Structure-based discovery of CFTR potentiators and inhibitors.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, whereas its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here, we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify CFTR modulators. We docked ∼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered mid-nanomolar potentiators, as well as inhibitors, that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

Structure-based discovery of CFTR potentiators and inhibitors.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, while its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify novel CFTR modulators. We docked ~155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered novel mid-nanomolar potentiators as well as inhibitors that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

Structure-Based Discovery of Inhibitors of the SARS-CoV-2 Nsp14 N7-Methyltransferase.

An under-explored target for SARS-CoV-2 is the S-adenosyl methionine (SAM)-dependent methyltransferase Nsp14, which methylates the N7-guanosine of viral RNA at the 5'-end, allowing the virus to evade host immune response. We sought new Nsp14 inhibitors with three large library docking strategies. First, up to 1.1 billion lead-like molecules were docked against the enzyme's SAM site, leading to three inhibitors with IC50 values from 6 to 50 µM. Second, docking a library of 16 million fragments revealed 9 new inhibitors with IC50 values from 12 to 341 µM. Third, docking a library of 25 million electrophiles to covalently modify Cys387 revealed 7 inhibitors with IC50 values from 3.5 to 39 µM. Overall, 32 inhibitors encompassing 11 chemotypes had IC50 values < 50 µM and 5 inhibitors in 4 chemotypes had IC50 values < 10 µM. These molecules are among the first non-SAM-like inhibitors of Nsp14, providing starting points for future optimization.

Synthetic Ionizable Colloidal Drug Aggregates Enable Endosomal Disruption.

Colloidal drug aggregates enable the design of drug-rich nanoparticles; however, the efficacy of stabilized colloidal drug aggregates is limited by entrapment in the endo-lysosomal pathway. Although ionizable drugs are used to elicit lysosomal escape, this approach is hindered by toxicity associated with phospholipidosis. It is hypothesized that tuning the pKa of the drug would enable endosomal disruption while avoiding phospholipidosis and minimizing toxicity. To test this idea, 12 analogs of the nonionizable colloidal drug fulvestrant are synthesized with ionizable groups to enable pH-dependent endosomal disruption while maintaining bioactivity. Lipid-stabilized fulvestrant analog colloids are endocytosed by cancer cells, and the pKa of these ionizable colloids influenced the mechanism of endosomal and lysosomal disruption. Four fulvestrant analogs-those with pKa values between 5.1 and 5.7-disrupted endo-lysosomes without measurable phospholipidosis. Thus, by manipulating the pKa of colloid-forming drugs, a tunable and generalizable strategy for endosomal disruption is established.

Structures of the σ2 receptor enable docking for bioactive ligand discovery.

The σ2 receptor has attracted intense interest in cancer imaging1, psychiatric disease2, neuropathic pain3-5 and other areas of biology6,7. Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone2 and the tool compound PB288. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 µM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ1 receptor. Crystal structures of two ligands bound to the σ2 receptor confirmed the docked poses. To investigate the contribution of the σ2 receptor in pain, two potent σ2-selective ligands and one potent σ1/σ2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model9, suggesting that the σ2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology.

Bacterial metabolism rescues the inhibition of intestinal drug absorption by food and drug additives.

Food and drug products contain diverse and abundant small-molecule additives (excipients) with unclear impacts on human physiology, drug safety, and response. Here, we evaluate their potential impact on intestinal drug absorption. By screening 136 unique compounds for inhibition of the key intestinal transporter OATP2B1 we identified and validated 24 potent OATP2B1 inhibitors, characterized by higher molecular weight and hydrophobicity compared to poor or noninhibitors. OATP2B1 inhibitors were also enriched for dyes, including 8 azo (R-N=N-R') dyes. Pharmacokinetic studies in mice confirmed that FD&C Red No. 40, a common azo dye excipient and a potent inhibitor of OATP2B1, decreased the plasma level of the OATP2B1 substrate fexofenadine, suggesting that FD&C Red No. 40 has the potential to block drug absorption through OATP2B1 inhibition in vivo. However, the gut microbiomes of multiple unrelated healthy individuals as well as diverse human gut bacterial isolates were capable of inactivating the identified azo dye excipients, producing metabolites that no longer inhibit OATP2B1 transport. These results support a beneficial role for the microbiome in limiting the unintended effects of food and drug additives in the intestine and provide a framework for the data-driven selection of excipients. Furthermore, the ubiquity and genetic diversity of gut bacterial azoreductases coupled to experiments in conventionally raised and gnotobiotic mice suggest that variations in gut microbial community structure may be less important to consider relative to the high concentrations of azo dyes in food products, which have the potential to saturate gut bacterial enzymatic activity.

Discovery of Lysine-Targeted eIF4E Inhibitors through Covalent Docking.

Eukaryotic translation initiation factor 4E (eIF4E) binds the m7GTP cap structure at the 5'-end of mRNAs, stimulating the translation of proteins implicated in cancer cell growth and metastasis. eIF4E is a notoriously challenging target, and most of the reported inhibitors are negatively charged guanine analogues with negligible cell permeability. To overcome these challenges, we envisioned a covalent targeting strategy. As there are no cysteines near the eIF4E cap binding site, we developed a covalent docking approach focused on lysine. Taking advantage of a "make-on-demand" virtual library, we used covalent docking to identify arylsulfonyl fluorides that target a noncatalytic lysine (Lys162) in eIF4E. Guided by cocrystal structures, we elaborated arylsulfonyl fluoride 2 to 12, which to our knowledge is the first covalent eIF4E inhibitor with cellular activity. In addition to providing a new tool for acutely inactivating eIF4E in cells, our computational approach may offer a general strategy for developing selective lysine-targeted covalent ligands.

Interactions of Oral Molecular Excipients with Breast Cancer Resistance Protein, BCRP.

Mechanistic-understanding-based selection of excipients may improve formulation development strategies for generic drug products and potentially accelerate their approval. Our study aimed at investigating the effects of molecular excipients present in orally administered FDA-approved drug products on the intestinal efflux transporter, BCRP (ABCG2), which plays a critical role in drug absorption with potential implications on drug safety and efficacy. We determined the interactions of 136 oral molecular excipients with BCRP in isolated membrane vesicles and identified 26 excipients as BCRP inhibitors with IC50 values less than 5 µM using 3H-cholecystokinin octapeptide (3H-CCK8). These BCRP inhibitors belonged to three functional categories of excipients: dyes, surfactants, and flavoring agents. Compared with noninhibitors, BCRP inhibitors had significantly higher molecular weights and SLogP values. The inhibitory effects of excipients identified in membrane vesicles were also evaluated in BCRP-overexpressing HEK293 cells at similar concentrations. Only 1 of the 26 inhibitors of BCRP identified in vesicles inhibited BCRP-mediated 3H-oxypurinol uptake by more than 50%, consistent with the notion that BCRP inhibition depends on transmembrane or intracellular availability of the inhibitors. Collectively, the results of this study provide new information on excipient selection during the development of drug products with active pharmaceutical ingredients that are BCRP substrates.

Triggered Release Enhances the Cytotoxicity of Stable Colloidal Drug Aggregates.

Chemotherapeutics that self-assemble into colloids have limited efficacy above their critical aggregation concentration due to their inability to penetrate intact plasma membranes. Even when colloid uptake is promoted, issues with colloid escape from the endolysosomal pathway persist. By stabilizing acid-responsive lapatinib colloids through coaggregation with fulvestrant, and inclusion of transferrin, we demonstrate colloid internalization by cancer cells, where subsequent lapatinib ionization leads to endosomal leakage and increased cytotoxicity. These results demonstrate a strategy for triggered drug release from stable colloidal aggregates.

Structural identification of a hotspot on CFTR for potentiation.

Cystic fibrosis is a fatal disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Two main categories of drugs are being developed: correctors that improve folding of CFTR and potentiators that recover the function of CFTR. Here, we report two cryo-electron microscopy structures of human CFTR in complex with potentiators: one with the U.S. Food and Drug Administration (FDA)-approved drug ivacaftor at 3.3-angstrom resolution and the other with an investigational drug, GLPG1837, at 3.2-angstrom resolution. These two drugs, although chemically dissimilar, bind to the same site within the transmembrane region. Mutagenesis suggests that in both cases, hydrogen bonds provided by the protein are important for drug recognition. The molecular details of how ivacaftor and GLPG1837 interact with CFTR may facilitate structure-based optimization of therapeutic compounds.

Colloidal Drug Aggregate Stability in High Serum Conditions and Pharmacokinetic Consequence.

Colloidal drug aggregates have been a nuisance in drug screening, yet, because they inherently comprise drug-rich particles, they may be useful in vivo if issues of stability can be addressed. As the first step toward answering this question, we optimized colloidal drug aggregate formulations using a fluorescence-based assay to study fulvestrant colloidal formation and stability in high (90%) serum conditions in vitro. We show, for the first time, that the critical aggregation concentration of fulvestrant depends on media composition and increases with serum concentration. Excipients, such as polysorbate 80, stabilize fulvestrant colloids in 90% serum in vitro for over 48 h. Using fulvestrant and an investigational pro-drug, pentyloxycarbonyl-( p-aminobenzyl) doxazolidinylcarbamate (PPD), as proof-of-concept colloidal formulations, we demonstrate that the in vivo plasma half-life for stabilized colloids is greater than their respective monomeric forms. These studies demonstrate the potential of turning the nuisance of colloidal drug aggregation into an opportunity for drug-rich formulations.

Structure-inspired design of ß-arrestin-biased ligands for aminergic GPCRs.

Development of biased ligands targeting G protein-coupled receptors (GPCRs) is a promising approach for current drug discovery. Although structure-based drug design of biased agonists remains challenging even with an abundance of GPCR crystal structures, we present an approach for translating GPCR structural data into ß-arrestin-biased ligands for aminergic GPCRs. We identified specific amino acid-ligand contacts at transmembrane helix 5 (TM5) and extracellular loop 2 (EL2) responsible for Gi/o and ß-arrestin signaling, respectively, and targeted those residues to develop biased ligands. For these ligands, we found that bias is conserved at other aminergic GPCRs that retain similar residues at TM5 and EL2. Our approach provides a template for generating arrestin-biased ligands by modifying predicted ligand interactions that block TM5 interactions and promote EL2 interactions. This strategy may facilitate the structure-guided design of arrestin-biased ligands at other GPCRs, including polypharmacological biased ligands.

Reverse translation of adverse event reports paves the way for de-risking preclinical off-targets.

The Food and Drug Administration Adverse Event Reporting System (FAERS) remains the primary source for post-marketing pharmacovigilance. The system is largely un-curated, unstandardized, and lacks a method for linking drugs to the chemical structures of their active ingredients, increasing noise and artefactual trends. To address these problems, we mapped drugs to their ingredients and used natural language processing to classify and correlate drug events. Our analysis exposed key idiosyncrasies in FAERS, for example reports of thalidomide causing a deadly ADR when used against myeloma, a likely result of the disease itself; multiplications of the same report, unjustifiably increasing its importance; correlation of reported ADRs with public events, regulatory announcements, and with publications. Comparing the pharmacological, pharmacokinetic, and clinical ADR profiles of methylphenidate, aripiprazole, and risperidone, and of kinase drugs targeting the VEGF receptor, demonstrates how underlying molecular mechanisms can emerge from ADR co-analysis. The precautions and methods we describe may enable investigators to avoid confounding chemistry-based associations and reporting biases in FAERS, and illustrate how comparative analysis of ADRs can reveal underlying mechanisms.

A New Spin on Antibody-Drug Conjugates: Trastuzumab-Fulvestrant Colloidal Drug Aggregates Target HER2-Positive Cells.

While the formation of colloidal aggregates leads to artifacts in early drug discovery, their composition makes them attractive as nanoparticle formulations for targeted drug delivery as the entire nanoparticle is composed of drug. The typical transient stability of colloidal aggregates has inhibited exploiting this property. To overcome this limitation, we investigated a series of proteins to stabilize colloidal aggregates of the chemotherapeutic, fulvestrant, including the following: bovine serum albumin, a generic human immunoglobulin G, and trastuzumab, a therapeutic human epidermal growth factor receptor 2 antibody. Protein coronas reduced colloid size to <300 nm and improved their stability to over 48 h in both buffered saline and media containing serum protein. Unlike colloids stabilized with other proteins, trastuzumab-fulvestrant colloids were taken up by HER2 overexpressing cells and were cytotoxic. This new targeted formulation reimagines antibody-drug conjugates, delivering mM concentrations of drug to a cell.

Internal Structure and Preferential Protein Binding of Colloidal Aggregates.

Colloidal aggregates of small molecules are the most common artifact in early drug discovery, sequestering and inhibiting target proteins without specificity. Understanding their structure and mechanism has been crucial to developing tools to control for, and occasionally even exploit, these particles. Unfortunately, their polydispersity and transient stability have prevented exploration of certain elementary properties, such as how they pack. Dye-stabilized colloidal aggregates exhibit enhanced homogeneity and stability when compared to conventional colloidal aggregates, enabling investigation of some of these properties. By small-angle X-ray scattering and multiangle light scattering, pair distance distribution functions suggest that the dye-stabilized colloids are filled, not hollow, spheres. Stability of the coformulated colloids enabled investigation of their preference for binding DNA, peptides, or folded proteins, and their ability to purify one from the other. The coformulated colloids showed little ability to bind DNA. Correspondingly, the colloids preferentially sequestered protein from even a 1600-fold excess of peptides that are themselves the result of a digest of the same protein. This may reflect the avidity advantage that a protein has in a surface-to-surface interaction with the colloids. For the first time, colloids could be shown to have preferences of up to 90-fold for particular proteins over others. Loaded onto the colloids, bound enzyme could be spun down, resuspended, and released back into buffer, regaining most of its activity. Implications of these observations for colloid mechanisms and utility will be considered.

Design, Synthesis, and Biological Evaluation of Novel Tetrahydroprotoberberine Derivatives (THPBs) as Selective α1A-Adrenoceptor Antagonists.

A novel series of tetrahydroprotoberberine derivatives (THPBs) were designed, synthesized, and evaluated as selective α1A-adrenergic receptors (AR) antagonists for the treatment of benign prostatic hyperplasia. On the basis of the pharmacophore model of the marketed drug silodosin, THPBs were modified by introducing an indole segment into their core scaffolds. In calcium assays, 7 out of 32 compounds displayed excellent antagonistic activities against α1A-ARs, with IC50 less than 250 nM. Among them, compound (S)-27 had the most potent biological activity; its IC50 toward α1A-AR was 12.8 ± 2.2 nM, which is 781 and 20 times more selective than that toward α1B- and α1D-AR, respectively. In the functional assay using isolated rat tissues, compound (S)-27 inhibited norepinephrine-induced urethra smooth muscle contraction potently (IC50 = 0.5 ± 0.3 nM), without inhibiting the aortic contraction (IC50 > 1000 nM), displaying a better tissue selectivity than the marketed drug silodosin. Additional results of preliminary safety studies (acute toxicity and hERG inhibition) and pharmacokinetics studies indicated the potential druggability for compound (S)-27 which is a promising lead for the development of selective α1A-AR antagonists for the treatment of BPH.

Identification of Novel Smoothened Ligands Using Structure-Based Docking.

The seven transmembrane protein Smoothened is required for Hedgehog signaling during embryonic development and adult tissue homeostasis. Inappropriate activation of the Hedgehog signalling pathway leads to cancers such as basal cell carcinoma and medulloblastoma, and Smoothened inhibitors are now available clinically to treat these diseases. However, resistance to these inhibitors rapidly develops thereby limiting their efficacy. The determination of Smoothened crystal structures enables structure-based discovery of new ligands with new chemotypes that will be critical to combat resistance. In this study, we docked 3.2 million available, lead-like molecules against Smoothened, looking for those with high physical complementarity to its structure; this represents the first such campaign against the class Frizzled G-protein coupled receptor family. Twenty-one high-ranking compounds were selected for experimental testing, and four, representing three different chemotypes, were identified to antagonize Smoothened with IC50 values better than 50 µM. A screen for analogs revealed another six molecules, with IC50 values in the low micromolar range. Importantly, one of the most active of the new antagonists continued to be efficacious at the D473H mutant of Smoothened, which confers clinical resistance to the antagonist vismodegib in cancer treatment.

Docking and Linking of Fragments To Discover Jumonji Histone Demethylase Inhibitors.

Development of tool molecules that inhibit Jumonji demethylases allows for the investigation of cancer-associated transcription. While scaffolds such as 2,4-pyridinedicarboxylic acid (2,4-PDCA) are potent inhibitors, they exhibit limited selectivity. To discover new inhibitors for the KDM4 demethylases, enzymes overexpressed in several cancers, we docked a library of 600,000 fragments into the high-resolution structure of KDM4A. Among the most interesting chemotypes were the 5-aminosalicylates, which docked in two distinct but overlapping orientations. Docking poses informed the design of covalently linked fragment compounds, which were further derivatized. This combined approach improved affinity by ∼ 3 log-orders to yield compound 35 (Ki = 43 nM). Several hybrid inhibitors were selective for KDM4C over the related enzymes FIH, KDM2A, and KDM6B while lacking selectivity against the KDM3 and KDM5 subfamilies. Cocrystal structures corroborated the docking predictions. This study extends the use of structure-based docking from fragment discovery to fragment linking optimization, yielding novel KDM4 inhibitors.

Covalent docking of large libraries for the discovery of chemical probes.

Chemical probes that form a covalent bond with a protein target often show enhanced selectivity, potency and utility for biological studies. Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns. Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small molecules. We apply this method prospectively to discover reversible covalent fragments that target distinct protein nucleophiles, including the catalytic serine of AmpC ß-lactamase and noncatalytic cysteines in RSK2, MSK1 and JAK3 kinases. We identify submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including what are to our knowledge the first reported reversible covalent inhibitors of JAK3. Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization. As covalent virtual screening may have broad utility for the rapid discovery of chemical probes, we have made the method freely available through an automated web server (http://covalent.docking.org/).