15,208 (cyclo[L-Phe-D-Pro-L-Phe-Trp]) is a natural product that was reported to be a novel KOR antagonist (Saito et al., J. Antiobiot. 2002, 55, 847). We used modifications to our initial synthetic protocol (Ross et al., Tetrahedron Lett. 2010, 51, 5020) to prepare larger quantities of the synthetically challenging CJ-15,208 Trp isomers and their alanine scan analogs for detailed pharmacological evaluation in vivo. As expected, both isomers exhibited activity after oral administration. The D-Trp macrocyclic tetrapeptide exhibited potent KOR selective antagonism in vivo in mice and prevented stress-induced reinstatement of extinguished cocaine-seeking behavior (Ross et al., Br. J. Pharmacol. 2012, 165, 1097). The Ala scan analogs of the promising D-Trp isomer, which exhibit a range of affinities for opioid receptors (Dolle et al., Bioorg. Med. Chem. Lett. 2009, 19, 3647), showed distinct pharmacological profiles in vivo in the 55 °C warm water tail-withdrawal assay, surprisingly exhibiting either mixed agonist/antagonist activity or exclusive agonist activity. One analog that shows mixed agonist/ KOR antagonist activity in the antinociceptive assay prevented stress-induced, but not cocaine-induced, reinstatement in mice, similar to the parent peptide. These novel macrocyclic tetrapeptides are promising candidates for the development of potential peptide KOR therapeutic ligands. Research supported by NIDA grants R01 DA018832 and R01 DA023924.
E11. High-Throughput Screening and SAR Analysis for Novel Small Molecule Beclin-1-mimetics Modulating Autophagy
Jia Yu*1, Lan Lan*1, Anuradha Roy2, Steven A. Rogers3, Philip Gao4, Na Zhang4, John Karanicolas1,5, Jeffrey Aubé3,6, Liang Xu1
1Department of Molecular Biosciences, 2University of Kansas high throughput screening laboratory, 3Center of Biomedical Research Excellence, 4Protein Production Group, NIH COBRE in Protein Structure and Function, 5Center for Bioinformatics, 6Department of Medicinal Chemistry, University of Kansas, Lawrence, KS. (*equal contribution)
Anti-apoptotic Bcl-2 family members, especially Bcl-2 and Bcl-XL, are overexpressed in many types of cancer and contribute to tumor initiation, progression and resistance to therapy. Bcl-2 family members are key regulators in apoptosis and/or autophagy pathway. Bcl-2 and Bcl-XL have recently been shown to inhibit autophagy by binding to and antagonizing the BH3-only protein, Beclin 1, an essential inducer of autophagy. Here, we used a fluorescence polarization-based assay to identify small molecules that could induce autophagy via blocking Bcl-2-Becin 1 interaction at the endoplasmic reticulum (ER). High throughput screening of several libraries (50,316 compounds total) was carried out with Z’ score 0.8±0.05, hit rate 0.14%. 69 inhibitors were identified as Beclin-mimetics and validated by surface plasmon resonance (SPR) assay and cell growth inhibition assay. As a result, we found several promising hits for lead optimization, especially some acridine analogues which showed potent binding affinity and cytotoxicity. We are currently working on structure−activity relationship (SAR) analysis for further computer-aided rational design and lead optimization.
E12. Copper-mediated deoxygenative trifluoromethylation of benzylic xanthates: Formation of C(sp3)–CF3 through nucleophilic substitution
Lingui Zhu; Shasha Liu; Ryan A. Altman
Department of Medicinal Chemistry, The University of Kansas, Lawrence, Kansas, USA
The incorporation of trifluoromethyl group (CF3) into organic molecules can impart profound changes in physical, chemical, and biological properties of molecules. Owing to high electronegativity, lipophilicity, and metabolic stability of the trifluoromethyl group, trifluoromethylated compounds have been widely employed in the fields of pharmaceuticals agrochemicals, and material science. Accordingly, various protocols for the introduction of CF3 into organic molecules have increasingly emerged. Recently, transition-metal-catalyzed or -mediated trifluoromethylation have made remarkable progress. However, most methods for trifluoromethylation reactions employing transition-metals form C(sp2)–CF3 bond. Comparably, transition-metal mediated C(sp3)–CF3 bonding-forming processes have been less explored.
Inspired by the potential to develop a C(sp3)–CF3 bonding-forming reaction, a copper-mediated deoxygenative trifluoromethylation of benzylic xanthates using Umemoto’s reagent as the source of CF3 has been developed in our laboratory. The protocol is compatible with a variety of benzylic and heterobenzylic xanthates bearing sensitive functional group and provides trifluoromethylated products in moderate to good yields. Due to the sensitivity of some xanthates to Lewis acidic conditions, substrates possessing electron-rich aromatic rings were not generally compatible with the trifluoromethylation process. A preliminary mechanistic investigation is underway in our laboratory.