Fragment Screening Research Articles

Design and Evaluation of a Low Hydrogen Bond Donor Count Fragment Screening Set to Aid Hit Generation of PROTACs Intended for Oral Delivery.

The development of orally bioavailable PROTACs presents a significant challenge due to the inflated physicochemical properties of such heterobifunctional molecules. Molecules occupying this "beyond rule of five" space often demonstrate limited oral bioavailability due to the compounding effects of elevated molecular weight and hydrogen bond donor count (among other properties), but it is possible to achieve sufficient oral bioavailability through physicochemical optimization. Herein, we disclose the design and evaluation of a low hydrogen bond donor count (≤1 HBD) fragment screening set to aid hit generation of PROTACs intended for an oral route of delivery. We demonstrate that application of this library can enhance fragment screens against PROTAC proteins of interest and ubiquitin ligases, yielding fragment hits containing ≤1 HBD suitable for optimizing toward orally bioavailable PROTACs.

An Analysis of Successful Hit-to-Clinical Candidate Pairs.

An analysis of 156 published clinical candidates from the Journal of Medicinal Chemistry between 2018 and 2021 was conducted to identify lead generation strategies most frequently employed leading to drug candidates. As in a previous publication, the most frequent lead generation strategies resulting in clinical candidates were from known compounds (59%) followed by random screening approaches (21%). The remainder of the approaches included directed screening, fragment screening, DNA-encoded library screening (DEL), and virtual screening. An analysis of similarity was also conducted based on Tanimoto-MCS and revealed most clinical candidates were distant from their original hits; however, most shared a key pharmacophore that translated from hit-to-clinical candidate. An examination of frequency of oxygen, nitrogen, fluorine, chlorine, and sulfur incorporation in clinical candidates was also conducted. The three most similar and least similar hit-to-clinical pairs from random screening were examined to provide perspective on changes that occur that lead to successful clinical candidates.

An Automated, Open-Source Workflow for the Generation of (3D) Fragment Libraries.

The recent success of fragment-based drug discovery (FBDD) is inextricably linked to adequate library design. To guide the design of our fragment libraries, we have constructed an automated workflow in the open-source KNIME software. The workflow considers chemical diversity and novelty of the fragments, and can also take into account the three-dimensional (3D) character. This design tool can be used to create large and diverse libraries but also to select a small number of representative compounds as a focused set of unique screening compounds to enrich existing fragment libraries. To illustrate the procedures, the design and synthesis of a 10-membered focused library is reported based on the cyclopropane scaffold, which is underrepresented in our existing fragment screening library. Analysis of the focused compound set indicates significant shape diversity and a favorable overall physicochemical profile. By virtue of its modular setup, the workflow can be readily adjusted to design libraries that focus on properties other than 3D shape.

Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 NSP15 endoribonuclease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.

Halo Library, a Tool for Rapid Identification of Ligand Binding Sites on Proteins Using Crystallographic Fragment Screening.

X-ray crystallographic fragment screening (XCFS) uses fragment-sized molecules (∼60 to 300 Da) to access binding sites on proteins that may be inaccessible to larger drug-like molecules (>300 Da). Previous studies have shown that fragments containing halogen atoms bind more often to proteins than non-halogenated fragments. Here, we designed the Halo Library containing 46 halogenated fragments (including the "universal fragment" 4-bromopyrazole), a majority of which have been reported to bind to or inhibit one or more targets. The library was screened against the crystals of HIV-1 reverse transcriptase with the drug rilpivirine, yielding an overall hit rate of 26%. Two new binding sites were discovered, and several hot spots were identified. This small library may thus provide a convenient tool for rapidly assessing the feasibility of a target for XCFS, mapping hot spots and cryptic sites, as well as finding fragment binders that can be useful for developing drug leads.

Novel perfluoroalkyl substances (PFAS) discovered in whole blood using automated non-targeted analysis of dried blood spots

A small subset of per- and polyfluoroalkyl substances (PFAS) are routinely screened in human blood. These compounds generally explain <50 % of the total PFAS in human blood. The percentage of known PFAS in human blood has been decreasing as replacement PFAS and more complex PFAS chemistries are introduced to the market. Most of these novel PFAS have not been previously identified. Non-targeted methods are required to characterize this “dark matter” PFAS. Our objective was to apply non-targeted PFAS analysis to human blood to gain an understanding about the sources, concentrations, and toxicity of these compounds. A high-resolution tandem mass spectrometry (HRMS) and software workflow for PFAS characterization in dried blood spots is reported. Dried blood spots are a less invasive collection technique compared to venous blood draws, allowing collection from vulnerable populations. Biorepositories of archived dried blood spots are available internationally from newborns and present opportunities to study prenatal exposure to PFAS. In this study, dried blood spot cards were analyzed using iterative MS/MS by liquid chromatography HRMS. Data processing was conducted using FluoroMatch Suite including a visualizer tool that presents homologous series, retention time vs m/z plots, MS/MS spectra, feature tables, annotations, and fragments for fragment screening. The researcher performing data-processing and annotation was blinded to the fact that standards were spiked in, and was able to annotate 95 % of standards spiked on dried blood spot samples, signifying a low false negative rate using FluoroMatch Suite. A total of 28 PFAS (20 standards and 4 exogenous compounds) were detected across five homologous series with Schymanski Level 2 confidence. Of these 4, 3 were perfluoroalkyl ether carboxylic acids (PFECA), a chemical class of PFAS which is increasingly being detected in environmental and biological matrices but is not currently screened in most targeted analysese. A further 86 potential PFAS were detected using fragment screening. PFAS are extremely persistent and widespread yet remain largely unregulated. Our findings will contribute to an improved an understanding of exposures. Application of these methods in environmental epidemiology studies have the potential to inform policy with regards to PFAS monitoring, regulation, and individual-level mitigation strategies.

Rapid Elaboration of Fragments into Leads Applied to Bromodomain-3 Extra-Terminal Domain.

The development of low-affinity fragment hits into higher-affinity leads is a major hurdle in fragment-based drug design. Here, we demonstrate the Rapid Elaboration of Fragments into Leads (REFiL) by applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure-activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. After a fragment screen against bromodomain-3 extra-terminal (BRD3-ET) domain, we applied the REFiL workflow, which allowed us to develop a series of ligands that bind to BRD3-ET. With REFiL, we were able to rapidly improve binding affinity > 30-fold. REFiL can be applied readily to a broad range of proteins without the need for a structure, allowing the efficient evolution of low-affinity fragments into higher-affinity leads and chemical probes.

An asymmetric structure of bacterial TrpRS supports the half-of-the-sites catalytic mechanism and facilitates antimicrobial screening.

Tryptophanyl-tRNA synthetase (TrpRS) links tryptophan to tRNATrp, thereby playing an indispensable role in protein translation. Unlike most class I aminoacyl-tRNA synthetases (AARSs), TrpRS functions as a homodimer. Herein, we captured an 'open-closed' asymmetric structure of Escherichia coli TrpRS (EcTrpRS) with one active site occupied by a copurified intermediate product and the other remaining empty, providing structural evidence for the long-discussed half-of-the-sites reactivity of bacterial TrpRS. In contrast to its human counterpart, bacterial TrpRS may rely on this asymmetric conformation to functionally bind with substrate tRNA. As this asymmetric conformation is probably a dominant form of TrpRS purified from bacterial cells, we performed fragment screening against asymmetric EcTrpRS to support antibacterial discovery. Nineteen fragment hits were identified, and 8 of them were successfully cocrystallized with EcTrpRS. While a fragment named niraparib bound to the L-Trp binding site of the 'open' subunit, the other 7 fragments all bound to an unprecedented pocket at the interface between two TrpRS subunits. Binding of these fragments relies on residues specific to bacterial TrpRS, avoiding undesired interactions with human TrpRS. These findings improve our understanding of the catalytic mechanism of this important enzyme and will also facilitate the discovery of bacterial TrpRS inhibitors with therapeutic potential.

SDU - software for high-throughput automated data collection at the Swiss Light Source.

Recent advances in automation have fostered the development of unattended data collection services at a handful of synchrotron facilities worldwide. At the Swiss Light Source, the installation of new high-throughput sample changers at all three macromolecular crystallography beamlines and the commissioning of the Fast Fragment and Compound Screening pipeline created a unique opportunity to automate data acquisition. Here, the DA+ microservice software stack upgrades, implementation of an automatic loop-centering service and deployment of the Smart Digital User (SDU) software for unattended data collection are reported. The SDU software is the decision-making software responsible for communications between services, sample and device safety, sample centering, sample alignment with grid based X-ray diffraction and, finally, data collection.

Abstract 6286: Targeting Polycomb Repressive Complex 1 with small-molecule inhibitors as a potential therapeutic approach to combat leukemia

Abstract Background: Polycomb repressive complex 1 (PRC1) is an epigenetic regulatory complex that monoubiquitinates lysine 119 on histone H2A, resulting in the repression of target genes. The canonical core of PRC1 responsible for its E3 ligase activity is a heterodimeric unit comprised of RING1B and BMI1. Emerging data implicates RING1B in various cancers and highlights the importance of PRC1 function in the maintenance and proliferation of leukemic stem cells. Studies demonstrate that loss of PRC1 activity through RING1B knockdown impairs leukemic stem cell proliferation, causes leukemic stem cell differentiation, and enhances survival in MLL-AF9 mouse models. Therefore, development of potent small molecules targeting the activity of PRC1 might offer a new approach for generating new therapeutics for leukemia. Results: Here, we report the discovery and development of the first-in-class small-molecule inhibitors directly binding to RING1B in PRC1. We performed an NMR-based fragment screen against RING1B-BMI1, identifying fragment hit, RB-1, with low-millimolar binding affinity. RB-1 was extensively optimized into a class of inhibitors with considerably enhanced binding affinity to the low-nanomolar range. These inhibitors have been shown to diminish the H2A ubiquitination activity of PRC1 E3 ligase cores. Structural studies revealed that our PRC1 inhibitors bind directly to the RING domain of RING1B and ultimately block the protein-protein interaction between RING1B and the nucleosome. In leukemic cell lines and primary AML samples, PRC1 inhibitor treatment elicits an anti-proliferative effect on cell growth and displays on-target action seen by the reduction of the H2A ubiquitination mark. Additionally, small-molecule inhibition of PRC1 results in the induction of cellular differentiation of leukemic cells, as well as apoptosis. Gene expression analysis in leukemic cells treated with our PRC1 inhibitors reveals upregulation of multiple genes that are repressed by PRC1, which is also supported by the displacement of PRC1 from chromatin assessed by CHIP-seq studies. Furthermore, our PRC1 inhibitors are active in vivo in mouse models of leukemia, demonstrating reduction of leukemia progression upon treatment. Conclusions: We developed first-in class inhibitors directly targeting the E3 ligase core of PRC1. Treatment with our PRC1 inhibitors in leukemic cells elicit an anti-proliferative effect, blocks ubiquitination of H2A, and results in the de-repression of many genes repressed by PRC1. Additionally, our PRC1 inhibitors elicit an anti-proliferative effect on leukemia progression in vivo, demonstrating the traction for developing anti-leukemia therapeutics that inhibit PRC1. Citation Format: Miranda L. Simes, Yiwu Yao, Se Ra Park, Trupta Purohit, Hongzhi Miao, Xiaotian Zhang, Weijiang Ying, Felicia Gray, Shirish Shukla, Qingjie Zhao, Caroline Nikolaidis, Alyssa Winkler, Jolanta Grembecka, Tomasz Cierpicki. Targeting Polycomb Repressive Complex 1 with small-molecule inhibitors as a potential therapeutic approach to combat leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6286.

Electroacupuncture for the management of symptom clusters in cancer patients and survivors (EAST)

BackgroundNeuropsychiatric symptoms, comprising cognitive impairment, fatigue, insomnia, depression, and anxiety, are prevalent and may co-occur during and after chemotherapy treatment for cancer. Electroacupuncture (EA), which involves mild electrical stimulation with acupuncture, holds great potential in addressing the management of individual symptoms. However, there is a lack of studies evaluating if EA can manage concurrent neuropsychiatric symptoms in cancer (i.e., symptom cluster). Hence, we designed a trial to evaluate the efficacy, safety, and feasibility of administering EA as an intervention to mitigate neuropsychiatric symptom clusters amongst cancer patients and survivors.MethodsThe EAST study is a randomized, sham-controlled, patient- and assessor-blinded clinical trial. Sixty-four cancer patients and survivors with complaints of one or more neuropsychiatric symptom(s) in the seven days prior to enrollment are recruited from the University of California Irvine (UCI) and Children’s Hospital of Orange County (CHOC). Individuals with needle phobia, metastases, bleeding disorders, electronic implants, epilepsy, exposure to acupuncture in the three months prior to enrollment, and who are breastfeeding, pregnant, or planning to get pregnant during the duration of the study will be excluded. Screening for metal fragments and claustrophobia are performed prior to the optional neuroimaging procedures. Recruited patients will be randomized (1:1) in random blocks of four or six to receive either ten weekly verum EA (treatment arm, vEA) or weekly sham EA (control arm, sEA) treatment visits with a follow-up appointment four to twelve weeks after their last treatment visit. The treatment arm will receive EA at 13 acupuncture points (acupoints) chosen for their therapeutic effects, while the control arm receives minimal EA at 7 non-disease-related acupoints. Questionnaires and cognitive assessments are administered, and blood drawn to assess changes in symptom clusters and biomarkers, respectively.ConclusionThe EAST study can provide insight into the efficacy of EA, an integrative medicine modality, in the management of cancer symptom clusters in routine clinical practice.Trial registrationThis trial is registered with clinicaltrials.gov NCT05283577.

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.

Direct Measurement of Nucleoside Ribohydrolase Enzyme Activities in Trichomonas vaginalis Cells Using 19F and 13C-Edited 1H NMR Spectroscopy.

Trichomoniasis is the most common nonviral sexually transmitted infection, affecting an estimated 275 million people worldwide. The causative agent is the parasitic protozoan Trichomonas vaginalis. Although the disease itself is typically mild, individuals with trichomonal infections have a higher susceptibility to more serious conditions. The emergence of parasite strains resistant to current therapies necessitates the need for novel treatment strategies. Since T. vaginalis is an obligate parasite that requires nucleoside salvage pathways, essential nucleoside ribohydrolase enzymes are promising new drug targets. Fragment screening and X-ray crystallography have enabled structure-guided design of inhibitors for two of these enyzmes. Linkage of enzymatic and antiprotozoal activity would be a transformative step toward designing novel, mechanism-based therapeutic agents. While a correlation with inhibition of purified enzyme would be mechanistically suggestive, a correlation with inhibition of in-cell enzyme activity would definitively establish this linkage. To demonstrate this linkage, we have translated our NMR-based activity assays that measure the activity of purified enzymes for use in T. vaginalis cells. The 19F NMR-based activity assay for the pyrimidine-specific enzyme translated directly to in-cell assays. However, the 1H NMR-based activity assay for the purine-specific enzyme required a switch from adenosine to guanosine substrate and the use of 13C-editing to resolve the substrate 1H signals from cell and growth media background signals. The in-cell NMR assays are robust and have been demonstrated to provide inhibition data on test compounds. The results described here represent the first direct measurement of enzyme activity in protozoan parasite cells.

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD <20 μM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a K D <20 μM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.

Crystal structure of the CoV-Y domain of SARS-CoV-2 nonstructural protein 3

Replication of the coronavirus genome starts with the formation of viral RNA-containing double-membrane vesicles (DMV) following viral entry into the host cell. The multi-domain nonstructural protein 3 (nsp3) is the largest protein encoded by the known coronavirus genome and serves as a central component of the viral replication and transcription machinery. Previous studies demonstrated that the highly-conserved C-terminal region of nsp3 is essential for subcellular membrane rearrangement, yet the underlying mechanisms remain elusive. Here we report the crystal structure of the CoV-Y domain, the most C-terminal domain of the SARS-CoV-2 nsp3, at 2.4 Å-resolution. CoV-Y adopts a previously uncharacterized V-shaped fold featuring three distinct subdomains. Sequence alignment and structure prediction suggest that this fold is likely shared by the CoV-Y domains from closely related nsp3 homologs. NMR-based fragment screening combined with molecular docking identifies surface cavities in CoV-Y for interaction with potential ligands and other nsps. These studies provide the first structural view on a complete nsp3 CoV-Y domain, and the molecular framework for understanding the architecture, assembly and function of the nsp3 C-terminal domains in coronavirus replication. Our work illuminates nsp3 as a potential target for therapeutic interventions to aid in the on-going battle against the COVID-19 pandemic and diseases caused by other coronaviruses.

Covalent fragment mapping of KRasG12C revealed novel chemotypes with in vivo potency

G12C mutant KRas is considered druggable by allele-specific covalent inhibitors due to the nucleophilic character of the oncogenic mutant cysteine at position 12. Discovery of these inhibitors requires the optimization of both covalent and noncovalent interactions. Here, we report covalent fragment screening of our electrophilic fragment library of diverse non-covalent scaffolds equipped with 40 different electrophilic functionalities to identify fragments as suitable starting points targeting Cys12. Screening the library against KRasG12C using Ellman's free thiol assay, followed by protein NMR and cell viability assays, resulted in two potential inhibitor chemotypes. Characterization of these scaffolds in in vitro cellular- and in vivo xenograft models revealed them as promising starting points for covalent drug discovery programs.

Inhibitors against Two PDZ Domains of MDA-9 Suppressed Migration of Breast Cancer Cells.

Melanoma differentiation-associated gene 9 (MDA-9) is a small adaptor protein with tandem PDZ domains that promotes tumor progression and metastasis in various human cancers. However, it is difficult to develop drug-like small molecules with high affinity due to the narrow groove of the PDZ domains of MDA-9. Herein, we identified four novel hits targeting the PDZ1 and PDZ2 domains of MDA-9, namely PI1A, PI1B, PI2A, and PI2B, using a protein-observed nuclear magnetic resonance (NMR) fragment screening method. We also solved the crystal structure of the MDA-9 PDZ1 domain in complex with PI1B and characterized the binding poses of PDZ1-PI1A and PDZ2-PI2A, guided by transferred paramagnetic relaxation enhancement. The protein-ligand interaction modes were then cross-validated by the mutagenesis of the MDA-9 PDZ domains. Competitive fluorescence polarization experiments demonstrated that PI1A and PI2A blocked the binding of natural substrates to the PDZ1 and PDZ2 domains, respectively. Furthermore, these inhibitors exhibited low cellular toxicity, but suppressed the migration of MDA-MB-231 breast carcinoma cells, which recapitulated the phenotype of MDA-9 knockdown. Our work has paved the way for the development of potent inhibitors using structure-guided fragment ligation in the future.

Computational fragment screening of dimeric SARS-CoV-2 main protease.

During the coronavirus pandemic the lack of antiviral therapeutics drove intense interest in rapid drug discovery by the scientific, and medical communities. The Voelz lab took part in the COVID Moonshot initiative, which focused on virtually screening small drug-like fragments as leads for inhibitors of the main protease (Mpro) of SARS-CoV-2, identified using high throughput crystallographic techniques. The Voelz lab performed absolute binding free energy calculations on the [email protected] distributed computing platform to massively parallelize the molecular dynamic (MD) simulations. Crystallographic fragment-based screening techniques can be time consuming, costly, and dependent on the availability of protein crystals. Could fragment based drug discovery be performed completely in silico? To address this question, we aim to perform simulations to virtually screen the original fragment library used in the COVID Moonshot, and assess the accuracy of the predictions. The GROMACS simulation package was used to construct a simulation topology of the apo form of dimeric Mpro from its crystal structure. The correct protonation states of titratable residues were taken from published experimental results. As a first step, we prepared and performed a set of simulations of Mpro in solution, each with one of 11 drug fragments selected from the crystallographic fragment screen, all of which are known to co-crystallize with Mpro experimentally. The simulations are analyzed to determine where the drug fragments like to bind to the Mpro receptor. These results are compared results to the experimental co-crystal structures to determine how accurately MD simulations can predict the known binding pose of small drug like fragments.

Novel approaches to understand function and explore inhibition within protein-membrane interfaces.

Despite their importance in biology and disease, peripheral membrane proteins (PMPs) have presented a significant challenge. This class of proteins reversibly binds to membranes to perform function. High-resolution functional and structural studies of PMPs are often limited to their water-solubilized state due to technical limitations. Large blind spots in functional interactions within membranes are therefore common and require methodological and technological advancement to overcome. Additionally, inhibitor development for PMPs in their functionally relevant, membrane embedded state is difficult, often impossible, using current methods. We address these barriers by utilizing newly developed membrane mimicking reverse micelles (mmRMs), which house PMPs within a spherical, nanoscale assembly of lipids. Studies using mmRMs have been applied to several PMPs, including glutathione peroxidase 4 (GPx4). Use of mmRMs to investigate PMPs has proven advantageous over other membrane models for various NMR-based experimental approaches. Observations of protein-lipid interactions and lipid specificity determinations are greatly enhanced. Applying mmRMs to PMP structural analysis promises to reveal greater detail of the effects of membrane binding. Finally, mmRMs are helping to overcome challenges associated with inhibitor discovery and design for membrane embedded PMPs. mmRM-based screening for fragments in the protein-membrane interface reveals building blocks that may be advanced to inhibitors. Furthermore, discovery of small-molecule binders within membrane interfaces promises to reveal fundamental properties of this largely unexplored chemical space. Together, these approaches promise to advance our knowledge and add to the tool belt for the important and elusive peripheral membrane category of proteins.