The diversity of splicing modifiers acting on A-1 bulged 5'-splice sites reveals rules for rational drug design.

Pharmacological modulation of RNA splicing by small molecules is an emerging facet of drug discovery. In this context, the SMN2 splicing modifier SMN-C5 was used as a prototype to understand the mode of action of small molecule splicing modifiers and propose the concept of 5'-splice site bulge repair. In this study, we combined in vitro binding assays and structure determination by NMR spectroscopy to identify the binding modes of four other small molecule splicing modifiers that switch the splicing of either the SMN2 or the HTT gene. Here, we determined the solution structures of risdiplam, branaplam, SMN-CX and SMN-CY bound to the intermolecular RNA helix epitope containing an unpaired adenine within the G-2A-1G+1U+2 motif of the 5'-splice site. Despite notable differences in their scaffolds, risdiplam, SMN-CX, SMN-CY and branaplam contact the RNA epitope similarly to SMN-C5, suggesting that the 5'-splice site bulge repair mechanism can be generalised. These findings not only deepen our understanding of the chemical diversity of splicing modifiers that target A-1 bulged 5'-splice sites, but also identify common pharmacophores required for modulating 5'-splice site selection with small molecules.

A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2.

The pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further fragment expansion and drug development.

Conformations of Macrocyclic Peptides Sampled by Nuclear Magnetic Resonance: Models for Cell-Permeability.

The biological activities and pharmacological properties of peptides and peptide mimetics are determined by their conformational states. Therefore, a detailed understanding of the conformational landscape is crucial for rational drug design. Nuclear magnetic resonance (NMR) is the only method for structure determination in solution. However, it remains challenging to determine the structures of peptides using NMR because of very weak nuclear Overhauser effects (NOEs), the semiquantitative nature of the rotating frame Overhauser effect (ROE), and the low number of NOEs/ROEs in N-methylated peptides. In this study, we introduce a new approach to investigating the structures of modified macrocyclic peptides. We utilize exact NOEs (eNOEs) in viscous solvent mixtures to replicate various cellular environments. eNOEs provide detailed structural information for highly dynamic modified peptides. Structures of high precision were obtained for cyclosporin A, with a backbone atom rmsd of 0.10 Å. Distinct conformational states in different environments were identified for omphalotin A (OmphA), a fungal nematotoxic and multiple backbone N-methylated macrocyclic peptides. A model for cell-permeation is presented for OmphA, based on its structures in polar, apolar, and mixed polarity solvents. During the transition from a polar to an apolar environment, OmphA undergoes a rearrangement of its H-bonding network, accompanied by a cis to trans isomerization of the ω torsion angle within a type VIa ß-turn. We hypothesize that the kinetics of these conformational transitions play a crucial role in determining the membrane-permeation capabilities of OmphA.

Modular Oxime Formation by a trans-AT Polyketide Synthase.

Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.

Structural basis of a small molecule targeting RNA for a specific splicing correction.

Splicing modifiers promoting SMN2 exon 7 inclusion have the potential to treat spinal muscular atrophy, the leading genetic cause of infantile death. These small molecules are SMN2 exon 7 selective and act during the early stages of spliceosome assembly. Here, we show at atomic resolution how the drug selectively promotes the recognition of the weak 5' splice site of SMN2 exon 7 by U1 snRNP. The solution structure of the RNA duplex formed following 5' splice site recognition in the presence of the splicing modifier revealed that the drug specifically stabilizes a bulged adenine at this exon-intron junction and converts the weak 5' splice site of SMN2 exon 7 into a stronger one. The small molecule acts as a specific splicing enhancer cooperatively with the splicing regulatory network. Our investigations uncovered a novel concept for gene-specific alternative splicing correction that we coined 5' splice site bulge repair.

Efficient affinity ranking of fluorinated ligands by 19F NMR: CSAR and FastCSAR.

Fluorine NMR has recently gained high popularity in drug discovery as it allows efficient and sensitive screening of large numbers of ligands. However, the positive hits found in screening must subsequently be ranked according to their affinity in order to prioritize them for follow-up chemistry. Unfortunately, the primary read-out from the screening experiments, namely the increased relaxation rate upon binding, is not proportional to the affinity of the ligand, as it is polluted by effects such as exchange broadening. Here we present the method CSAR (Chemical Shift-anisotropy-based Affinity Ranking) for reliable ranking of fluorinated ligands by NMR, without the need of isotope labeled protein, titrations or setting up a reporter format. Our strategy is to produce relaxation data that is directly proportional to the binding affinity. This is achieved by removing all other contributions to relaxation as follows: (i) exchange effects are efficiently suppressed by using high power spin lock pulses, (ii) dipolar relaxation effects are approximately subtracted by measuring at two different magnetic fields and (iii) differences in chemical shift anisotropy are normalized using calculated values. A similar ranking can be obtained with the simplified approach FastCSAR that relies on a measurement of a single relaxation experiment at high field (preferably > 600 MHz). An affinity ranking obtained in this simple way will enable prioritizing ligands and thus improve the efficiency of fragment-based drug design.

Small-molecule factor D inhibitors targeting the alternative complement pathway.

Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.

Enzymatically Crosslinked Collagen as a Versatile Matrix for In Vitro and In Vivo Co-Engineering of Blood and Lymphatic Vasculature.

Adequate vascularization is required for the successful translation of many in vitro engineered tissues. This study presents a novel collagen derivative that harbors multiple recognition peptides for orthogonal enzymatic crosslinking based on sortase A (SrtA) and Factor XIII (FXIII). SrtA-mediated crosslinking enables the rapid co-engineering of human blood and lymphatic microcapillaries and mesoscale capillaries in bulk hydrogels. Whereas tuning of gel stiffness determines the extent of neovascularization, the relative number of blood and lymphatic capillaries recapitulates the ratio of blood and lymphatic endothelial cells originally seeded into the hydrogel. Bioengineered capillaries readily form luminal structures and exhibit typical maturation markers both in vitro and in vivo. The secondary crosslinking enzyme Factor XIII is used for in situ tethering of the VEGF mimetic QK peptide to collagen. This approach supports the formation of blood and lymphatic capillaries in the absence of exogenous VEGF. Orthogonal enzymatic crosslinking is further used to bioengineer hydrogels with spatially defined polymer compositions with pro- and anti-angiogenic properties. Finally, macroporous scaffolds based on secondary crosslinking of microgels enable vascularization independent from supporting fibroblasts. Overall, this work demonstrates for the first time the co-engineering of mature micro- and meso-sized blood and lymphatic capillaries using a highly versatile collagen derivative.

Discovery and Design of First Benzylamine-Based Ligands Binding to an Unlocked Conformation of the Complement Factor D.

Complement Factor D, a serine protease of the S1 family and key component of the alternative pathway amplification loop, represents a promising target for the treatment of several prevalent and rare diseases linked to the innate immune system. Previously reported FD inhibitors have been shown to bind to the FD active site in its self-inhibited conformation characterized by the presence of a salt bridge at the bottom of the S1 pocket between Asp189 and Arg218. We report herein a new set of small-molecule FD ligands that harbor a basic S1 binding moiety directly binding to the carboxylate of Asp189, thereby displacing the Asp189-Arg218 ionic interaction and significantly changing the conformation of the self-inhibitory loop.

Structure-Based Library Design and Fragment Screening for the Identification of Reversible Complement Factor D Protease Inhibitors.

Chronic dysregulation of alternative complement pathway activation has been associated with diverse clinical disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinurea. Factor D is a trypsin-like serine protease with a narrow specificity for arginine in the P1 position, which catalyzes the first enzymatic reaction of the amplification loop of the alternative pathway. In this article, we describe two hit finding approaches leading to the discovery of new chemical matter for this pivotal protease of the complement system: in silico active site mapping for hot spot identification to guide rational structure-based design and NMR screening of focused and diverse fragment libraries. The wealth of information gathered by these complementary approaches enabled the identification of ligands binding to different subpockets of the latent Factor D conformation and was instrumental for understanding the binding requirements for the generation of the first known potent noncovalent reversible Factor D inhibitors.

Discovery of Highly Potent and Selective Small-Molecule Reversible Factor D Inhibitors Demonstrating Alternative Complement Pathway Inhibition in Vivo.

The highly specific S1 serine protease factor D (FD) plays a central role in the amplification of the complement alternative pathway (AP) of the innate immune system. Genetic associations in humans have implicated AP activation in age-related macular degeneration (AMD), and AP dysfunction predisposes individuals to disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The combination of structure-based hit identification and subsequent optimization of the center (S)-proline-based lead 7 has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties. The orally bioavailable compound 2 exerted excellent potency in 50% human whole blood in vitro and blocked AP activity ex vivo after oral administration to monkeys as demonstrated by inhibition of membrane attack complex (MAC) formation. Inhibitor 2 demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide (LPS)-induced systemic AP activation in mice expressing human FD.

NMR and in silico screening.

NMR-based screening and virtual, or in silico, screening can be highly complementary and synergistic. NMR-based screening is a rapid and reliable method for validating hits that come from in silico screens. In addition, ligand-binding data derived from NMR-based screens can focus and direct subsequent in silico screening. We will first give a short overview of existing NMR and in silico screening methods, discuss the drawbacks associated with each, and finally present applications that highlight the combination of the two technologies.