Structural basis of HIV-1 inhibitor activity, and resistance, and characterization of active pharmaceutical ingredients in drugs and microcrystalline proteins by multinuclear magic angle spinning NMR spectroscopy
Date
2025
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Publisher
University of Delaware
Abstract
Magic angle spinning (MAS) NMR is a powerful structural biology technique that provides three-dimensional structures of large biological systems with atomic-level resolution and offers insights into site-specific dynamics across various experimental conditions. It can be seamlessly integrated with other structural biology techniques to tackle some of the most complex scientific questions of our time. In this dissertation, I describe newly developed MAS NMR-based methods for determining the structures of proteins and protein assemblies in the presence of naturally abundant small molecules. These distinct applications range from the characterization of active pharmaceutical ingredients (APIs) in pharmaceutical formulations to the assignment of absolute configurations of chiral small molecules based on the differences in orientation of another symmetric small molecule that is simultaneously bound in a protein assembly. The overall approach utilizes strategically designed experiments and leverages multinuclear probes, including 1H, 13C, 15N, 19F, and 31P, alongside the unique chemical shifts of the small molecules under investigation. The MAS NMR data are integrated with information from X-ray crystallography, cryogenic electron microscopy, solution NMR, density functional theory calculations, molecular dynamics simulations, and virological data, yielding atomic-level insights that cannot be achieved by any method in isolation. ☐ Chapter 1 offers an overview of integrative approaches for characterizing protein dynamics. Proteins are inherently dynamic, and their internal movements are crucial for biological functions. Protein motions span a wide range of timescales: 10−14 to 10 seconds, encompassing sub-picosecond vibrational motions of atoms, microsecond loop conformational rearrangements, and millisecond large amplitude domain reorientations. Observing protein dynamics across all timescales and linking motions and structures to biological mechanisms necessitates the integration of multiple experimental and computational techniques. This chapter discusses state-of-the-art methods for analyzing dynamics in biological systems, using recent examples of viral assemblies, enzymes, and molecular machines. By combining NMR spectroscopy in both solution and solid state, cryo-electron microscopy, and molecular dynamics simulations, detailed atomistic views of protein motions are achieved—insights that cannot be acquired from any single method alone. This information yields fundamental insights into protein behavior, which can inform the development of future therapeutics. ☐ Chapter 2 describes the elucidation of the structural basis for HIV-1 maturation inhibitor PF-46396 activity, resistance, and enantiomer-dependent binding in the immature Gag lattice. Among the different types of HIV-1 maturation inhibitors, those that stabilize the junction between the capsid protein C-terminal domain (CACTD) and the spacer peptide 1 (SP1) within the immature Gag lattice are promising candidates for antiretroviral therapies. Here, we report atomic-resolution structure of CACTD-SP1 assemblies with the small-molecule maturation inhibitor PF-46396 and the assembly cofactor inositol hexakisphosphate (IP6), determined by magic angle spinning (MAS) NMR spectroscopy. Our results reveal that although the two PF-46396 enantiomers exhibit distinct binding modes, they both possess similar anti-HIV potency. PF-46396 binding arrests IP6 dynamics in the six-helix bundle pore, and the two enantiomers induce unique IP6 orientations in the pore. Our study establishes the structural basis for PF-46396 action and suggests a mechanistic model for drug resistance. ☐ Chapter 3 explores methods for determining the protonation states of histidine residues in proteins. Histidine residues hold significant structural and functional importance in proteins, acting as metal-binding ligands, facilitating enzyme catalysis, and regulating proton channel activity. Many of these functions are modulated by the ionization state of the imidazole ring. Here, we introduce a fast MAS NMR approach for identifying the protonation and tautomeric states of His at frequencies between 40 and 62 kHz. The experiments integrate 1H detection with selective magnetization inversion techniques and transferred echo double resonance (TEDOR)-based filters in 2D heteronuclear correlation experiments. This method is demonstrated using microcrystalline assemblies of the HIV-1 CACTD-SP1 protein. ☐ In Chapter 4, the benefits of 19F distance restraints for protein structure determination are discussed. Traditional protein structure determination using MAS solid-state NMR spectroscopy primarily relies on interatomic distances up to 8 Å, extracted from 13C-, 15N-, and 1H-based dipolar correlation experiments. Here, we show that 19F fast MAS NMR spectroscopy can provide additional, longer distances. Utilizing 4F-Trp, U-13C,15N crystalline Oscillatoria agardhii agglutinin (OAA), we demonstrate that carefully designed 2D and 3D 19F-based dipolar correlation experiments, such as (H)CF, (H)CHF, and FF, can yield interatomic distances in the 8-16 Å range. The incorporation of fluorine-based restraints into structure calculations improves the precision of Trp side chain conformations as well as regions in the protein around the fluorine-containing residues, with notable improvements observed for residues near the Trp pairs (W10/W17 and W77/W84) in the carbohydrate-binding loops, which lacked sufficient long-range 13C-13C distance restraints. Our work highlights the use of fluorine and 19F fast MAS NMR spectroscopy as a powerful structural biology tool. ☐ In Chapter 5, the use of fast 19F MAS NMR spectroscopy for the structural characterization of APIs in pharmaceuticals is examined. Fluorinated drugs represent a significant and growing share of the pharmaceutical market. We explore high-frequency 19F MAS NMR spectroscopy for the structural analysis of fluorinated active pharmaceutical ingredients in commercial formulations of seven blockbuster drugs: Celebrex, Cipro, Crestor, Levaquin, Lipitor, Prozac, and Zyvox. 19F signals can be detected in a single scan, and spectra with high signal-to-noise ratios can be obtained in minutes. 19F spectral parameters, including chemical shifts and line widths, are sensitive to both the nature of the fluorine moiety and the formulation. It is anticipated that the fast 19F MAS NMR-based approach presented here will be beneficial for the rapid assessment of fluorine-containing drugs across a wide range of formulations.
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Keywords
Magic angle spinning, Active pharmaceutical ingredients, NMR spectroscopy, Structural biology, Inhibitor activity