Macrocyclic Drugs and Clinical Candidates: What Can Medicinal Chemists Learn from Their Properties?

Macrocycles are ideal in efforts to tackle “difficult” targets, but our understanding of what makes them cell permeable and orally bioavailable is limited. Analysis of approximately 100 macrocyclic drugs and clinical candidates revealed that macrocycles are predominantly used for infectious disease and in oncology and that most belong to the macrolide or cyclic peptide class. A significant number (<i>N</i> = 34) of these macrocycles are administered orally, revealing that oral bioavailability can be obtained at molecular weights up to and above 1 kDa and polar surface areas ranging toward 250 Ų. Moreover, insight from a group of “de novo designed” oral macrocycles in clinical studies and understanding of how cyclosporin A and model cyclic hexapeptides cross cell membranes may unlock wider opportunities in drug discovery. However, the number of oral macrocycles is still low and it remains to be seen if they are outliers or if macrocycles will open up novel oral druggable space

How Beyond Rule of 5 Drugs and Clinical Candidates Bind to Their Targets

To improve discovery of drugs for difficult targets, the opportunities of chemical space beyond the rule of 5 (bRo5) were examined by retrospective analysis of a comprehensive set of structures for complexes between drugs and clinical candidates and their targets. The analysis illustrates the potential of compounds far beyond rule of 5 space to modulate novel and difficult target classes that have large, flat, and groove-shaped binding sites. However, ligand efficiencies are significantly reduced for flat- and groove-shape binding sites, suggesting that adjustments of how to use such metrics are required. Ligands bRo5 appear to benefit from an appropriate balance between rigidity and flexibility to bind with sufficient affinity to their targets, with macrocycles and nonmacrocycles being found to have similar flexibility. However, macrocycles were more disk- and spherelike, which may contribute to their superior binding to flat sites, while rigidification of nonmacrocycles lead to rodlike ligands that bind well to groove-shaped binding sites. These insights should contribute to altering perceptions of what targets are considered “druggable” and provide support for drug design in beyond rule of 5 space

Predictive Modeling of PROTAC Cell Permeability with Machine Learning

Approaches for predicting proteolysis targeting chimera (PROTAC) cell permeability are of major interest to reduce resource-demanding synthesis and testing of low-permeable PROTACs. We report a comprehensive investigation of the scope and limitations of machine learning-based binary classification models developed using 17 simple descriptors for large and structurally diverse sets of cereblon (CRBN) and von Hippel–Lindau (VHL) PROTACs. For the VHL PROTAC set, kappa nearest neighbor and random forest models performed best and predicted the permeability of a blinded test set with >80% accuracy (k ≥ 0.57). Models retrained by combining the original training and the blinded test set performed equally well for a second blinded VHL set. However, models for CRBN PROTACs were less successful, mainly due to the imbalanced nature of the CRBN datasets. All descriptors contributed to the models, but size and lipophilicity were the most important. We conclude that properly trained machine learning models can be integrated as effective filters in the PROTAC design process

Linker-Dependent Folding Rationalizes PROTAC Cell Permeability

Proteolysis-targeting chimeras (PROTACs) must be cell permeable to reach their target proteins. This is challenging as the bivalent structure of PROTACs puts them in chemical space at, or beyond, the outer limits of oral druggable space. We used NMR spectroscopy and molecular dynamics (MD) simulations independently to gain insights into the origin of the differences in cell permeability displayed by three flexible cereblon PROTACs having closely related structures. Both methods revealed that the propensity of the PROTACs to adopt folded conformations with a low solvent-accessible 3D polar surface area in an apolar environment is correlated to high cell permeability. The chemical nature and the flexibility of the linker were essential for the PROTACs to populate folded conformations stabilized by intramolecular hydrogen bonds, π–π interactions, and van der Waals interactions. We conclude that MD simulations may be used for the prospective ranking of cell permeability in the design of cereblon PROTACs

Linker-Dependent Folding Rationalizes PROTAC Cell Permeability

Proteolysis-targeting chimeras (PROTACs) must be cell permeable to reach their target proteins. This is challenging as the bivalent structure of PROTACs puts them in chemical space at, or beyond, the outer limits of oral druggable space. We used NMR spectroscopy and molecular dynamics (MD) simulations independently to gain insights into the origin of the differences in cell permeability displayed by three flexible cereblon PROTACs having closely related structures. Both methods revealed that the propensity of the PROTACs to adopt folded conformations with a low solvent-accessible 3D polar surface area in an apolar environment is correlated to high cell permeability. The chemical nature and the flexibility of the linker were essential for the PROTACs to populate folded conformations stabilized by intramolecular hydrogen bonds, π–π interactions, and van der Waals interactions. We conclude that MD simulations may be used for the prospective ranking of cell permeability in the design of cereblon PROTACs

Dynamics Determine Signaling in a Multicomponent System Associated with Rheumatoid Arthritis

Strategies that target multiple components are usually required for treatment of diseases originating from complex biological systems. The multicomponent system consisting of the DR4 major histocompatibility complex type II molecule, the glycopeptide CII259–273 from type II collagen, and a T-cell receptor is associated with development of rheumatoid arthritis (RA). We introduced non-native amino acids and amide bond isosteres into CII259–273 and investigated the effect on binding to DR4 and the subsequent T-cell response. Molecular dynamics simulations revealed that complexes between DR4 and derivatives of CII259–273 were highly dynamic. Signaling in the overall multicomponent system was found to depend on formation of an appropriate number of dynamic intramolecular hydrogen bonds between DR4 and CII259–273, together with the positioning of the galactose moiety of CII259–273 in the DR4 binding groove. Interestingly, the system tolerated modifications at several positions in CII259–273, indicating opportunities to use analogues to increase our understanding of how rheumatoid arthritis develops and for evaluation as vaccines to treat RA

Dynamics Determine Signaling in a Multicomponent System Associated with Rheumatoid Arthritis

Strategies that target multiple components are usually required for treatment of diseases originating from complex biological systems. The multicomponent system consisting of the DR4 major histocompatibility complex type II molecule, the glycopeptide CII259–273 from type II collagen, and a T-cell receptor is associated with development of rheumatoid arthritis (RA). We introduced non-native amino acids and amide bond isosteres into CII259–273 and investigated the effect on binding to DR4 and the subsequent T-cell response. Molecular dynamics simulations revealed that complexes between DR4 and derivatives of CII259–273 were highly dynamic. Signaling in the overall multicomponent system was found to depend on formation of an appropriate number of dynamic intramolecular hydrogen bonds between DR4 and CII259–273, together with the positioning of the galactose moiety of CII259–273 in the DR4 binding groove. Interestingly, the system tolerated modifications at several positions in CII259–273, indicating opportunities to use analogues to increase our understanding of how rheumatoid arthritis develops and for evaluation as vaccines to treat RA

Macrocyclic Prodrugs of a Selective Nonpeptidic Direct Thrombin Inhibitor Display High Permeability, Efficient Bioconversion but Low Bioavailability

The only oral direct thrombin inhibitors that have reached the market, ximelagatran and dabigatran etexilat, are double prodrugs with low bioavailability in humans. We have evaluated an alternative strategy: the preparation of a nonpeptidic, polar direct thrombin inhibitor as a single, macrocyclic esterase-cleavable (acyloxy)­alkoxy prodrug. Two homologous prodrugs were synthesized and displayed high solubilities and Caco-2 cell permeabilities, suggesting high absorption from the intestine. In addition, they were rapidly and completely converted to the active zwitterionic thrombin inhibitor in human hepatocytes. Unexpectedly, the most promising prodrug displayed only moderately higher oral bioavailability in rat than the polar direct thrombin inhibitor, most likely due to rapid metabolism in the intestine or the intestinal wall. To the best of our knowledge, this is the first in vivo ADME study of macrocyclic (acyloxy)­alkoxy prodrugs, and it remains to be established if the modest increase in bioavailability is a general feature of this category of prodrugs or not

3-Aminopiperidine-Based Peptide Analogues as the First Selective Noncovalent Inhibitors of the Bacterial Cysteine Protease IdeS

A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes, IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies

Impact of Dynamically Exposed Polarity on Permeability and Solubility of Chameleonic Drugs Beyond the Rule of 5

Conformational flexibility has been proposed to significantly affect drug properties outside rule-of-5 (Ro5) chemical space. Here, we investigated the influence of dynamically exposed polarity on cell permeability and aqueous solubility for a structurally diverse set of drugs and clinical candidates far beyond the Ro5, all of which populated multiple distinct conformations as revealed by X-ray crystallography. Efflux-inhibited (passive) Caco-2 cell permeability correlated strongly with the compounds’ minimum solvent-accessible 3D polar surface areas (PSA), whereas aqueous solubility depended less on the specific 3D conformation. Inspection of the crystal structures highlighted flexibly linked aromatic side chains and dynamically forming intramolecular hydrogen bonds as particularly effective in providing “chameleonic” properties that allow compounds to display both high cell permeability and aqueous solubility. These structural features, in combination with permeability predictions based on the correlation to solvent-accessible 3D PSA, should inspire drug design in the challenging chemical space far beyond the Ro5