A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules.

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.

Discovery of a First-in-Class Inhibitor of the PRMT5-Substrate Adaptor Interaction.

PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. Mode of action studies revealed the formation of a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts PRMT5-RIOK1 complexes, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive inhibitor that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.

Multimodal small-molecule screening for human prion protein binders.

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including 19F-observed and saturation transfer difference (STD) NMR spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and in silico screening. A single benzimidazole compound was confirmed in concentration-response, but affinity was very weak (Kd > 1 mm), and it could not be advanced further. The exceptionally low hit rate observed here suggests that PrP is a difficult target for small-molecule binders. Whereas orthogonal binder discovery methods could yield high-affinity compounds, non-small-molecule modalities may offer independent paths forward against prion disease.

A drug discovery toolbox for Nuclear Magnetic Resonance (NMR) characterization of ligands and their targets.

Information about the structure, dynamics, and ligand-binding properties of biomolecules can be derived from Nuclear Magnetic Resonance (NMR) spectroscopy and provides valuable information for drug discovery. A multitude of experimental approaches provides a wealth of information that can be tailored to the system of interest. Methods to study the behavior of ligands upon target binding enable the identification of weak binders in a robust manner that is critical for the identification of truly novel binding interactions. This is particularly important for challenging targets. Observing the solution behavior of biomolecules yields information about their structure, dynamics, and interactions. This review describes the breadth of approaches that are available, many of which are under-utilized in a drug-discovery environment, and focuses on recent advances that continue to emerge.

Characterization of the Prion Protein Binding Properties of Antisense Oligonucleotides.

Antisense oligonucleotides (ASOs) designed to lower prion protein (PrP) expression in the brain through RNase H1-mediated degradation of PrP RNA are in development as prion disease therapeutics. ASOs were previously reported to sequence-independently interact with PrP and inhibit prion accumulation in cell culture, yet in vivo studies using a new generation of ASOs found that only PrP-lowering sequences were effective at extending survival. Cerebrospinal fluid (CSF) PrP has been proposed as a pharmacodynamic biomarker for trials of such ASOs, but is only interpretable if PrP lowering is indeed the relevant mechanism of action in vivo and if measurement of PrP is unconfounded by any PrP-ASO interaction. Here, we examine the PrP-binding and antiprion properties of ASOs in vitro and in cell culture. Binding parameters determined by isothermal titration calorimetry were similar across all ASOs tested, indicating that ASOs of various chemistries bind full-length recombinant PrP with low- to mid-nanomolar affinity in a sequence-independent manner. Nuclear magnetic resonance, dynamic light scattering, and visual inspection of ASO-PrP mixtures suggested, however, that this interaction is characterized by the formation of large aggregates, a conclusion further supported by the salt dependence of the affinity measured by isothermal titration calorimetry. Sequence-independent inhibition of prion accumulation in cell culture was observed. The inefficacy of non-PrP-lowering ASOs against prion disease in vivo may be because their apparent activity in vitro is an artifact of aggregation, or because the concentration of ASOs in relevant compartments within the central nervous system (CNS) quickly drops below the effective concentration for sequence-independent antiprion activity after bolus dosing into CSF. Measurements of PrP concentration in human CSF were not impacted by the addition of ASO. These findings support the further development of PrP-lowering ASOs and of CSF PrP as a pharmacodynamic biomarker.

A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9.

The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.

The Autophagy-Related Beclin-1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity.

Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3-5 mg/L in an Escherichia coli culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (KD ∼ 0.45 µM) that interacts with Bcl-2 (KD = 4.3 ± 1.2 µM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer KD 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by ∼5-fold.

Discovery of Pyrazolopyridones as a Novel Class of Gyrase B Inhibitors Using Structure Guided Design.

The ATPase subunit of DNA gyrase B is an attractive antibacterial target due to high conservation across bacteria and the essential role it plays in DNA replication. A novel class of pyrazolopyridone inhibitors was discovered by optimizing a fragment screening hit scaffold using structure guided design. These inhibitors show potent Gram-positive antibacterial activity and low resistance incidence against clinically important pathogens.

Fragment-based discovery of DNA gyrase inhibitors targeting the ATPase subunit of GyrB.

Inhibitors of the ATPase function of bacterial DNA gyrase, located in the GyrB subunit and its related ParE subunit in topoisomerase IV, have demonstrated antibacterial activity. In this study we describe an NMR fragment-based screening effort targeting Staphylococcus aureus GyrB that identified several attractive and novel starting points with good ligand efficiency. Fragment hits were further characterized using NMR binding studies against full-length S. aureus GyrB and Escherichia coli ParE. X-ray co-crystal structures of select fragment hits confirmed binding and suggested a path for medicinal chemistry optimization. The identification, characterization, and elaboration of one of these fragment series to a 0.265 µM inhibitor is described herein.

Targeting Bacterial Cell Wall Peptidoglycan Synthesis by Inhibition of Glycosyltransferase Activity.

Synthesis of bacterial cell wall peptidoglycan requires glycosyltransferase enzymes that transfer the disaccharide-peptide from lipid II onto the growing glycan chain. The polymerization of the glycan chain precedes cross-linking by penicillin-binding proteins and is essential for growth for key bacterial pathogens. As such, bacterial cell wall glycosyltransferases are an attractive target for antibiotic drug discovery. However, significant challenges to the development of inhibitors for these targets include the development of suitable assays and chemical matter that is suited to the nature of the binding site. We developed glycosyltransferase enzymatic activity and binding assays using the natural products moenomycin and vancomycin as model inhibitors. In addition, we designed a library of disaccharide compounds based on the minimum moenomycin fragment with peptidoglycan glycosyltransferase inhibitory activity and based on a more drug-like and synthetically versatile disaccharide building block. A subset of these disaccharide compounds bound and inhibited the glycosyltransferase enzymes, and these compounds could serve as chemical entry points for antibiotic development.

Structure-Activity Relationship Studies of a Series of Semisynthetic Lipopeptides Leading to the Discovery of Surotomycin, a Novel Cyclic Lipopeptide Being Developed for the Treatment of Clostridium difficile-Associated Diarrhea.

Novel cyclic lipopeptides with different acyl tails were synthesized via a semisynthetic approach. Structure-activity relationship studies revealed that lipophilicity, chain length, and the location of key aromatic functionalities of the tail modulated activity. The lead compound surotomycin exhibited significantly improved in vitro activity compared with daptomycin (MIC90 0.5 vs 2 µg/mL) against Clostridium difficile including NAP1 epidemic strains. In hamster efficacy studies, surotomycin protected animals at a dose of 0.5 mg/kg, PO.

An efficient chemical synthesis of carboxylate-isostere analogs of daptomycin.

Herein we report a direct and efficient method for the synthesis of four new carboxylate-isostere analogs of daptomycin. The side chain carboxylic acid moieties of the aspartic acids (Asp-3, Asp-7 and Asp-9) and ß-methyl glutamic acid (MeGlu-12) were all converted into the corresponding carboxylate isosteres using direct synthetic procedures. The present study also describes an esterification protocol to overcome the possible backbone cyclization of the activated side chain carboxylic acid group of either Asp or Glu onto the backbone amide.

NMR structural characterization of a minimal peptide antagonist bound to the extracellular domain of the corticotropin-releasing factor1 receptor.

Natural peptide agonists of corticotrophin-releasing factor (CRF) receptors bind to the receptor by a two-site mechanism as follows: the carboxyl end of the ligand binds the N-terminal extracellular domain (ECD) of the receptor and the amino portion of the ligand binds the extracellular face of the seven transmembrane region. Recently, peptide antagonists homologous to the 12 C-terminal residues of CRF have been derived, which bind the CRF(1) receptor through an interaction with the ECD. Here we characterized the binding of a minimal 12-residue peptide antagonist while bound to the isolated ECD of the CRF(1) receptor. We have expressed and purified soluble and properly folded ECD independent from the seven-transmembrane region as a thioredoxin fusion protein in Escherichia coli. A model of the peptide antagonist, cyclic corticotrophin-releasing factor residues 30-41 (cCRF(30-41)), was calculated while bound to the recombinant ECD using transferred nuclear Overhauser effect spectroscopy. Although the peptide is unstructured in solution, it adopts an alpha-helical conformation when bound to the ECD. Residues of cCRF(30-41) comprising the binding interface with the ECD were mapped using saturation transfer difference NMR. Two hydrophobic residues (Met(38) and Ile(41)) as well as two amide groups (Asn(34) and the C-terminal amide) on one face of the helix defined the binding epitope of the antagonist. This epitope may be used as a starting point for development of non-peptide antagonists targeting the ECD of this receptor.

Determination of the binding mode of thienopyrimidinedione antagonists to the human gonadotropin releasing hormone receptor using structure-activity relationships, site-directed mutagenesis, and homology modeling.

We have investigated the specific interactions of a series thienopyrimidinediones with the gonadotropin-releasing hormone receptor (GnRH-R). Competitive radioligand binding assays were used to determine the effect of several mutants on nonpeptide binding. Distinct interactions were observed in two separate regions: the N-terminal end of TM7 and the C-terminal end of TM6. The effects of mutants at D302((7.32)) and H306((7.36)) suggest that these residues are part of a hydrogen-bond network important for anchoring the nonpeptides. Structure-activity relationships indicated urea substituents on the 6-(4-aminophenyl) group with a trans conformational preference bind with high affinity and are sensitive to D302((7.32)) mutations. Another interaction area was found between the N-benzyl-N-methylamino substituent and L300((6.68)) and Y290((6.58)). These interaction sites facilitated the derivation of a model in which a representative member of the series was docked into GnRH-R. The model is consistent with known SAR and illuminates inconsistencies with previous hypotheses regarding how this series interacts with the receptor.

Atropisomeric property of 1-(2,6-difluorobenzyl)-3-[(2R)-amino-2-phenethyl]-5-(2-fluoro-3-methoxyphenyl)-6-methyluracil.

1-(2,6-Difluorobenzyl)-3-[(2R)-amino-2-phenethyl]-5-(2-fluoro-3-methoxyphenyl)-6-methyluracil (6), a potent and orally active antagonist of the human gonadotropin-releasing hormone receptor, exists as a pair of atropisomers in solution, which was detected by NMR spectroscopy, and separable by HPLC. In addition to a (R)-configured benzylamine, there is a second stereogenic element due to the presence of a chiral axis between the substituted 5-phenyl group and the uracil core. The rate constant of the interconversion (k = 5.07 x 10(-5) s(-1)) of these two atropisomers was determined by proton NMR analysis of a diastereoisomer-enriched sample in aqueous solution at 25 degrees C, and the corresponding Gibbs free energy DeltaG(#) of rotation barrier (97.4 kJ mol(-1)) was calculated using the Eyring equation. The diastereoisomer half-life at physiological temperature (37 degrees C) in aqueous media was estimated to be about 46 min.

NMR structure determination of a membrane protein with two transmembrane helices in micelles: MerF of the bacterial mercury detoxification system.

The three-dimensional backbone structure of a membrane protein with two transmembrane helices in micelles was determined using solution NMR methods that rely on the measurement of backbone (1)H-(15)N residual dipolar couplings (RDCs) from samples of two different constructs that align differently in stressed polyacrylamide gels. Dipolar wave fitting to the (1)H-(15)N RDCs determines the helical boundaries based on periodicity and was utilized in the generation of supplemental dihedral restraints for the helical segments. The (1)H-(15)N RDCs and supplemental dihedral restraints enable the determination of the structure of the helix-loop-helix core domain of the mercury transport membrane protein MerF with a backbone RMSD of 0.58 A. Moreover, the fold of this polypeptide demonstrates that the two vicinal pairs of cysteine residues, shown to be involved in the transport of Hg(II) across the membrane, are exposed to the cytoplasm. This finding differs from earlier structural and mechanistic models that were based primarily on the somewhat atypical hydropathy plot for MerF and related transport proteins.

Structure determination of aligned samples of membrane proteins by NMR spectroscopy.

The paper briefly reviews the process of determining the structures of membrane proteins by NMR spectroscopy of aligned samples, describes the integration of recent developments in the interpretation of spectra of aligned proteins and illustrates the application of these methods to the trans-membrane helical domain of a protein. The emerging methods of interpreting the spectral parameters from aligned samples of isotopically labeled proteins provide opportunities for simultaneously assigning the spectra and determining the structures of the proteins, and also for comparing the results from solid-state NMR experiments on completely aligned samples with those of solution NMR experiments on weakly aligned samples.

Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1.

The three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) of HIV-1 was determined by NMR spectroscopy in micelle and bilayer samples. Vpu(2-30+) is a 36-residue polypeptide that consists of residues 2-30 from the N terminus of Vpu and a six-residue "solubility tag" at its C terminus that facilitates the isolation, purification, and sample preparation of this highly hydrophobic minimal channel-forming domain. Nearly all of the resonances in the two-dimensional 1H/15N HSQC spectrum of uniformly 15N labeled Vpu(2-30+) in micelles are superimposable on those from the corresponding residues in the spectrum of full-length Vpu, which indicates that the structure of the trans-membrane domain is not strongly affected by the presence of the cytoplasmic domain at its C terminus. The two-dimensional 1H/15N PISEMA spectrum of Vpu(2-30+) in lipid bilayers aligned between glass plates has been fully resolved and assigned. The "wheel-like" pattern of resonances in the spectrum is characteristic of a slightly tilted membrane-spanning helix. Experiments were also performed on weakly aligned micelle samples to measure residual dipolar couplings and chemical shift anisotropies. The analysis of the PISA wheels and Dipolar Waves obtained from both weakly and completely aligned samples show that Vpu(2-30+) has a trans-membrane alpha-helix spanning residues 8-25 with an average tilt of 13 degrees. The helix is kinked slightly at Ile17, which results in tilts of 12 degrees for residues 8-16 and 15 degrees for residues 17-25. A structural fit to the experimental solid-state NMR data results in a three-dimensional structure with precision equivalent to an RMSD of 0.4 A. Vpu(2-30+) exists mainly as an oligomer on PFO-PAGE and forms ion-channels, a most frequent conductance of 96(+/- 6) pS in lipid bilayers. The structural features of the trans-membrane domain are determinants of the ion-channel activity that may be associated with the protein's role in facilitating the budding of new virus particles from infected cells.

Dipolar Waves as NMR maps of helices in proteins.

Dipolar Waves describe the periodic variation in the magnitudes of dipolar couplings in the backbone of a protein as a function of residue number. They provide a direct link between experimental measurements of dipolar couplings in aligned samples and the periodicity inherent in regular secondary structure elements. It is possible to identify the residues in a helix and the type of helix, deviations from ideality, and to orient the helices relative to an external axis in completely aligned samples and relative to each other in a common frame in weakly aligned samples with Dipolar Waves. They provide a tool for accurately describing helices and a step towards high throughput structure determination of proteins.

Dipolar waves map the structure and topology of helices in membrane proteins.

Dipolar waves describe the structure and topology of helices in membrane proteins. The fit of sinusoids with the 3.6 residues per turn period of ideal alpha-helices to experimental measurements of dipolar couplings as a function of residue number makes it possible to simultaneously identify the residues in the helices, detect kinks or curvature in the helices, and determine the absolute rotations and orientations of helices in completely aligned bilayer samples and relative rotations and orientations of helices in a common molecular frame in weakly aligned micelle samples. Since as much as 80% of the structured residues in a membrane protein are in helices, the analysis of dipolar waves provides a significant step toward structure determination of helical membrane proteins by NMR spectroscopy.