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.

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.

Antibiotic-resistant bacteria are an emerging threat to global public health. New classes of antibiotics and tools for antimicrobial discovery are urgently needed. Type III secretion systems (T3SS), which are required by dozens of Gramnegative bacteria for virulence but largely absent from nonpathogenic bacteria, are promising virulence blocker targets. The ability of mammalian cells to recognize the presence of a functional T3SS and trigger NF-B activation provides a rapid and sensitive method for identifying chemical inhibitors of T3SS activity. In this study, we generated a HEK293 stable cell line expressing green fluorescent protein (GFP) driven by a promoter containing NF-B enhancer elements to serve as a readout of T3SS function. We identified a family of synthetic cyclic peptide-peptoid hybrid molecules (peptomers) that exhibited dose-dependent inhibition of T3SS effector secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa without affecting bacterial growth or motility. Among these inhibitors, EpD-3=N, EpD-1,2N, EpD-1,3=N, EpD-1,2,3=N, and EpD-1,2,4=N exhibited strong inhibitory effects on translocation of the Yersinia YopM effector protein into mammalian cells (Ͼ40% translocation inhibition at 7.5 M) and showed no toxicity to mammalian cells at 240 M. In addition, EpD-3=N and EpD-1,2,4=N reduced the rounding of HeLa cells caused by the activity of Yersinia effector proteins that target the actin cytoskeleton. In summary, we have discovered a family of novel cyclic peptomers that inhibit the injectisome T3SS but not the flagellar T3SS.

Installation of methyl groups can significantly improve the binding of small-molecule drugs to protein targets; however, siteselective methylation often presents a significant synthetic challenge. Metal-and S-adenosyl-methionine (SAM)-dependent methyltransferases (MTs) in natural-product biosynthetic pathways are powerful enzymatic tools for selective or chemically challenging C-methylation reactions. Each of these MTs selectively catalyzes one or two methyl transfer reactions. Crystal structures and biochemical assays of the Mn 2+ -dependent monomethyltransferase from the saxitoxin biosynthetic pathway (SxtA MT) revealed the structural basis for control of methylation extent. The SxtA monomethyltransferase was converted to a dimethyltransferase by modification of the metal binding site, addition of an active site base, and an amino acid substitution to provide space in the substrate pocket for two methyl substituents. A reciprocal change converted a related dimethyltransferase into a monomethyltransferase, supporting our hypothesis that steric hindrance can prevent a second methylation event. A novel understanding of MTs will accelerate the development of MT-based catalysts and MT engineering for use in small-molecule synthesis.

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