Structure-based Design Approach Research Articles

Assessment of Nucleobase Protomeric and Tautomeric States in Nucleic Acid Structures for Interaction Analysis and Structure-Based Ligand Design.

With increasing interest in RNA as a therapeutic and a potential target, the role of RNA structures has become more important. Even slight changes in nucleobases, such as modifications or protomeric and tautomeric states, can have a large impact on RNA structure and function, while local environments in turn affect protonation and tautomerization. In this work, the application of empirical tools for pKa and tautomer prediction for RNA modifications was elucidated and compared with ab initio quantum mechanics (QM) methods and expanded toward macromolecular RNA structures, where QM is no longer feasible. In this regard, the Protonate3D functionality within the molecular operating environment (MOE) was expanded for nucleobase protomer and tautomer predictions and applied to reported examples of altered protonation states depending on the local environment. Overall, observations of nonstandard protomers and tautomers were well reproduced, including structural C+G:C(A) and A+GG motifs, several mismatches, and protonation of adenosine or cytidine as the general acid in nucleolytic ribozymes. Special cases, such as cobalt hexamine-soaked complexes or the deprotonation of guanosine as the general base in nucleolytic ribozymes, proved to be challenging. The collected set of examples shall serve as a starting point for the development of further RNA protonation prediction tools, while the presented Protonate3D implementation already delivers reasonable protonation predictions for RNA and DNA macromolecules. For cases where higher accuracy is needed, like following catalytic pathways of ribozymes, incorporation of QM-based methods can build upon the Protonate3D-generated starting structures. Likewise, this protonation prediction can be used for structure-based RNA-ligand design approaches.

Hepatitis C Virus E1E2 Structure, Diversity, and Implications for Vaccine Development.

Hepatitis C virus (HCV) is a major medical health burden and the leading cause of chronic liver disease and cancer worldwide. More than 58 million people are chronically infected with HCV, with 1.5 million new infections occurring each year. An effective HCV vaccine is a major public health and medical need as recognized by the World Health Organization. However, due to the high variability of the virus and its ability to escape the immune response, HCV rapidly accumulates mutations, making vaccine development a formidable challenge. An effective vaccine must elicit broadly neutralizing antibodies (bnAbs) in a consistent fashion. After decades of studies from basic research through clinical development, the antigen of choice is considered the E1E2 envelope glycoprotein due to conserved, broadly neutralizing antigenic domains located in the constituent subunits of E1, E2, and the E1E2 heterodimeric complex itself. The challenge has been elicitation of robust humoral and cellular responses leading to broad virus neutralization due to the relatively low immunogenicity of this antigen. In view of this challenge, structure-based vaccine design approaches to stabilize key antigenic domains have been hampered due to the lack of E1E2 atomic-level resolution structures to guide them. Another challenge has been the development of a delivery platform in which a multivalent form of the antigen can be presented in order to elicit a more robust anti-HCV immune response. Recent nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both the cellular and humoral components of the immune system. This review focuses on recent advances in understanding the E1E2 heterodimeric structure to facilitate a rational design approach and the potential for development of a multivalent nanoparticle-based HCV E1E2 vaccine. Both aspects are considered important in the development of an effective HCV vaccine that can effectively address viral diversity and escape.

Identifying p56lck SH2 Domain Inhibitors Using Molecular Docking and In Silico Scaffold Hopping

Bacterial infections are the second-leading cause of death, globally. The prevalence of antibacterial resistance has kept the demand strong for the development of new and potent drug candidates. It has been demonstrated that Src protein tyrosine kinases (TKs) play an important role in the regulation of inflammatory responses to tissue injury, which can trigger the onset of several severe diseases. We carried out a search for novel Src protein TK inhibitors, commencing from reported highly potent anti-bacterial compounds obtained using the Mannich reaction, using a combination of e-pharmacophore modeling, virtual screening, ensemble docking, and core hopping. The top-scoring compounds from ligand-based virtual screening were modified using protein structure-based design approaches, and their binding to the Src homology-2 domain of p56lck TK was predicted using ensemble molecular docking. We have prepared a database of 202 small molecules and have identified six novel top hits that can be subjected to further investigation. We have also performed in silico ADMET property prediction for the hit compounds. This combined computer-aided drug design approach can serve as a starting point for identifying novel TK inhibitors that could be further subjected to in vitro studies and validation of antimicrobial activity.

Identification of Novel Potent NSD2-PWWP1 Ligands Using Structure-Based Design and Computational Approaches.

Dysregulation of histone methyl transferase nuclear receptor-binding SET domain 2 (NSD2) has been implicated in several hematological and solid malignancies. NSD2 is a large multidomain protein that carries histone writing and histone reading functions. To date, identifying inhibitors of the enzymatic activity of NSD2 has proven challenging in terms of potency and SET domain selectivity. Inhibition of the NSD2-PWWP1 domain using small molecules has been considered as an alternative approach to reduce NSD2-unregulated activity. In this article, we present novel computational chemistry approaches, encompassing free energy perturbation coupled to machine learning (FEP/ML) models as well as virtual screening (VS) activities, to identify high-affinity NSD2 PWWP1 binders. Through these activities, we have identified the most potent NSD2-PWWP1 binder reported so far in the literature: compound 34 (pIC50 = 8.2). The compounds identified herein represent useful tools for studying the role of PWWP1 domains for inhibition of human NSD2.

Abstract 4066: Generation of tumor targeted self-assembling split IL-12 subunits for the treatment of cancer

Abstract A major milestone in immuno-oncology was the development of immune checkpoint inhibitors, resulting in significant benefits for numerous patients. However, many patients are not responsive, become refractory, or cannot tolerate the treatment due to toxicity or side effects, demonstrating the need for additional immunotherapies. Cytokines are a class of promising immunomodulatory proteins being explored as therapeutics, but their success has been limited due to their rapid clearance or pleiotropic properties. Classically known as ‘signal 3’ of an endogenous immune response, IL-12p70 is a heterodimeric cytokine comprising p35 and p40 subunits. When administered exogenously, IL-12 is a potent stimulator of the immune system and can have profound anti-tumor activity; however, the clinical use of IL-12 has been limited due to poor systemic tolerability. Therefore, developing an immunotherapy that can be systemically administered and improve the therapeutic index could capitalize on the potential clinical benefit of IL-12. Mural Oncology has developed an innovative approach to mitigate the toxicity of IL-12 by splitting the heterodimer into inactive p35 and p40 monomers. The individual subunits are separately fused to two non-competitive antibody fragments targeting a highly expressed tumor-associated antigen. The goal of this approach is to conditionally activate IL-12 preferentially in the tumor microenvironment (TME). This is achieved by sequential administration of the targeted subunits, which will drive assembly and activity of the IL-12 heterodimer primarily at the tumor site, reducing systemic exposure and thereby potentially reducing associated toxicities. To engineer the split IL-12 subunits, a structure-based rational design approach was used to remove the natural intra-molecular disulfide bond and generate variants that maintained affinity between the p35 and p40 monomers. The engineered subunits were characterized using analytical methods and screened for the pair with the strongest affinity. Additionally, the candidate pair was assessed for receptor binding and functional activity on human T cells through phosphorylation of STAT4 and secretion of IFN-γ. Pharmacokinetics and pharmacodynamics were evaluated in mouse models with and without tumors demonstrating that dual targeting led to an increase in tumor retention, rapid clearance from circulation, and dose dependent release of IFN-γ. The results thus far demonstrate that the innovative approach of tumor targeted self-assembling split IL-12 has the potential to enhance the overall therapeutic potential of this cytokine. A development candidate will be identified and moved into clinical testing in cancer patients to enable us to fully understand if this design approach holds true in the clinical setting. Citation Format: Joshua Heiber, Pinar Gurel, Robert G. Newman, Kristiana Dreaden, Yanchun Zhao, Chunhua Wang, Su-Ping Pearson, Jean Chamoun. Generation of tumor targeted self-assembling split IL-12 subunits for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4066.

Targeting staphylococcal enterotoxin B binding to CD28 as a new strategy for dampening superantigen-mediated intestinal epithelial barrier dysfunctions.

Staphylococcus aureus is a gram-positive bacterium that may cause intestinal inflammation by secreting enterotoxins, which commonly cause food-poisoning and gastrointestinal injuries. Staphylococcal enterotoxin B (SEB) acts as a superantigen (SAg) by binding in a bivalent manner the T-cell receptor (TCR) and the costimulatory receptor CD28, thus stimulating T cells to produce large amounts of inflammatory cytokines, which may affect intestinal epithelial barrier integrity and functions. However, the role of T cell-mediated SEB inflammatory activity remains unknown. Here we show that inflammatory cytokines produced by T cells following SEB stimulation induce dysfunctions in Caco-2 intestinal epithelial cells by promoting actin cytoskeleton remodelling and epithelial cell-cell junction down-regulation. We also found that SEB-activated inflammatory T cells promote the up-regulation of epithelial-mesenchymal transition transcription factors (EMT-TFs) in a nuclear factor-κB (NF-κB)- and STAT3-dependent manner. Finally, by using a structure-based design approach, we identified a SEB mimetic peptide (pSEB116-132) that, by blocking the binding of SEB to CD28, dampens inflammatory-mediated dysregulation of intestinal epithelial barrier.

Structure-based approaches for the design of 6-aryl-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d][1,2,3]triazoles as tubulin polymerization inhibitors

The colchicine binding site on tubulin has been widely acknowledged as an attractive target for anticancer drug exploitation. Here, we reported the structural optimization of the lead compound 4, which was proved in our previous work as a colchicine binding site inhibitor (CBSI). Based on docking researches for the active binding conformation of compound 4, a series of novel 6-aryl-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d][1,2,3]triazole derivatives (9a–9x) were developed by replacing a CH group in the 1H-benzo[d]imidazole skeleton of compound 4 with a nitrogen atom as a hydrogen bond acceptor. Among them, compound 9a showed the strongest antiproliferative activity with IC50 values ranging from 14 to 45 nM against three human cancer cell lines (MCF-7, SGC-7901 and A549), lower than that of compound 4. Mechanistic studies indicated that compound 9a could inhibit tubulin polymerization, destroy the microtubule skeleton, block the cell cycle in G2/M phase, induce cancer cell apoptosis, prevent cancer cell migration and colony formation. Moreover, compound 9a significantly inhibited tumor growth in vivo without observable toxicity in the mice 4T1 xenograft tumor model. In conclusion, this report shows a successful case of the structure-based design approach of a potent tubulin polymerization inhibitor for cancer treatment.

SiteMine: Large-scale binding site similarity searching in protein structure databases.

Drug discovery and design challenges, such as drug repurposing, analyzing protein-ligand and protein-protein complexes, ligand promiscuity studies, or function prediction, can be addressed by protein binding site similarity analysis. Although numerous tools exist, they all have individual strengths and drawbacks with regard to run time, provision of structure superpositions, and applicability to diverse application domains. Here, we introduce SiteMine, an all-in-one database-driven, alignment-providing binding site similarity search tool to tackle the most pressing challenges of binding site comparison. The performance of SiteMine is evaluated on the ProSPECCTs benchmark, showing a promising performance on most of the data sets. The method performs convincingly regarding all quality criteria for reliable binding site comparison, offering a novel state-of-the-art approach for structure-based molecular design based on binding site comparisons. In a SiteMine showcase, we discuss the high structural similarity between cathepsin L and calpain 1 binding sites and give an outlook on the impact of this finding on structure-based drug design. SiteMine is available at https://uhh.de/naomi.

Development of 2-Aminotetralin-Type Serotonin 5-HT1 Agonists: Molecular Determinants for Selective Binding and Signaling at 5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1F Receptors.

The serotonin (5-hydroxytryptamine, 5-HT) 5-HT1 G-protein coupled receptor subtypes (5-HT1A/1B/1D/1E/1F) share a high sequence homology, confounding development of subtype-specific ligands. This study used a 5-HT1 structure-based ligand design approach to develop subtype-selective ligands using a 5-substituted-2-aminotetralin (5-SAT) chemotype, leveraging results from pharmacological, molecular modeling, and mutagenesis studies to delineate molecular determinants for 5-SAT binding and function at 5-HT1 subtypes. 5-SATs demonstrated high affinity (Ki ≤ 25 nM) and at least 50-fold stereoselective preference ([2S] > [2R]) at 5-HT1A, 5-HT1B, and 5-HT1D receptors but essentially nil affinity (Ki > 1 μM) at 5-HT1F receptors. The 5-SATs tested were agonists with varying degrees of potency and efficacy, depending on chemotype substitution and 5-HT1 receptor subtype. Models were built from the 5-HT1A (cryo-EM), 5-HT1B (crystal), and 5-HT1D (cryo-EM) structures, and 5-SATs underwent docking studies with up to 1 μs molecular dynamics simulations. 5-SAT interactions observed at positions 3.33, 5.38, 5.42, 5.43, and 7.39 of 5-HT1 subtypes were confirmed with point mutation experiments. Additional 5-SATs were designed and synthesized to exploit experimental and computational results, yielding a new full efficacy 5-HT1A agonist with 100-fold selectivity over 5-HT1B/1D receptors. The results presented lay the foundation for the development of additional 5-HT1 subtype selective ligands for drug discovery purposes.

Novel Pan-ERR Agonists Ameliorate Heart Failure Through Enhancing Cardiac Fatty Acid Metabolism and Mitochondrial Function.

Cardiac metabolic dysfunction is a hallmark of heart failure (HF). Estrogen-related receptors ERRα and ERRγ are essential regulators of cardiac metabolism. Therefore, activation of ERR could be a potential therapeutic intervention for HF. However, in vivo studies demonstrating the potential usefulness of ERR agonist for HF treatment are lacking, because compounds with pharmacokinetics appropriate for in vivo use have not been available. Using a structure-based design approach, we designed and synthesized 2 structurally distinct pan-ERR agonists, SLU-PP-332 and SLU-PP-915. We investigated the effect of ERR agonist on cardiac function in a pressure overload-induced HF model in vivo. We conducted comprehensive functional, multi-omics (RNA sequencing and metabolomics studies), and genetic dependency studies both in vivo and in vitro to dissect the molecular mechanism, ERR isoform dependency, and target specificity. Both SLU-PP-332 and SLU-PP-915 significantly improved ejection fraction, ameliorated fibrosis, and increased survival associated with pressure overload-induced HF without affecting cardiac hypertrophy. A broad spectrum of metabolic genes was transcriptionally activated by ERR agonists, particularly genes involved in fatty acid metabolism and mitochondrial function. Metabolomics analysis showed substantial normalization of metabolic profiles in fatty acid/lipid and tricarboxylic acid/oxidative phosphorylation metabolites in the mouse heart with 6-week pressure overload. ERR agonists increase mitochondria oxidative capacity and fatty acid use in vitro and in vivo. Using both in vitro and in vivo genetic dependency experiments, we show that ERRγ is the main mediator of ERR agonism-induced transcriptional regulation and cardioprotection and definitively demonstrated target specificity. ERR agonism also led to downregulation of cell cycle and development pathways, which was partially mediated by E2F1 in cardiomyocytes. ERR agonists maintain oxidative metabolism, which confers cardiac protection against pressure overload-induced HF in vivo. Our results provide direct pharmacologic evidence supporting the further development of ERR agonists as novel HF therapeutics.

Improving glucose tolerance of a fungal β-glucosidase: structure-based rational design approach

Improving glucose tolerance of a fungal β-glucosidase: structure-based rational design approach

Rational design and evaluation of 2-((pyrrol-2-yl)methylene)thiophen-4-ones as RNase L inhibitors

Ribonuclease L (RNase L) plays a crucial role in an antiviral pathway of interferon-induced innate immunity by degrading RNAs to prevent viral replication. Modulating RNase L activity thus mediates the innate immune responses and inflammation. Although a few small molecule-based RNase L modulators have been reported, only limited molecules have been mechanistically investigated. This study explored the strategy of RNase L targeting by using a structure-based rational design approach and evaluated the RNase L-binding and inhibitory activities of the yielded 2-((pyrrol-2-yl)methylene)thiophen-4-ones, which exhibited improved inhibitory effect as determined by in vitro FRET and gel-based RNA cleavage assay. A further structural optimization study yielded selected thiophenones that showed >30-fold more potent inhibitory activity than that of sunitinib, the approved kinase inhibitor with reported RNase L inhibitory activity. The binding mode with RNase L for the resulting thiophenones was analyzed by using docking analysis. Furthermore, the obtained 2-((pyrrol-2-yl)methylene)thiophen-4-ones exhibited efficient inhibition of RNA degradation in cellular rRNA cleavage assay. The newly designed thiophenones are the most potent synthetic RNase L inhibitors reported to date and the results revealed in our study lay the foundation for the development of future RNase L-modulating small molecules with new scaffold and improved potency.

Optimization of an α-aminonaphthylmethylphosphonic acid inhibitor of purple acid phosphatase using rational structure-based design approaches

Purple acid phosphatases (PAPs) are ubiquitous binuclear metallohydrolases that have been isolated from various animals, plants and some types of fungi. In humans and mice, elevated PAP activity in osteoclasts is associated with osteoporosis, making human PAP an attractive target for the development of anti-osteoporotic drugs. Based on previous studies focusing on phosphonate scaffolds, as well as a new crystal structure of a PAP in complex with a derivative of a previously synthesized α-aminonaphthylmethylphosphonic acid, phosphonates 24–40 were designed as new PAP inhibitor candidates. Subsequent docking studies predicted that all of these compounds are likely to interact strongly with the active site of human PAP and most are likely to interact strongly with the active site of pig PAP. The seventeen candidates were synthesized with good yields and nine of them (26–28, 30, 33–36 and 38) inhibit in the sub-micromolar to nanomolar range against pig PAP, with 28 and 35 being the most potent mammalian PAP inhibitors reported with Ki values of 168 nM and 186 nM, respectively. This study thus paves the way for the next stage of drug development for phosphonate inhibitors of PAPs as anti-osteoporotic agents.

A Structure-based Design Approach to Catharanthus roseus Phytoconstituents as Potential Inhibitors of B-Cell Lymphoma 6 Protein

A Structure-based Design Approach to Catharanthus roseus Phytoconstituents as Potential Inhibitors of B-Cell Lymphoma 6 Protein

In Silico Discovery and Optimisation of a Novel Structural Class of Hsp90 C-Terminal Domain Inhibitors.

Hsp90 is a promising target for the development of novel agents for cancer treatment. The N-terminal Hsp90 inhibitors have several therapeutic limitations, the most important of which is the induction of heat shock response, which can be circumvented by targeting the allosteric binding site on the C-terminal domain (CTD) of Hsp90. In the absence of an Hsp90—CTD inhibitor co-crystal structure, the use of structure-based design approaches for the Hsp90 CTD is difficult and the structural diversity of Hsp90 CTD inhibitors is limited. In this study, we describe the discovery of a novel structural class of Hsp90 CTD inhibitors. A structure-based virtual screening was performed by docking a library of diverse compounds to the Hsp90β CTD binding site. Three selected virtual hits were tested in the MCF-7 breast cancer cell line, with compound TVS-23 showing antiproliferative activity with an IC50 value of 26.4 ± 1.1 µM. We report here the optimisation, synthesis and biological evaluation of TVS-23 analogues. Several analogues showed significantly enhanced antiproliferative activities in MCF-7 breast cancer and SK-N-MC Ewing sarcoma cell lines, with 7l being the most potent (IC50 = 1.4 ± 0.4 µM MCF-7; IC50 = 2.8 ± 0.4 µM SK-N-MC). The results of this study highlight the use of virtual screening to expand the structural diversity of Hsp90 CTD inhibitors and provide new starting points for further development.

Structure-Based Ligand Design Targeting Pseudomonas aeruginosa LpxA in Lipid A Biosynthesis.

Enzymes involved in lipid A biosynthesis are promising antibacterial drug targets in Gram-negative bacteria. In this study, we use a structure-based design approach to develop a series of novel tetrazole ligands with low μM affinity for LpxA, the first enzyme in the lipid A pathway. Aided by previous structural data, X-ray crystallography, and surface plasmon resonance bioanalysis, we identify 17 hit compounds. Two of these hits were subsequently modified to optimize interactions with three regions of the LpxA active site. This strategy ultimately led to the discovery of ligand L13, which had a KD of 3.0 μM. The results reveal new chemical scaffolds as potential LpxA inhibitors, important binding features for ligand optimization, and protein conformational changes in response to ligand binding. Specifically, they show that a tetrazole ring is well-accommodated in a small cleft formed between Met169, the "hydrophobic-ruler" and His156, both of which demonstrate significant conformational flexibility. Furthermore, we find that the acyl-chain binding pocket is the most tractable region of the active site for realizing affinity gains and, along with a neighboring patch of hydrophobic residues, preferentially binds aliphatic and aromatic groups. The results presented herein provide valuable chemical and structural information for future inhibitor discovery against this important antibacterial drug target.

Self-derived peptides from the SARS-CoV-2 spike glycoprotein disrupting shaping and stability of the homotrimer unit

The structural spike (S) protein from the SARS-CoV-2 β-coronavirus is shown to make different pre- and post-fusion conformations within its homotrimer unit. To support the ongoing novel vaccine design and development strategies, we report the structure-based design approach to develop self-derived S peptides. A dataset of crucial regions from the S protein were transformed into linear motifs that could act as the blockers or stabilizers for the S protein homotrimer unit. Among these distinct S peptides, the pep02 (537-QQFGRDIAD-545) and pep07 (821-RDLICAQKFNGLTVLPPLLTDE-842) were found making stable folded binding with the S protein (550–750 and 950–1050 regions). Upon inserting SARS-CoV-2 S variants in the peptide destabilized the complexed S protein structure, resulting an allosteric effect in different functional regions of the protein. Particularly, the molecular dynamics revealed that A544D mutation in the pep02 peptide induced instability for the complexed S protein, whereas the N943K variant from pep09 exhibited an opposite behavior. An increased protein-peptide binding affinity and the stable structural folding were observed in mutated systems, compared to that of the wild type systems. The presence of mutation has induced an “up” active conformation of the spike (RBD) domain, responsible for interacting the host cell receptor. Among the lower affinity peptide datasets (e.g., pep01), the S1 and S2 subunit in the protein formed an “open” conformation, whereas with higher affinity peptides (e.g., pep07) these domains gained a “closed” conformation. These findings propose that our designed self-derived S peptides could replace a single S protein monomer, blocking the homotrimer formation or inducing stability.

Discovery of Novel 7-Azaindole Derivatives as Selective Covalent Fibroblast Growth Factor Receptor 4 Inhibitors for the Treatment of Hepatocellular Carcinoma.

Fibroblast growth factor receptor 4 (FGFR4) has been identified as a potential target for the treatment of hepatocellular carcinoma (HCC) with aberrant FGFR4 signaling because of its important role in HCC progression and development. Several FGFR4 inhibitors are under clinical development. Using a 7-azaindole scaffold, we discovered a series of novel selective and covalent FGFR4 inhibitors by performing a structure-based design approach. Representative compounds 24 and 30 exhibited potent FGFR4 inhibition and high selectivity among kinases. Western blot analysis showed that compounds 24 and 30 significantly inhibited the FGF19/FGFR4 signaling pathway in HuH-7 cells and effectively suppressed the proliferation of HuH-7 HCC cells and MDA-MB-453 breast cancer cells. Moreover, compound 30 exhibited significant in vivo antitumor activity in a mouse HuH-7 xenograft model. Thus, compound 30 and the 7-azaindole scaffold can be applied to develop anticancer agents for the treatment of cancers characterized by aberrant FGFR4 signaling.

Discovery of a Highly Selective and H435R-Sensitive Thyroid Hormone Receptor β Agonist.

The design and development of agonists selectively targeting thyroid hormone receptor β (TRβ) and TRβ mutants remain challenging tasks. In this study, we first adopted the strategy of breaking the "His-Phe switch" to solve two problems, simultaneously. A structure-based design approach was successfully utilized to obtain compound 16g, which is a potent TRβ agonist (EC50: 21.0 nM, 85.0% of the maximum efficacy of 1) with outstanding selectivity for TRβ over TRα and also effectively activates the TRβH435R mutant. Then, we developed a highly efficient synthetic method for 16g. Our serials of cocrystal structures revealed detailed structural mechanisms in overcoming subtype selectivity and rescuing the H435R mutation. 16g also showed excellent lipid metabolism, safety, metabolic stability, and pharmacokinetic properties. Collectively, 16g is a well-characterized selective and mutation-sensitive TRβ agonist for further investigating its function in treating dyslipidemia, nonalcoholic steatohepatitis (NASH), and resistance to thyroid hormone (RTH).

Driving E3 Ligase Substrate Specificity for Targeted Protein Degradation: Lessons from Nature and the Laboratory.

Methods to direct the degradation of protein targets with proximity-inducing molecules that coopt the cellular degradation machinery are advancing in leaps and bounds, and diverse modalities are emerging. The most used and well-studied approach is to hijack E3 ligases of the ubiquitin-proteasome system. E3 ligases use specific molecular recognition to determine which proteins in the cell are ubiquitinated and degraded. This review focuses on the structural determinants of E3 ligase recruitment of natural substrates and neo-substrates obtained through monovalent molecular glues and bivalent proteolysis-targeting chimeras. We use structures to illustrate the different types of substrate recognition and assess the basis for neo-protein-protein interactions in ternary complex structures. The emerging structural and mechanistic complexity is reflective of the diverse physiological roles of protein ubiquitination. This molecular insight is also guiding the application of structure-based design approaches to the development of new and existing degraders as chemical tools and therapeutics.