Geometrically Diverse Lariat Peptide Scaffolds Reveal an Untapped Chemical Space of High Membrane Permeability.

Constrained, membrane-permeable peptides offer the possibility of engaging challenging intracellular targets. Structure-permeability relationships have been extensively studied in cyclic peptides whose backbones are cyclized from head to tail, like the membrane permeable and orally bioavailable natural product cyclosporine A. In contrast, the physicochemical properties of lariat peptides, which are cyclized from one of the termini onto a side chain, have received little attention. Many lariat peptide natural products exhibit interesting biological activities, and some, such as griselimycin and didemnin B, are membrane permeable and have intracellular targets. To investigate the structure-permeability relationships in the chemical space exemplified by these natural products, we generated a library of scaffolds using stable isotopes to encode stereochemistry and determined the passive membrane permeability of over 1000 novel lariat peptide scaffolds with molecular weights around 1000. Many lariats were surprisingly permeable, comparable to many known orally bioavailable drugs. Passive permeability was strongly dependent on N-methylation, stereochemistry, and ring topology. A variety of structure-permeability trends were observed including a relationship between alternating stereochemistry and high permeability, as well as a set of highly permeable consensus sequences. For the first time, robust structure-permeability relationships are established in synthetic lariat peptides exceeding 1000 compounds.

Retrospective analysis of natural products provides insights for future discovery trends.

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.

Drug-Like Properties in Macrocycles above MW 1000: Backbone Rigidity versus Side-Chain Lipophilicity.

Large macrocyclic peptides can achieve surprisingly high membrane permeability, although the properties that govern permeability in this chemical space are only beginning to come into focus. We generated two libraries of cyclic decapeptides with stable cross-ß conformations, and found that peptoid substitutions within the ß-turns of the macrocycle preserved the rigidity of the parent scaffold, whereas peptoid substitutions in the opposing ß-strands led to "chameleonic" species that were rigid in nonpolar media but highly flexible in water. Both rigid and chameleonic compounds showed high permeability over a wide lipophilicity range, with peak permeabilities differing significantly depending on scaffold rigidity. Our findings indicate that modulating lipophilicity can be used to engineer favorable ADME properties into both rigid and flexible macrocyclic peptides, and that scaffold rigidity can be used to tune optimal lipophilicity.

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.

Cell-permeable cyclic peptides from synthetic libraries inspired by natural products.

Drug design efforts are turning to a new generation of therapeutic targets, such as protein-protein interactions (PPIs), that had previously been considered "undruggable" by typical small molecules. There is an emerging view that accessing these targets will require molecules that are larger and more complex than typical small molecule drugs. Here, we present a methodology for the discovery of geometrically diverse, membrane permeable cyclic peptide scaffolds based on the synthesis and permeability screening of a combinatorial library, followed by deconvolution of membrane-permeable scaffolds to identify cyclic peptides with good to excellent passive cell permeabilities. We use a combination of experimental and computational approaches to investigate structure-permeability relationships in one of these scaffolds, and uncover structural and conformational factors that govern passive membrane diffusion in a related set of cyclic peptide diastereomers. Further, we investigate the dependency of permeability on side-chain identity of one of these scaffolds through single-point diversifications to show the adaptability of these scaffolds toward development of permeability-biased libraries suitable for bioactivity screens. Overall, our results demonstrate that many novel, cell permeable scaffolds exist beyond those found in extant natural products, and that such scaffolds can be rapidly identified using a combination of synthesis and deconvolution which can, in principle, be applied to any type of macrocyclic template.

DNA-Encoded Macrocyclic Peptide Libraries Enable the Discovery of a Neutral MDM2-p53 Inhibitor.

Synthetic macrocyclic peptides are an emerging molecular class for both targeting intracellular protein-protein interactions (PPIs) and providing an oral modality for drug targets typically addressed by biologics. Display technologies, such as mRNA and phage display, often yield peptides that are too large and too polar to achieve passive permeability or oral bioavailability without substantial off-platform medicinal chemistry. Herein, we use DNA-encoded cyclic peptide libraries to discover a neutral nonapeptide, UNP-6457, that inhibits MDM2-p53 interaction with an IC50 of 8.9 nM. X-ray structural analysis of the MDM2-UNP-6457 complex revealed mutual binding interactions and identified key ligand modification points which may be tuned to enhance its pharmacokinetic profile. These studies showcase how tailored DEL libraries can directly yield macrocyclic peptides benefiting from low MW, TPSA, and HBD/HBA counts that are capable of potently inhibiting therapeutically relevant protein-protein interactions.

NP Analyst: An Open Online Platform for Compound Activity Mapping.

Few tools exist in natural products discovery to integrate biological screening and untargeted mass spectrometry data at the library scale. Previously, we reported Compound Activity Mapping as a strategy for predicting compound bioactivity profiles directly from primary screening results on extract libraries. We now present NP Analyst, an open online platform for Compound Activity Mapping that accepts bioassay data of almost any type, and is compatible with mass spectrometry data from major instrument manufacturers via the mzML format. In addition, NP Analyst will accept processed mass spectrometry data from the MZmine 2 and GNPS open-source platforms, making it a versatile tool for integration with existing discovery workflows. We demonstrate the utility of this new tool for both the dereplication of known compounds and the discovery of novel bioactive natural products using a challenging low-resolution antimicrobial bioassay data set. This new platform is available at www.npanalyst.org.

Lipophilic Permeability Efficiency Reconciles the Opposing Roles of Lipophilicity in Membrane Permeability and Aqueous Solubility.

As drug discovery moves increasingly toward previously "undruggable" targets such as protein-protein interactions, lead compounds are becoming larger and more lipophilic. Although increasing lipophilicity can improve membrane permeability, it can also incur serious liabilities, including poor water solubility, increased toxicity, and faster metabolic clearance. Here we introduce a new efficiency metric, especially relevant to "beyond rule of 5" molecules, that captures, in a simple, unitless value, these opposing effects of lipophilicity on molecular properties. Lipophilic permeability efficiency (LPE) is defined as log D7.4dec/w - mlipocLogP + bscaffold, where log D7.4dec/w is the experimental decadiene-water distribution coefficient (pH 7.4), cLogP is the calculated octanol-water partition coefficient, and mlipo and bscaffold are scaling factors to standardize LPE values across different cLogP metrics and scaffolds. Using a variety of peptidic and nonpeptidic macrocycle drugs, we show that LPE provides a functional assessment of the efficiency with which a compound achieves passive membrane permeability at a given lipophilicity.

Nonclassical Size Dependence of Permeation Defines Bounds for Passive Adsorption of Large Drug Molecules.

Macrocyclic peptides are considered large enough to inhibit "undruggable" targets, but the design of passively cell-permeable molecules in this space remains a challenge due to the poorly understood role of molecular size on passive membrane permeability. Using split-pool combinatorial synthesis, we constructed a library of cyclic, per-N-methlyated peptides spanning a wide range of calculated lipohilicities (0 < AlogP < 8) and molecular weights (∼800 Da < MW < ∼1200 Da). Analysis by the parallel artificial membrane permeability assay revealed a steep drop-off in apparent passive permeability with increasing size in stark disagreement with current permeation models. This observation, corroborated by a set of natural products, helps define criteria for achieving permeability in larger molecular size regimes and suggests an operational cutoff, beyond which passive permeability is constrained by a sharply increasing penalty on membrane permeation.

Passive Membrane Permeability in Cyclic Peptomer Scaffolds Is Robust to Extensive Variation in Side Chain Functionality and Backbone Geometry.

Synthetic and natural cyclic peptides provide a testing ground for studying membrane permeability in nontraditional drug scaffolds. Cyclic peptomers, which incorporate peptide and N-alkylglycine (peptoid) residues, combine the stereochemical and geometric complexity of peptides with the functional group diversity accessible to peptoids. We synthesized cyclic peptomer libraries by split-pool techniques, separately permuting side chain and backbone geometry, and analyzed their membrane permeabilities using the parallel artificial membrane permeability assay. Nearly half of the side chain permutations had permeability coefficients (Papp) > 1 × 10-6 cm/s. Some backbone geometries enhanced permeability due to their ability to form more stable intramolecular hydrogen bond networks compared with other scaffolds. These observations suggest that hexameric cyclic peptomers can have good passive permeability even in the context of extensive side chain and backbone variation, and that high permeability can generally be achieved within a relatively wide lipophilicity range.

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.

Stereochemistry Balances Cell Permeability and Solubility in the Naturally Derived Phepropeptin Cyclic Peptides.

Cyclic peptide (CP) natural products provide useful model systems for mapping "beyond-Rule-of-5" (bRo5) space. We identified the phepropeptins as natural product CPs with potential cell permeability. Synthesis of the phepropeptins and epimeric analogues revealed much more rapid cellular permeability for the natural stereochemical pattern. Despite being more cell permeable, the natural compounds exhibited similar aqueous solubility as the corresponding epimers, a phenomenon explained by solvent-dependent conformational flexibility among the natural compounds. When analyzing the polarity of the solution structures we found that neither the number of hydrogen bonds nor the total polar surface area accurately represents the solvation energies of the high and low dielectric conformations. This work adds to a growing number of natural CPs whose solvent-dependent conformational behavior allows for a balance between aqueous solubility and cell permeability, highlighting structural flexibility as an important consideration in the design of molecules in bRo5 chemical space.

Going Out on a Limb: Delineating The Effects of ß-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 ß-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 ß-branching, N-methylation, and side chain size to passive membrane permeability and aqueous solubility. We demonstrate that ß-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.

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.