Adam K. Charnley

Pyrrolinone-Based Peptidomimetics: “Let the Enzyme or Receptor be the Judge”

Peptides and proteins, evolved by nature to perform vital biological functions, would constitute ideal candidates for therapeutic intervention were it not for their generally poor pharmacokinetic profiles. Nonpeptide peptidomimetics have thus been pursued because they might overcome these limitations while maintaining both the potency and selectivity of the parent peptide or protein. Since the late 1980s, we have sought to design, synthesize, and evaluate a novel, proteolytically stable nonpeptide peptidomimetic scaffold consisting of a repeating structural unit amenable to iterative construction; a primary concern is maintaining both the appropriate peptide-like side-chains and requisite hydrogen bonding. In this Account, we detail how efforts in the Smith-Hirschmann laboratories culminated in the identification of the 3,5-linked polypyrrolinone scaffold. We developed effective synthetic protocols, both in solution and on solid supports, for iterative construction of diverse polypyrrolinones that present functionalized peptide-like side-chains. As a result of the rigid nature of the pyrrolinone scaffold, control over the backbone conformation could be exerted by modulation of the stereogenicity of the constituent monomers and the network of intramolecular hydrogen bonding. The extended conformation of the homochiral 3,5-linked polypyrrolinone scaffold proved to be an excellent mimic for β-strands and β-sheets. Application to enzyme inhibitor design and synthesis led not only to modest inhibitors of the aspartic acid protease renin and the matrix metalloprotease class of enzymes, but importantly to bioavailable HIV-1 protease inhibitors with subnanomolar binding constants. The design and synthesis of a competent peptide-pyrrolinone hybrid ligand for the class II major histocompatibility complex (MHC) antigen protein HLA-DR1 further demonstrated the utility of the 3,5-polypyrrolinone motif as a mimic for the extended polyproline type II peptide backbone. Equally important, we sought to define, by synthesis, the additional conformational space accessible to the polypyrrolinone structural motif, with the ultimate goal of accessing pyrrolinone-based turn and helix mimetics. Toward this end, a mono-N-methylated bispyrrolinone was found to adopt an extended helical array in the solid state. Subsequent synthesis of d,l-alternating (heterochiral) tetrapyrrolinones both validated the expected turn conformations in solution and led to a functionally active mimetic of a peptidal β-turn (similar to somatostatin). Finally, the design, synthesis, and structural evaluation of both acyclic and cyclic heterochiral (that is, d,l-alternating) hexapyrrolinones yielded nanotube-like assemblies in the solid state. Taken together, these results illustrate the remarkable potential of the 3,5-linked polypyrrolinone scaffold as β-strand, β-sheet, β-turn, and potentially helical peptidomimetics.

The Identification and Pharmacological Characterization of 6-(tert-Butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine (GSK583), a Highly Potent and Selective Inhibitor of RIP2 Kinase

RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.

Design, Synthesis, and Structural Analysis of d,l-Mixed Polypyrrolinones. 1. From Nonpeptide Peptidomimetics to Nanotubes

To expand the potential conformational space available to the polypyrroline structural motif, an open chain, D,L-alternating hexapyrrolinone was designed and synthesized. Structural studies, including solution NMR and X-ray crystallographic analysis, revealed that the hexapyrrolinone adopts a turn conformation both in solution and in the solid state, with aggregation in solution and a nanotube-like quaternary structure in the crystal.

Design, synthesis, and structural analysis of D,L-mixed polypyrrolinones. 2. Macrocyclic hexapyrrolinones.

The design, synthesis, and structural analysis of two macrocyclic D,L-alternating hexapyrrolinones have been achieved. These cyclic peptide mimics adopt a flat, hexagonal conformation, stabilized by intramolecular hydrogen bonding between adjacent pyrrolinone rings. Extensive NMR studies and X-ray analysis reveal, respectively, that the macrocyclic hexapyrrolinones aggregate in solution and in the solid state form staggered stacked nanotube-like assemblies.

Multidimensional and dual frequency infrared analogues of NMR: Applications to peptides and isotopomers of secondary structures

The development of methods to time resolve structural changes in complex systems is an important challenge. Because of its time resolution, multidimensional infrared spectroscopies 2D-IR and 3D-IR, can contribute to this challenge complementing the knowledge on average structures obtained by the established methods of structural biology and their time dependent variants. The multidimensional infrared experiment arose from many years of theoretical and experimental research on laser technology, nonlinear optics, and infrared spectroscopy. The manipulation of multidimensional data sets in time or frequency domains are also basic material in NMR even though the practical aspects of the two experiments are quite different and they arose quite independently. The heterodyned 2D-IR approach has proven useful in determining structures of peptides in solution and the anharmonic nature of the potential surfaces of peptides and secondary structures, as have polarized photon echo or pump-probe techniques. A full 2D-IR spectrum is obtained in which the two frequency axes correspond to the quite different spectral regions at 3300 cm -1 and 1650 cm -1 : the amide-A and amide-I modes influence each other in N-H O=C hydrogen bonding configurations.