An Amphiphilic Cell-Penetrating Macrocycle for Efficient Cytosolic Delivery of Proteins, DNA, and CRISPR Cas9.

The discovery of safe platforms that can circumvent the endocytic pathway is of great significance for biological therapeutics that are usually degraded during endocytosis. Here we show that a self-assembled and dynamic macrocycle can passively diffuse through the cell membrane and deliver a broad range of biologics, including proteins, CRISPR Cas9, and ssDNA, directly to the cytosol while retaining their bioactivity. Cell-penetrating macrocycle CPM can be easily prepared from the room temperature condensation of diketopyrrolopyrrole lactams with diamines. We attribute the high cellular permeability of CPM to its amphiphilic nature and chameleonic properties. It adopts conformations that partially bury polar groups and expose hydrophobic side chains, thus self-assembling into micellar-like structures. Its superior fluorescence makes CPM trackable inside cells where it follows the endomembrane system. CPM outperformed commercial reagents for biologics delivery and showed high RNA knockdown efficiency of CRISPR Cas9. We envisage that this macrocycle will be an ideal starting point to design and synthesize biomimetic macrocyclic tags that can readily facilitate the interaction and uptake of biomolecules and overcome endosomal digestion.

In Vitro Selection of Foldamer-Like Macrocyclic Peptides Containing 2-Aminobenzoic Acid and 3-Aminothiophene-2-Carboxylic Acid.

Aromatic cyclic ß2,3-amino acids (cßAAs), such as 2-aminobenzoic acid and 3-aminothiophene-2-carboxylic acid, are building blocks that can induce unique folding propensities of peptides. Although their ribosomal elongation had been a formidable task due to the low nucleophilicity of their amino groups, we have recently overcome this issue by means of an engineered tRNAPro1E2 that enhances their incorporation efficiency into nascent peptide chains. Here we report ribosomal synthesis of a random macrocyclic peptide library containing aromatic and aliphatic cßAAs, and its application to de novo discovery of binders against human IFNGR1 and FXIIa as model targets. The potent binding peptides showed not only high inhibitory activity but also high protease resistance in human serum. Moreover, these cßAAs play a critical role in exhibiting their properties, establishing a discovery platform for de novo foldamer-like macrocycles containing such unique building blocks.

Synthesis of 20-Membered Macrocyclic Pseudo-Natural Products Yields Inducers of LC3 Lipidation.

Design and synthesis of pseudo-natural products (PNPs) through recombination of natural product (NP) fragments in unprecedented arrangements enables the discovery of novel biologically relevant chemical matter. With a view to wider coverage of NP-inspired chemical and biological space, we describe the combination of this principle with macrocycle formation. PNP-macrocycles were synthesized efficiently in a stereoselective one-pot procedure including the 1,3-dipolar cycloadditions of different dipolarophiles with dimeric cinchona alkaloid-derived azomethine ylides formed in situ. The 20-membered bis-cycloadducts embody 18 stereocenters and an additional fragment-sized NP-structure. After further functionalization, a collection of 163 macrocyclic PNPs was obtained. Biological investigation revealed potent inducers of the lipidation of the microtubule associated protein 1 light chain 3 (LC3) protein, which plays a prominent role in various autophagy-related processes.

Coassembly of hypoxia-sensitive macrocyclic amphiphiles and extracellular vesicles for targeted kidney injury imaging and therapy.

BACKGROUND: Hypoxia is a major contributor to global kidney diseases. Targeting hypoxia is a promising therapeutic option against both acute kidney injury and chronic kidney disease; however, an effective strategy that can achieve simultaneous targeted kidney hypoxia imaging and therapy has yet to be established. Herein, we fabricated a unique nano-sized hypoxia-sensitive coassembly (Pc/C5A@EVs) via molecular recognition and self-assembly, which is composed of the macrocyclic amphiphile C5A, the commercial dye sulfonated aluminum phthalocyanine (Pc) and mesenchymal stem cell-excreted extracellular vesicles (MSC-EVs). RESULTS: In murine models of unilateral or bilateral ischemia/reperfusion injury, MSC-EVs protected the Pc/C5A complex from immune metabolism, prolonged the circulation time of the complex, and specifically led Pc/C5A to hypoxic kidneys via surface integrin receptor α4ß1 and αLß2, where Pc/C5A released the near-infrared fluorescence of Pc and achieved enhanced hypoxia-sensitive imaging. Meanwhile, the coassembly significantly recovered kidney function by attenuating cell apoptosis, inhibiting the progression of renal fibrosis and reducing tubulointerstitial inflammation. Mechanistically, the Pc/C5A coassembly induced M1-to-M2 macrophage transition by inhibiting the HIF-1α expression in hypoxic renal tubular epithelial cells (TECs) and downstream NF-κB signaling pathway to exert their regenerative effects. CONCLUSION: This synergetic nanoscale coassembly with great translational potential provides a novel strategy for precise kidney hypoxia diagnosis and efficient kidney injury treatment. Furthermore, our strategy of coassembling exogenous macrocyclic receptors with endogenous cell-derived membranous structures may offer a functional platform to address multiple clinical needs.

Actinomadura graeca sp. nov.: A novel producer of the macrocyclic antibiotic zelkovamycin.

As part of a screening programme for antibiotic-producing bacteria, a novel Actinomadura species was discovered from a soil sample collected in Santorini, Greece. Preliminary 16S rRNA gene sequence comparisons highlighted Actinomadura macra as the most similar characterised species. However, whole-genome sequencing revealed an average nucleotide identity (ANI) value of 89% with A. macra, the highest among related species. Further phenotypic and chemotaxonomic analyses confirmed that the isolate represents a previously uncharacterised species in the genus Actinomadura, for which the name Actinomadura graeca sp. nov. is proposed (type strain 32-07T). The G+C content of A. graeca 32-07 is 72.36%. The cell wall contains DL-diaminopimelic acid, intracellular sugars are glucose, ribose and galactose, the predominant menaquinone is MK-9(H6), the major cellular lipid is phosphatidylinositol and fatty acids consist mainly of hexadecanoic acid. No mycolic acid was detected. Furthermore, A. graeca 32-07 has been confirmed as a novel producer of the non-ribosomal peptide antibiotic zelkovamycin and we report herein a provisional description of the unique biosynthetic gene cluster.

Theoretical and experimental study of interaction of macroheterocyclic compounds with ORF3a of SARS-CoV-2.

The pandemic infectious disease (Covid-19) caused by the coronavirus (SARS-CoV2) is spreading rapidly around the world. Covid-19 does an irreparable harm to the health and life of people. It also has a negative financial impact on the economies of most countries of the world. In this regard, the issue of creating drugs aimed at combating this disease is especially acute. In this work, molecular docking was used to study the docking of 23 compounds with QRF3a SARS-CoV2. The performed in silico modeling made it possible to identify leading compounds capable of exerting a potential inhibitory and virucidal effect. The leading compounds include chlorin (a drug used in PDT), iron(III)protoporphyrin (endogenous porphyrin), and tetraanthraquinone porphyrazine (an exogenous substance). Having taken into consideration the localization of ligands in the QRF3a SARS-CoV2, we have made an assumption about their influence on the pathogenesis of Covid-19. The interaction of chlorin, iron(III)protoporphyrin and protoporphyrin with the viral protein ORF3a were studied by fluorescence and UV-Vis spectroscopy. The obtained experimental results confirm the data of molecular docking. The results showed that a viral protein binds to endogenous porphyrins and chlorins, moreover, chlorin is a competitive ligand for endogenous porphyrins. Chlorin should be considered as a promising drug for repurposing.

Computational Insights into the Role of External and Local Electric Fields in Macrocyclic Chemical and Biological Systems.

The investigation of the role of the electric field in systems of widespread interest employing computational techniques is an emerging area of research. The outcome of applying an oriented external electric field (OEEF) on the geometric and electronic properties of the chemically unique π-conjugated cyclic carbon ring compounds has been explored with density functional theory (DFT). Distinct changes in the structural and electronic features of such ring compounds are observed upon the application of OEEFs. Importantly, the calculations indicate that a mixed aliphatic-aromatic conjugated ring converts from a singlet to a triplet after the application of an OEEF, suggesting potential applications in optoelectronics for such molecules, without the need for photochemically induced change in the spin state. Furthermore, the influence of built-in local electric fields (LEFs) present in naturally occurring macrocyclic systems such as valinomycin has also been explored. Static and ab initio molecular dynamics (AIMD) calculations indicate that LEFs are the primary driving factor in determining the energetically favoured position of counter anions such as chloride (Cl- ) in the potassium (K+ ) and sodium (Na+ ) coordinated valinomycin macrocycle structures: they exist inside the cage in the case of K+ sequestration by valinomycin and outside for Na+ . This divergence has been proposed to be the determining factor for the selectivity of the valinomycin macrocycle for binding a K+ cation over Na+ .

A Cryptic Plant Terpene Cyclase Producing Unconventional 18- and 14-Membered Macrocyclic C25 and C20 Terpenoids with Immunosuppressive Activity.

A versatile terpene synthase (LcTPS2) producing unconventional macrocyclic terpenoids was characterized from Leucosceptrum canum. Engineered Escherichia coli and Nicotiana benthamiana expressing LcTPS2 produced six 18-/14-membered sesterterpenoids including five new ones and two 14-membered diterpenoids. These products represent the first macrocyclic sesterterpenoids from plants and the largest sesterterpenoid ring system identified to date. Two variants F516A and F516G producing approximately 3.3- and 2.5-fold, respectively, more sesterterpenoids than the wild-type enzyme were engineered. Both 18- and 14-membered ring sesterterpenoids displayed significant inhibitory activity on the IL-2 and IFN-γ production of T cells probably via inhibition of the MAPK pathway. The findings will contribute to the development of efficient biocatalysts to create bioactive macrocyclic sesterterpenoids, and also herald a new potential in the well-trodden territory of plant terpenoid biosynthesis.

Semisynthetic Macrocyclic Lipo-lanthipeptides Display Antimicrobial Activity Against Bacterial Pathogens.

A large number of antimicrobial peptides depend on intramolecular disulfide bonds for their biological activity. However, the relative instability of disulfide bonds has limited the potential of some of these peptides to be developed into therapeutics. Conversely, peptides containing intramolecular (methyl)lanthionine-based bonds, lanthipeptides, are highly stable under a broader range of biological and physical conditions. Here, the class-II lanthipeptide synthetase CinM, from the cinnamycin gene cluster, was employed to create methyllanthionine stabilized analogues of disulfide-bond-containing antimicrobial peptides. The resulting analogues were subsequently modified in vitro by adding lipid tails of variable lengths through chemical addition. Finally, the created compounds were characterized by MIC tests against several relevant pathogens, killing assays, membrane permeability assays, and hemolysis assays. It was found that CinM could successfully install methyllanthionine bonds at the intended positions of the analogues and that the lipidated macrocyclic core peptides have bactericidal activity against tested Gram-positive and Gram-negative pathogenic bacteria. Additionally, fluorescence microscopy assays revealed that the lipidated compounds disrupt the bacterial membrane and lyse bacterial cells, hinting toward a potential mode of action. Notably, the semisynthesized macrocyclic lipo-lanthipeptides show low hemolytic activity. These results show that the methods developed here extend the toolbox for novel antimicrobial development and might enable the further development of novel compounds with killing activity against relevant pathogenic bacteria.

Discovery of Quinoxaline-Based P1-P3 Macrocyclic NS3/4A Protease Inhibitors with Potent Activity against Drug-Resistant Hepatitis C Virus Variants.

The three pan-genotypic HCV NS3/4A protease inhibitors (PIs) currently in clinical use-grazoprevir, glecaprevir, and voxilaprevir-are quinoxaline-based P2-P4 macrocycles and thus exhibit similar resistance profiles. Using our quinoxaline-based P1-P3 macrocyclic lead compounds as an alternative chemical scaffold, we explored structure-activity relationships (SARs) at the P2 and P4 positions to develop pan-genotypic PIs that avoid drug resistance. A structure-guided strategy was used to design and synthesize two series of compounds with different P2 quinoxalines in combination with diverse P4 groups of varying sizes and shapes, with and without fluorine substitutions. Our SAR data and cocrystal structures revealed the interplay between the P2 and P4 groups, which influenced inhibitor binding and the overall resistance profile. Optimizing inhibitor interactions in the S4 pocket led to PIs with excellent antiviral activity against clinically relevant PI-resistant HCV variants and genotype 3, providing potential pan-genotypic inhibitors with improved resistance profiles.

Rapid and selective detection of macrocyclic trichothecene producing Stachybotrys chartarum strains by loop-mediated isothermal amplification (LAMP).

Cytotoxic macrocyclic trichothecenes such as satratoxins are produced by chemotype S strains of Stachybotrys chartarum. Diseases such as stachybotryotoxicosis in animals and the sick building syndrome as a multifactorial disease complex in humans have been associated with this mold and its toxins. Less toxic non-chemotype S strains of S. chartarum are morphologically indistinguishable from chemotype S strains, which results in uncertainties in hazard characterization of isolates. To selectively identify macrocyclic trichothecene producing S. chartarum isolates, a set of sat14 gene-specific primers was designed and applied in a loop-mediated isothermal amplification (LAMP) assay using neutral red for visual signal detection. The assay was highly specific for S. chartarum strains of the macrocyclic trichothecene producing chemotype and showed no cross-reaction with non-macrocyclic trichothecene producing S. chartarum strains or 152 strains of 131 other fungal species. The assay's detection limit was 0.635 pg/rxn (picogram per reaction) with a reaction time of 60 min. Its high specificity and sensitivity as well as the cost-saving properties make the new assay an interesting and powerful diagnostic tool for easy and rapid testing.

Macrolide Inspired Macrocycles as Modulators of the IL-17A/IL-17RA Interaction.

Interleukin 17 (IL-17) cytokines promote inflammatory pathophysiology in many autoimmune diseases, including psoriasis, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Such broad involvement of IL-17 in various autoimmune diseases makes it an ideal target for drug discovery. Psoriasis is a chronic inflammatory disease characterized by numerous defective components of the immune system. Significantly higher levels of IL-17A have been noticed in lesions of psoriatic patients, if compared to non-lesion parts. Therefore, this paper is focused on the macrolide inspired macrocycles as potential IL-17A/IL-17RA modulators and covers the molecular design, synthesis, and in vitro profiling. Macrocycles are designed to diversify and enrich chemical space through different ring sizes and a variety of three-dimensional shapes. Inhibitors in the nM range were identified in both target-based and phenotypic assays. In vitro ADME as well as in vivo PK properties are reported.

Sense codon reassignment enables viral resistance and encoded polymer synthesis.

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.

Structural Basis for a Dual Function ATP Grasp Ligase That Installs Single and Bicyclic ω-Ester Macrocycles in a New Multicore RiPP Natural Product.

Among the ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, "graspetides" (formerly known as microviridins) contain macrocyclic esters and amides that are formed by ATP-grasp ligase tailoring enzymes using the side chains of Asp/Glu as acceptors and Thr/Ser/Lys as donors. Graspetides exhibit diverse patterns of macrocylization and connectivities exemplified by microviridins, that have a caged tricyclic core, and thuringin and plesiocin that feature a "hairpin topology" with cross-strand ω-ester bonds. Here, we characterize chryseoviridin, a new type of multicore RiPP encoded by Chryseobacterium gregarium DS19109 (Phylum Bacteroidetes) and solve a 2.44 Å resolution crystal structure of a quaternary complex consisting of the ATP-grasp ligase CdnC bound to ADP, a conserved leader peptide and a peptide substrate. HRMS/MS analyses show that chryseoviridin contains four consecutive five- or six-residue macrocycles ending with a microviridin-like core. The crystal structure captures respective subunits of the CdnC homodimer in the apo or substrate-bound state revealing a large conformational change in the B-domain upon substrate binding. A docked model of ATP places the γ-phosphate group within 2.8 Å of the Asp acceptor residue. The orientation of the bound substrate is consistent with a model in which macrocyclization occurs in the N- to C-terminal direction for core peptides containing multiple Thr/Ser-to-Asp macrocycles. Using systematically varied sequences, we validate this model and identify two- or three-amino acid templating elements that flank the macrolactone and are required for enzyme activity in vitro. This work reveals the structural basis for ω-ester bond formation in RiPP biosynthesis.

Ribosomal Synthesis of Macrocyclic Peptides with ß2- and ß2,3-Homo-Amino Acids for the Development of Natural Product-Like Combinatorial Libraries.

The development of large, natural-product-like, combinatorial macrocyclic peptide libraries is essential in the quest to develop therapeutics for "undruggable" cellular targets. Herein we report the ribosomal synthesis of macrocyclic peptides containing one or more ß2-homo-amino acids (ß2haa) to enable their incorporation into mRNA display-based selection libraries. We confirmed the compatibility of 14 ß2-homo-amino acids, (S)- and (R)-stereochemistry, for single incorporation into a macrocyclic peptide with low to high translation efficiency. Interestingly, N-methylation of the backbone amide of ß2haa prevented the incorporation of this amino acid subclass by the ribosome. Additionally, we designed and incorporated several α,ß-disubstituted ß2,3-homo-amino acids (ß2,3haa) with different R-groups on the α- and ß-carbons of the same amino acid. Incorporation of these ß2,3haa enables increased diversity in a single position of a macrocyclic peptide without significantly increasing the overall molecular weight, which is an important consideration for passive cell permeability. We also successfully incorporated multiple (S)-ß2hAla into a single macrocycle with other non-proteinogenic amino acids, confirming that this class of ß-amino acid is suitable for development of large scale macrocyclic peptide libraries.

Structure-Enabled Discovery of Novel Macrocyclic Inhibitors Targeting Glutaminase 1 Allosteric Binding Site.

The inhibition of glutaminase 1 (GLS1) represents a potential treatment of malignant tumors. Structural analysis led to the design of a novel series of macrocyclic GLS1 allosteric inhibitors. Through extensive structure-activity relationship studies, a promising candidate molecule 13b (LL202) was identified with robust GLS1 inhibitory activity (IC50 = 6 nM) and high GLS1 binding affinity (SPR, Kd = 24 nM; ITC, Kd = 37 nM). The X-ray crystal structure of the 13b-GLS1 complex was resolved, revealing a unique binding mode and providing a novel structural scaffold for GLS1 allosteric inhibitors. Importantly, 13b clearly adjusted the cellular metabolites and induced an increase in the ROS level by blocking glutamine metabolism. Furthermore, 13b exhibited a similar in vivo antitumor activity as CB839. This study adds to the growing body of evidence that macrocyclization provides an alternative and complementary approach for the design of small-molecule inhibitors, with the potential to improve the binding affinity to the targets.

Modulation of the Passive Permeability of Semipeptidic Macrocycles: N- and C-Methylations Fine-Tune Conformation and Properties.

Incorporating small modifications to peptidic macrocycles can have a major influence on their properties. For instance, N-methylation has been shown to impact permeability. A better understanding of the relationship between permeability and structure is of key importance as peptidic drugs are often associated with unfavorable pharmacokinetic profiles. Starting from a semipeptidic macrocycle backbone composed of a tripeptide tethered head-to-tail with an alkyl linker, we investigated two small changes: peptide-to-peptoid substitution and various methyl placements on the nonpeptidic linker. Implementing these changes in parallel, we created a collection of 36 compounds. Their permeability was then assessed in parallel artificial membrane permeability assay (PAMPA) and Caco-2 assays. Our results show a systematic improvement in permeability associated with one peptoid position in the cycle, while the influence of methyl substitution varies on a case-by-case basis. Using a combination of molecular dynamics simulations and NMR measurements, we offer hypotheses to explain such behavior.

Genetically Encoded Fragment-Based Discovery from Phage-Displayed Macrocyclic Libraries with Genetically Encoded Unnatural Pharmacophores.

Genetically encoded macrocyclic peptide libraries with unnatural pharmacophores are valuable sources for the discovery of ligands for many targets of interest. Traditionally, generation of such libraries employs "early stage" incorporation of unnatural building blocks into the chemically or translationally produced macrocycles. Here, we describe a divergent late-stage approach to such libraries starting from readily available starting material: genetically encoded libraries of peptides. A diketone linchpin 1,5-dichloropentane-2,4-dione converts peptide libraries displayed on phage to 1,3-diketone bearing macrocyclic peptides (DKMP): shelf-stable precursors for Knorr pyrazole synthesis. Ligation of diverse hydrazine derivatives onto DKMP libraries displayed on phage that carries silent DNA-barcodes yields macrocyclic libraries in which the amino acid sequence and the pharmacophore are encoded by DNA. Selection of this library against carbonic anhydrase enriched macrocycles with benzenesulfonamide pharmacophore and nanomolar Kd. The methodology described in this manuscript can graft diverse pharmacophores into many existing genetically encoded phage libraries and significantly increase the value of such libraries in molecular discoveries.

SAMPL7 Host-Guest Challenge Overview: assessing the reliability of polarizable and non-polarizable methods for binding free energy calculations.

The SAMPL challenges focus on testing and driving progress of computational methods to help guide pharmaceutical drug discovery. However, assessment of methods for predicting binding affinities is often hampered by computational challenges such as conformational sampling, protonation state uncertainties, variation in test sets selected, and even lack of high quality experimental data. SAMPL blind challenges have thus frequently included a component focusing on host-guest binding, which removes some of these challenges while still focusing on molecular recognition. Here, we report on the results of the SAMPL7 blind prediction challenge for host-guest affinity prediction. In this study, we focused on three different host-guest categories-a familiar deep cavity cavitand series which has been featured in several prior challenges (where we examine binding of a series of guests to two hosts), a new series of cyclodextrin derivatives which are monofunctionalized around the rim to add amino acid-like functionality (where we examine binding of two guests to a series of hosts), and binding of a series of guests to a new acyclic TrimerTrip host which is related to previous cucurbituril hosts. Many predictions used methods based on molecular simulations, and overall success was mixed, though several methods stood out. As in SAMPL6, we find that one strategy for achieving reasonable accuracy here was to make empirical corrections to binding predictions based on previous data for host categories which have been studied well before, though this can be of limited value when new systems are included. Additionally, we found that alchemical free energy methods using the AMOEBA polarizable force field had considerable success for the two host categories in which they participated. The new TrimerTrip system was also found to introduce some sampling problems, because multiple conformations may be relevant to binding and interconvert only slowly. Overall, results in this challenge tentatively suggest that further investigation of polarizable force fields for these challenges may be warranted.

Prediction of Transporter-Mediated Drug-Drug Interactions and Phenotyping of Hepatobiliary Transporters Involved in the Clearance of E7766, a Novel Macrocycle-Bridged Dinucleotide.

E7766 represents a novel class of macrocycle-bridged dinucleotides and is under clinical development for immuno-oncology. In this report, we identified mechanism of systemic clearance E7766 and investigated the hepatobiliary transporters involved in the disposition of E7766 and potential drug interactions of E7766 as a victim of organic anion-transporting polypeptide (OATP) inhibitors. In bile-duct cannulated rats and dogs, E7766 was mainly excreted unchanged in bile (>80%) and to a lesser extent in urine (<20%). Sandwich-cultured human hepatocytes (SCHHs), transfected cells, and vesicles were used to phenotype the hepatobiliary transporters involved in the clearance of E7766. SCHH data showed temperature-dependent uptake of E7766 followed by active biliary secretion. In vitro transport assays using transfected cells and membrane vesicles confirmed that E7766 was a substrate of OATP1B1, OATP1B3, and multidrug resistance-associated protein 2. Phenotyping studies suggested predominant contribution of OATP1B3 over OATP1B1 in the hepatic uptake of E7766. Studies in OATP1B1/1B3 humanized mice showed that plasma exposure of E7766 increased 4.5-fold when coadministered with Rifampicin. Physiologically based pharmacokinetic models built upon two independent bottom-up approaches predicted elevation of E7766 plasma exposure when administered with Rifampicin, a clinical OATP inhibitor. In conclusion, we demonstrate that OATP-mediated hepatic uptake is the major contributor to the clearance of E7766, and inhibition of OATP1B may increase its systemic exposure. Predominant contribution of OATP1B3 in the hepatic uptake of E7766 was observed, suggesting polymorphisms in OATP1B1 would be unlikely to cause variability in the exposure of E7766. SIGNIFICANCE STATEMENT: Understanding the clearance mechanisms of new chemical entities is critical to predicting human pharmacokinetics and drug interactions. A physiologically based pharmacokinetic model that incorporated parameters from mechanistic in vitro and in vivo experiments was used to predict pharmacokinetics and drug interactions of E7766, a novel dinucleotide drug. The findings highlighted here may shed a light on the pharmacokinetic profile and transporter-mediated drug interaction propensity of other dinucleotide drugs.