MicroED Structures of Fluticasone Furoate and Fluticasone Propionate Provide New Insights into Their Function.

The detailed understanding of the conformational pathway of fluticasone, a widely prescribed medicine for allergic rhinitis, asthma, and chronic obstructive pulmonary disease (COPD), from formulation to its protein-bound state, has been limited due to a lack of access to its high-resolution structures. The three-dimensional (3D) structure of fluticasone furoate 1 remains unpublished, and the deposited structure of fluticasone propionate 2 could be further refined due to refinement against new data. We applied microcrystal electron diffraction (MicroED) to determine the 3D structures of 1 and 2 in their solid states. The preferred geometries in solution were predicted by using density functional theory (DFT) calculations. A comparative analysis of the structures of 1 and 2 across three states (in solid state, in solution, and protein-bound conformation) revealed the course of the conformational changes during the entire transition. Potential energy plots were calculated for the most dynamic bonds, uncovering their rotational barriers. This study underscores the combined use of MicroED and DFT calculations to provide a comprehensive understanding of conformational and energy changes during drug administration. The quantitative comparison also highlights the subtle structural differences that may lead to significant changes in the pharmaceutical properties

Uncovering the Elusive Structures and Mechanisms of Prevalent Antidepressants.

Most treatments to alleviate major depression work by either inhibiting human monoamine transporters, vital for the reuptake of monoamine neurotransmitters, or by inhibiting monoamine oxidases, which are vital for their degradation. The analysis of the experimental 3D structures of those antidepressants in their drug formulation state is key to precision drug design and development. In this study, we apply microcrystal electron diffraction (MicroED) to reveal the atomic 3D structures for the first time of five of the most prevalent antidepressants (reboxetine, pipofezine, ansofaxine, phenelzine, bifemelane) directly from the commercially available powder of the active ingredients. Their modes of binding are investigated by molecular docking, revealing the essential contacts and conformational changes into the biologically active state. This study underscores the combined use of MicroED and molecular docking to uncover elusive drug structures and mechanisms to aid in further drug development pipelines

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19

Synthesis, X-ray Analysis, and Biological Evaluation of a New Class of Stereopure Lactam-Based HIV-1 Protease Inhibitors

In an effort to identify a new class of druglike HIV-1 protease inhibitors, four different stereopure beta-hydroxy gamma-lactam-containing inhibitors have been synthesized, biologically evaluated, and cocrystallized. The impact of the tether length of the central spacer (two or three carbons) was also investigated. A compound with a shorter tether and (3R,4S) absolute configuration exhibited high activity with a K-i of 2.1 nM and an EC50 of 0.64 mu M. Further optimization by decoration of the P1' side chain furnished an even more potent HIV-1 protease inhibitor (K-i = 0.8 nM, EC50 = 0.04 mu M). According to X-ray analysis, the new class of inhibitors did not fully succeed in forming two symmetric hydrogen bonds to the catalytic aspartates. The crystal structures of the complexes further explain the difference in potency between the shorter inhibitors (two-carbon spacer) and the longer inhibitors (three-carbon spacer)

Function via structure in vivo and in modeling

Knowledge of biomolecules three-dimensional structure has provided a detailed understanding of their function. In recent years the number of new protein sequences has far exceeded the number of protein experimental structure determinations. One goal of the new field of protein structural bioinformatics is to provide reliable and automated protein functional annotations and initial protein structure models based on sequence comparisons of target proteins of unknown structure to proteins of known structure. We present methods designed to address these goals, particularly for cases of sequence homology below what is needed by conventional sequence comparison methods. We found that combining structure and sequence information for families of evolutionarily-related proteins increases the sensitivity of sequence-to-structure comparison methods. The novelty of the method is in how the common structure elements in protein families are extracted and used in comparing sequences of target proteins with proteins where structure is known. The usefulness of the method was demonstrated in its ability to recognize distantly-related proteins with higher accuracy than when considering protein sequences only. The ribosome and its related factors are responsible for the synthesis of new proteins within cells. We have performed structural studies of two ribosomal proteins, L1 and L22, using X-ray crystallography. We verify a distinct movement between the two domains in L1 connected by a hinge region, from the comparison of the wild-type structure and a mutant structure. An open conformation of L1, with the two subunits considerably shifted from previous structures, was later verified to be active, confirming our results. The unusually elongated structure of L22 was determined for the first time and several clues were found that indicated extensive binding to ribosomal RNA. The structure and interaction with RNA was later confirmed in the structure of the complete large ribosomal subunit