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Cellular retinoic acid binding protein 2 (CRABP2) transports retinoic acid from the cytoplasm to the nucleus where it then transfers its cargo to retinoic acid receptor-containing complexes leading to activation of gene transcription. We demonstrate using purified proteins that CRABP2 is also a cyclin D3-specific binding protein and that the CRABP2 cyclin D3 binding site and the proposed CRABP2 nuclear localization sequence overlap. Both sequences are within the helix-loop-helix motif that forms a lid to the retinoic acid binding pocket. Mutations within this sequence that block both cyclin D3 and retinoic acid binding promote formation of a CRABP2 structure in which the retinoic acid binding pocket is occupied by an alternative lid conformation. Structural and functional analysis of CRABP2 and cyclin D3 mutants combined with AlphaFold models of the ternary CDK4/6-cyclin D3-CRABP2 complex supports the identification of an α-helical protein binding site on the cyclin D3 C-terminal cyclin box fold., Competing Interests: Declaration of interests Some work in the authors’ laboratory is supported by a research grant from Astex Pharmaceuticals., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent successes in developing small molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the recruitment of targets to LC3/GABARAP, a family of ubiquitin-like proteins that presumably bind to the autophagosome membrane and tether cargo-loaded autophagy receptors into the autophagosome. In this work, we rigorously tested the target engagement of the reported ATTECs to validate the existing LC3/GABARAP ligands. Surprisingly, we were unable to detect interaction with their designated target LC3 using a diversity of biophysical methods. Intrigued by the idea of developing ATTECs, we evaluated the ligandability of LC3/GABARAP by in silico docking and large-scale crystallographic fragment screening. Data based on approximately 1000 crystal structures revealed that most fragments bound to the HP2 but not to the HP1 pocket within the LIR docking site, suggesting a favorable ligandability of HP2. Through this study, we identified diverse validated LC3/GABARAP ligands and fragments as starting points for chemical probe and ATTEC development., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Chikungunya virus (CHIKV) causes severe fever, rash and debilitating joint pain that can last for months 1,2 or even years. Millions of people have been infected with CHIKV, mostly in low and middle-income countries, and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts. Its genome encodes a macrodomain, which functions as an ADP-ribosyl hydrolase, removing ADPr from viral and host-cell proteins interfering with the innate immune response. Mutational studies have shown that the CHIKV nsP3 macrodomain is necessary for viral replication, making it a potential target for the development of antiviral therapeutics. We, therefore, performed a high-throughput crystallographic fragment screen against the CHIKV nsP3 macrodomain, yielding 109 fragment hits covering the ADPr-binding site and two adjacent subsites that are absent in the homologous macrodomain of SARS-CoV-2 but may be present in other alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV). Finally, a subset of overlapping fragments was used to manually design three fragment merges covering the adenine and oxyanion subsites. The rich dataset of chemical matter and structural information discovered from this fragment screen is publicly available and can be used as a starting point for developing a CHIKV nsP3 macrodomain inhibitor.

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.

Zika virus (ZIKV) infections cause microcephaly in new-borns and Guillain-Barre syndrome in adults raising a significant global public health concern, yet no vaccines or antiviral drugs have been developed to prevent or treat ZIKV infections. The viral protease NS3 and its co-factor NS2B are essential for the cleavage of the Zika polyprotein precursor into individual structural and non-structural proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 48 binders with diverse chemical scaffolds were identified in the active site of the protease, with another 6 fragment hits observed in a potential allosteric binding site. Our work provides potential starting points for the development of potent NS2B-NS3 protease inhibitors. Furthermore, we have structurally characterized a potential allosteric binding pocket, identifying opportunities for allosteric inhibitor development.

The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3 Hel ) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3 Hel . Here we applied high-throughput crystallographic fragment screening on ZIKV NS3 Hel , which yielded structures that reveal 3D binding poses of 46 fragments at multiple sites of the protein, including 11 unique fragments in the RNA-cleft site. These fragment structures provide templates for direct design of hit compounds and should thus assist the development of novel direct-acting antivirals against ZIKV and related flaviviruses, thus opening a promising avenue for combating future outbreaks.

Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of these viruses is responsible for the self-cleavage of the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. These 2A proteases are highly conserved between Enterovirus species, such as Enterovirus A71 and Coxsackievirus A16 . Inhibition of the 2A protease deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. Herein, we describe a crystallographic fragment screening campaign that identified 75 fragments which bind to the 2A protease including 38 unique compounds shown to bind within the active site. These fragments reveal a path for the development of non-peptidomimetic inhibitors of the 2A protease with broad-spectrum anti-enteroviral activity.

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.

A detailed understanding of the interactions between small-molecule ligands and their proposed binding targets is of the utmost importance for modern drug-development programs. Cellular retinoic acid-binding proteins I and II (CRABPI and CRABPII) facilitate a number of vital retinoid signalling pathways in mammalian cells and offer a gateway to manipulation of signalling that could potentially reduce phenotypes in serious diseases, including cancer and neurodegeneration. Although structurally very similar, the two proteins possess distinctly different biological functions, with their signalling influence being exerted through both genomic and nongenomic pathways. In this article, crystal structures are presented of the L29C mutant of Homo sapiens CRABPI in complex with naturally occurring fatty acids (1.64 Å resolution) and with the synthetic retinoid DC645 (2.41 Å resolution), and of CRABPII in complex with the ligands DC479 (1.80 Å resolution) and DC645 (1.71 Å resolution). DC645 and DC479 are two potential drug compounds identified in a recent synthetic retinoid development program. In particular, DC645 has recently been shown to have disease-modifying capabilities in neurodegenerative disease models by activating both genomic and nongenomic signalling pathways. These co-crystal structures demonstrate a canonical binding behaviour akin to that exhibited with all-trans-retinoic acid and help to explain how the compounds are able to exert an influence on part of the retinoid signalling cascade., (open access.)

Retinoic acid receptors were discovered during early studies of the actions and mechanisms of essential vitamins. Vitamin A is metabolized in the body to retinoic acid (RA) which is a key compound in the control of many developmental processes in chordates. These functions are mediated by a subfamily of nuclear receptors, divided into two classes, the retinoic acid receptors (RAR) and the retinoid X receptors (RXR). Each class is encoded by three closely related genes that are located on different chromosomes. The three proteins in each class are designated α, β and γ, respectively. A wealth of structural studies have shown that they all share the same architecture including a DNA-binding domain connected by a flexible linker to the ligand and co-activator binding domain. Retinoic acid incorporation into the ligand-binding domain leads to a conformational change enabling the formation of RAR homodimers or RAR/RXR heterodimers that in turn bind specifically to target DNA sequences. The consensus sequences located on the promotors of regulated genes are known as retinoic acid response elements (RARE). The activated RAR/RXR homodimers recruit co-activators with histone acetylase activity leading to an opening of the chromatin structure and enabling downstream transcription of regulated genes. These canonical pathways describe the control mechanism for the majority of developmental processes mediated by retinoic acid and its derivatives., (© 2020 Elsevier Inc. All rights reserved.)

Assays for identifying the activity of retinoic acids and retinoic acid derivatives, have become increasingly accessible in the 21st century. Movement away from complex in cellulo expression and radiological systems toward accessible FRET or fluorescent compound led studies has increased the accessibility and high-throughput capabilities of the field. The improvement in assaying technologies will lead to both increased and faster screening of retinoids and related compounds, improving drug discovery workflows and implementation pipelines. This enabling technology will allow better understanding of the huge range of phenotypes closely linked to retinoid signaling, and through the development of improved treatments improve outlooks for diagnoses in these cases and those of closely related diseases. In this chapter we present an examination of historical and modern techniques for assaying the activity of retinoic acid derivatives against their binding proteins., (© 2020 Elsevier Inc. All rights reserved.)

Retinoids, such as all- trans-retinoic acid (ATRA), are endogenous signaling molecules derived from vitamin A that influence a variety of cellular processes through mediation of transcription events in the cell nucleus. Because of these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they control. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluorescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quantify their binding to their cellular targets, including cellular retinoid binding protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding ( K d = 34.0 ± 2.5 nM), and we further used X-ray crystallography to determine the structure of the DC360-CRABPII complex to 1.8 Å, which showed that DC360 occupies the known hydrophobic retinoid binding pocket. Finally, we used confocal fluorescence microscopy to image the cellular behavior of the compounds in cultured human epithelial cells, highlighting a fascinating nuclear localization, and used RNA sequencing to confirm that the compounds regulate cellular processes similar to those of ATRA. We anticipate that the unique properties of these fluorescent retinoids can now be used to cast new light on the vital and highly complex retinoid signaling pathway.

Vitamin A derived retinoid compounds have multiple, powerful roles in the cellular growth and development cycle and, as a result, have attracted significant attention from both academic and pharmaceutical research in developing and characterizing synthetic retinoid analogues. Simplifying the hit development workflow for retinoid signaling will improve options available for tackling related pathologies, including tumor growth and neurodegeneration. Here, we present a novel assay that employs an intrinsically fluorescent synthetic retinoid, DC271, which allows direct measurement of the binding of nonlabeled compounds to relevant proteins. The method allows for straightforward initial measurement of binding using existing compound libraries and is followed by calculation of binding constants using a dilution series of plausible hits. The ease of use, high throughput format, and measurement of both qualitative and quantitative binding offer a new direction for retinoid-related pharmacological development., Competing Interests: The authors declare no competing financial interest.