Cameron R. Pye

Probing the Physicochemical Boundaries of Cell Permeability and Oral Bioavailability in Lipophilic Macrocycles Inspired by Natural Products

Cyclic peptide natural products contain a variety of conserved, nonproteinogenic structural elements such as d-amino acids and amide N-methylation. In addition, many cyclic peptides incorporate γ-amino acids and other elements derived from polyketide synthases. We hypothesized that the position and orientation of these extended backbone elements impact the ADME properties of these hybrid molecules, especially their ability to cross cell membranes and avoid metabolic degradation. Here we report the synthesis of cyclic hexapeptide diastereomers containing γ-amino acids (e.g., statines) and systematically investigate their structure-permeability relationships. These compounds were much more water-soluble and, in many cases, were both more membrane permeable and more stable to liver microsomes than a similar non-statine-containing derivative. Permeability correlated well with the extent of intramolecular hydrogen bonding observed in the solution structures determined in the low-dielectric solvent CDCl3, and one compound showed an oral bioavailability of 21% in rat. Thus, the incorporation of γ-amino acids offers a route to increase backbone diversity and improve ADME properties in cyclic peptide scaffolds.

Retrospective analysis of natural products provides insights for future discovery trends

Significance Natural products research seems to be at a critical juncture in terms of its relevance to modern biological science. We have evaluated this landscape of chemical diversity to ask key questions, including the following. How has the rate of discovery of new natural products progressed over the past 70 y? Has natural product structural novelty changed as a function of time? Has the rate of novel discovery declined in recent years? Does exploring novel taxonomic space afford an advantage in terms of novel compound discovery? Is it possible to estimate how close we are to describing all of the chemical space covered by natural products? And, finally, is there still value in exploring natural products space for novel biologically active natural products? Understanding of the capacity of the natural world to produce secondary metabolites is important to a broad range of fields, including drug discovery, ecology, biosynthesis, and chemical biology, among others. Both the absolute number and the rate of discovery of natural products have increased significantly in recent years. However, there is a perception and concern that the fundamental novelty of these discoveries is decreasing relative to previously known natural products. This study presents a quantitative examination of the field from the perspective of both number of compounds and compound novelty using a dataset of all published microbial and marine-derived natural products. This analysis aimed to explore a number of key questions, such as how the rate of discovery of new natural products has changed over the past decades, how the average natural product structural novelty has changed as a function of time, whether exploring novel taxonomic space affords an advantage in terms of novel compound discovery, and whether it is possible to estimate how close we are to having described all of the chemical space covered by natural products. Our analyses demonstrate that most natural products being published today bear structural similarity to previously published compounds, and that the range of scaffolds readily accessible from nature is limited. However, the analysis also shows that the field continues to discover appreciable numbers of natural products with no structural precedent. Together, these results suggest that the development of innovative discovery methods will continue to yield compounds with unique structural and biological properties.

Going Out on a Limb: Delineating The Effects of β-Branching, N-Methylation, and Side Chain Size on the Passive Permeability, Solubility, and Flexibility of Sanguinamide A Analogues.

It is well established that intramolecular hydrogen bonding and N-methylation play important roles in the passive permeability of cyclic peptides, but other structural features have been explored less intensively. Recent studies on the oral bioavailability of the cyclic heptapeptide sanguinamide A have raised the question of whether steric occlusion of polar groups via β-branching is an effective, yet untapped, tool in cyclic peptide permeability optimization. We report the structures of 17 sanguinamide A analogues designed to test the relative contributions of β-branching, N-methylation, and side chain size to passive membrane permeability and aqueous solubility. We demonstrate that β-branching has little effect on permeability compared to the effects of aliphatic carbon count and N-methylation of exposed NH groups. We highlight a new N-methylated analogue of sanguinamide A with a Leu substitution at position 2 that exhibits solvent-dependent flexibility and improved permeability over that of the natural product.

Revisiting N-to-O Acyl Shift for Synthesis of Natural Product-like Cyclic Depsipeptides

Despite the prevalence of head-to-side chain threonine linkages in natural products, their incorporation has been underexplored in synthetic cyclic peptides. Herein we investigate a cyclic peptide scaffold able to undergo an N-O acyl rearrangement. Upon acylation of the amine with diverse carboxylic acids, the resulting cyclic depsipeptides displayed favorable cellular permeability and a conformation similar to the parent peptide. The rearrangement was found to be scaffold and conformation dependent as evidenced by molecular dynamics experiments.

Synthesis, structure, and spectroscopy of two benzil-based α-diimine ligands and their palladium(II) complexes

Two α-diimine ligands were prepared in 60–70% yield via p-toluenesulfonic acid-catalyzed condensation reactions from benzil with 4-bromoaniline and with p-anisidine. Palladium(II) complexes were prepared from both ligands in 70–80% yield. X-ray structures were obtained for the ligand prepared from p-anisidine and its palladium(II) complex. A notable feature observed in the former was its unconjugated C–N double bonds, both in the (E)-configuration. The latter structure possessed two molecules of the metal complex in its unit cell, both of which have diimine cores with a degree of conjugation and a nonideal square-planar geometry around palladium caused by the small bite angles (79.61(3) and 79.15(3)°) of the diimine ligands. Solution-phase electronic absorption spectra of the ligands in chloroform have two bands from π→π ∗ and n→π ∗ transitions at 269–345 nm. Absorption spectra of the complexes in chloroform exhibited bands attributed to ligand-centered transitions that were red-shifted as compared to free ligands. Only the spectrum obtained from a chloroform solution of the palladium(II) complex with the diimine ligand prepared from p-anisidine featured a band at approximately 520 nm, which was assigned to a combination of d π(Pd)→π ∗ and n(Cl)→π ∗ transitions.

Reply to Skinnider and Magarvey: Rates of novel natural product discovery remain high

It is encouraging that our recent article examining trends in discovery rates and structural diversity for natural products (NP) (1) is generating discussion in this fascinating area (2). However, we wish to correct several misconceptions presented in the comments from Skinnider and Magarvey (3). Skinnider and Magarvey’s (3) letter incorrectly summarizes the key conclusion of our work. The letter states that “[t]heir analysis suggests that the pace of structurally unique NP discovery is decreasing.” Our study makes precisely the opposite conclusion: “A cursory review of these data might suggest that the field of natural products is no longer discovering novel chemical entities…[However,] it is also important to evaluate the distribution of molecules with low similarity scores… Overall, this … [↵][1]1To whom correspondence may be addressed. Email: wgerwick{at}ucsd.edu or rliningt{at}sfu.ca. [1]: #xref-corresp-1-1

A Strategy for Direct Chemical Activation of the Retinoblastoma Protein

The retinoblastoma (Rb) tumor suppressor protein negatively regulates cell proliferation by binding and inhibiting E2F transcription factors. Rb inactivation occurs in cancer cells upon cyclin-dependent kinase (Cdk) phosphorylation, which induces E2F release and activation of cell cycle genes. We present a strategy for activating phosphorylated Rb with molecules that bind Rb directly and enhance affinity for E2F. We developed a fluorescence polarization assay that can detect the effect of exogenous compounds on modulating affinity of Rb for the E2F transactivation domain. We found that a peptide capable of disrupting the compact inactive Rb conformation increases affinity of the repressive Rb-E2F complex. Our results demonstrate the feasibility of discovering novel molecules that target the cell cycle and proliferation through directly targeting Rb rather than upstream kinase activity.

CycLS: Accurate, whole-library sequencing of cyclic peptides using tandem mass spectrometry

Cyclic peptides are of great interest as therapeutic compounds due to their potential for specificity and intracellular activity, but specific compounds can be difficult to identify from large libraries without resorting to molecular encoding techniques. Large libraries of cyclic peptides are often DNA-encoded or linearized before sequencing, but both of those deconvolution strategies constrain the chemistry, assays, and quantification methods which can be used. We developed an automated sequencing program, CycLS, to identify cyclic peptides contained within large synthetic libraries. CycLS facilitates quick and easy identification of all library-members via tandem mass spectrometry data without requiring any specific chemical moieties or modifications within the library. Validation of CycLS against a library of 400 cyclic hexapeptide peptoid hybrids (peptomers) of unique mass yielded a result of 95% accuracy when compared against a simulated library size of 234,256 compounds. CycLS was also evaluated by resynthesizing pure compounds from a separate 1800-member library of cyclic hexapeptides and hexapeptomers with high mass redundancy. Of 22 peptides resynthesized, 17 recapitulated the retention times and fragmentation patterns assigned to them from the whole-library bulk assay results. Implementing a database-matching approach, CycLS is fast and provides a robust method for sequencing cyclic peptides that is particularly applicable to the deconvolution of synthetic libraries.