Novel Furin Inhibitors with Potent Anti‐infectious Activity

New peptidomimetic furin inhibitors with unnatural amino acid residues in the P3 position were synthesized. The most potent compound 4‐guanidinomethyl‐phenylacteyl‐Arg‐Tle‐Arg‐4‐amidinobenzylamide (MI‐1148) inhibits furin with a K_i value of 5.5 pM . The derivatives also strongly inhibit PC1/3, whereas PC2 is less affected. Selected inhibitors were tested in cell culture for antibacterial and antiviral activity against infectious agents known to be dependent on furin activity. A significant protective effect against anthrax and diphtheria toxin was observed in the presence of the furin inhibitors. Furthermore, the spread of the highly pathogenic H5N1 and H7N1 avian influenza viruses and propagation of canine distemper virus was strongly inhibited. Inhibitor MI‐1148 was crystallized in complex with human furin. Its N‐terminal guanidinomethyl group in the para position of the P5 phenyl ring occupies the same position as that found previously for a structurally related inhibitor containing this substitution in the meta position, thereby maintaining all of the important P5 interactions. Our results confirm that the inhibition of furin is a promising strategy for a short‐term treatment of acute infectious diseases.     

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

A series of new substrate analogue inhibitors of the WNV NS2B–NS3 protease containing decarboxylated arginine mimetics at the P1 position was developed. Among the various analogues, trans‐(4‐guanidino)cyclohexylmethylamide (GCMA) was identified as the most suitable P1 residue. In combination with dichloro‐substituted phenylacetyl groups at the P4 position, three inhibitors with inhibition constants of <0.2 μM were obtained. These GCMA inhibitors have a better selectivity profile than the previously described agmatine analogues, and possess negligible affinity for the trypsin‐like serine proteases thrombin, factor Xa, and matriptase. A crystal structure in complex with the WNV protease was determined for one of the most potent inhibitors, 3,4‐dichlorophenylacetyl‐Lys‐Lys‐GCMA (K_i=0.13 μM ). The inhibitor adopts a horseshoe‐like conformation, most likely due to a hydrophobic contact between the P4 phenyl ring and the P1 cyclohexyl group, which is further stabilized by an intramolecular hydrogen bond between the P1 guanidino group and the P4 carbonyl oxygen atom. These inhibitors are stable, readily accessible, and have a noncovalent binding mode. Therefore, they may serve as suitable lead structures for further development.     

Substrate Selection Influences Molecular Recognition in a Screen for Lymphoid Tyrosine Phosphatase Inhibitors

Assay design is an important variable that influences the outcome of an inhibitor screen. Here, we have investigated the hypothesis that protein tyrosine phosphatase inhibitors with improved biological activity could be identified from a screen by using a biologically relevant peptide substrate, rather than traditional phosphotyrosine mimetic substrates. A 2000‐member library of drugs and drug‐like compounds was screened for inhibitors of lymphoid tyrosine phosphatase (LYP) by using both a peptide substrate (Ac‐ARLIEDNE‐pCAP‐TAREG‐NH₂, peptide 1) and a small‐molecule phosphotyrosine mimetic substrate (difluoromethyl umbelliferyl phosphate, DiFMUP). The results demonstrate that compounds that inhibited enzyme activity on the peptide substrate had greater biological activity than compounds that only inhibited enzyme activity on DiFMUP. Finally, epigallocatechin‐3,5‐digallate was identified as the most potent inhibitor of lymphoid tyrosine phosphatase activity to date, with an IC₅₀ of 50 nM and significant activity in T‐cells. Molecular docking simulations provided a first model for binding of this potent inhibitor to LYP; this will constitute the platform for ongoing lead optimization efforts.     

Inhibitors of Matriptase‐2 Based on the Trypsin Inhibitor SFTI‐1

A series of 17 new analogues of trypsin inhibitor SFTI‐1 were designed and synthesized to obtain matriptase‐2 inhibitors. A number of the modified bicyclic peptides displayed much higher affinity towards matriptase‐2 than towards the highly homologous matriptase‐1. Replacement of Lys5 by Arg in the wild‐type SFTI‐1 led to an 11‐fold increase in the matriptase‐2 inhibitory activity. Replacement of Arg2 by its enantiomer (D ‐arginine) slightly lowered the inhibition of matriptase‐2, but almost completely abolished the affinity towards matriptase‐1, thus yielding the most selective matriptase‐2 inhibitor. This is the first report describing inhibitors of the recently discovered matriptase‐2 based on the SFTI‐1 structure. The results showed that SFTI‐1 is a promising scaffold for the design of potent and selective inhibitors of this enzyme.     

Beyond Heparinization: Design of Highly Potent Thrombin Inhibitors Suitable for Surface Coupling

During extracorporeal circulation, when blood comes in contact with artificial surfaces, patients receive a standard treatment with anticoagulants to avoid blood coagulation. Dialysis patients in particular are systemically treated with heparin up to four times a week, causing a high burden for the body. For potential anticoagulant modification of external materials, such as dialysis equipment, a series of highly potent thrombin inhibitors was developed. All inhibitors share the general formula arylsulfonyl‐P3‐Pro‐4‐amidinobenzylamide, where P3 is glycyl or a trifunctional amino acid residue in L ‐configuration. Among this series, several derivatives inhibit thrombin with K_i values of less than 1 nM . Specificity measurements revealed that this inhibitor type is highly specific for thrombin with negligible activity against related trypsin‐like serine proteases. X‐ray analysis of the most potent analogue in complex with thrombin demonstrated that the N‐terminal arylsulfonyl group occupies the aryl binding site, whereas the P3 side chain is directed into the solvent and therefore is well suited for further coupling. Based on their in vitro profile, these inhibitors are suitable candidates for the development of hemocompatible materials with anticoagulant properties.     

Targeted Delivery of an Activatable Fluorescent Probe for the Detection of Furin Activity in Living Cells

Furin, a protein convertase, plays a crucial role in the progression of tumors. In this work, a new fluorescent probe consisting of a peptide, Arg‐Val‐Arg‐Arg (RVRR), and an aminoluciferin fluorophore was designed and prepared for the responsive and activatable detection of furin activity in vitro and in living cells. We demonstrated that this probe could be responsive toward furin with an “off–on” florescence signal and generated an approximately 3.58‐fold enhancement in the fluorescence intensity in vitro. Fluorescence imaging in MDA‐MB‐468 and LoVo cells showed that the probe could be cleaved by overexpressed furin with fluorescence turn‐on in MDA‐MB‐468 cells, and this probe was also found to be capable of discriminating between furin‐overexpressing and furin‐deficient cell lines. Our research indicates that this probe has great potential for the detection of furin activity in living cells.     

Synthesis and in vitro Evaluation of West Nile Virus Protease Inhibitors Based on the 2‐{6‐[2‐(5‐Phenyl‐4H‐{1,2,4]triazol‐3‐ylsulfanyl)acetylamino]benzothiazol‐2‐ylsulfanyl}acetamide Scaffold

In recent years, clinical symptoms resulting from West Nile virus (WNV) infection have worsened in severity, with an increased frequency in neuroinvasive diseases among the elderly. As there are presently no successful therapies against WNV for use in humans, continual efforts to develop new chemotherapeutics against this virus are highly desired. The viral NS2B‐NS3 protease is a promising target for viral inhibition due to its importance in viral replication and its unique substrate preference. In this study, a WNV NS2B‐NS3 protease inhibitor with a 2‐{6‐[2‐(5‐phenyl‐4H‐[1,2,4]triazol‐3‐ylsulfanyl)acetylamino]benzothiazol‐2‐ylsulfanyl}acetamide scaffold was identified during screening. Optimization of this initial hit by synthesis and screening of a focused compound library with this scaffold led to the identification of a novel uncompetitive inhibitor ( 1 a24 , IC₅₀=3.4±0.2 μM ) of the WNV NS2B‐NS3 protease. Molecular docking of 1 a24 into the WNV protease showed that the compound interferes with productive interactions of the NS2B cofactor with the NS3 protease and is an allosteric inhibitor of the WNV NS3 protease.     

Reactive Centre Loop Mutagenesis of SerpinB3 to Target TMPRSS2 and Furin: Inhibition of SARS-CoV-2 Cell Entry and Replication

The SARS-CoV-2 virus can utilize host cell proteases to facilitate cell entry, whereby the Spike (S) protein is cleaved at two specific sites to enable membrane fusion. Furin, transmembrane protease serine 2 (TMPRSS2), and cathepsin L (CatL) are the major proteases implicated, and are thus targets for anti-viral therapy. The human serpin (serine protease inhibitor) alpha-1 antitrypsin (A1AT) shows inhibitory activity for TMPRSS2, and has previously been found to suppress cell infection with SARS-CoV-2. Here, we have generated modified serpin inhibitors with increased specificity for these cellular proteases. Using SerpinB3 (SCCA-1), a cross-class inhibitor of CatL, as a scaffold, we have designed and produced reactive centre loop (RCL) variants to more specifically target both furin and TMPRSS2. Two further variants were generated by substituting the RCL P7–P1 with the spike protein S1/S2 cleavage site from either SARS-CoV-2 alpha or delta (P681R) sequences. Altered inhibitory specificity of purified recombinant proteins was verified in protease assays, with attenuated CatL inhibition and gain of furin or TMPRSS2 inhibition, as predicted, and modified serpins were shown to block S protein cleavage in vitro. Furthermore, the serpin variants were able to inhibit S-pseudoparticle entry into A549-ACE2-TMPRSS2 cells and suppress SARS-CoV-2 replication in Vero E6 cells expressing TMPRSS2. The construct designed to inhibit TMPRSS2 (B3-TMP) was most potent. It was more effective than A1AT for TMPRSS2 enzyme inhibition (with an eighteen-fold improvement in the second order inhibition rate constant) and for blocking SARS-CoV-2 viral replication. These findings advance the potential for serpin RCL mutagenesis to generate new inhibitors, and may lead to novel anti-viral biological molecules.     

Alzheimer’s Disease: Identification and Development of β‐Secretase (BACE‐1) Binding Fragments and Inhibitors by Dynamic Ligation Screening (DLS)

The application of dynamic ligation screening (DLS), a methodology for fragment‐based drug discovery (FBDD), to the aspartic protease β‐secretase (BACE‐1) is reported. For this purpose, three new fluorescence resonance energy transfer (FRET) substrates were designed and synthesized. Their kinetic parameters (V_max, K_M, and k_cat) were determined and compared with a commercial substrate. Secondly, a peptide aldehyde was designed as a chemically reactive inhibitor (CRI) based on the Swedish mutation substrate sequence. Incubation of this CRI with the protease, a FRET substrate, and one amine per well taken from an amine library, which was assembled by a maximum common substructure (MCS) approach, revealed the fragment 3‐(3‐aminophenyl)‐2H‐chromen‐2‐one ( 1 ) to be a competitive BACE‐1 inhibitor that enhanced the activity of the CRI. Irreversibly formed fragment combination products of 1 with the initial peptide sequence were active and confirmed the targeting of the active site through the ethane‐1,2‐diamine isostere. Finally, structure‐assisted combination of fragment 1 with secondary fragments that target the S1 site in hit optimization yielded novel, entirely fragment‐based BACE‐1 inhibitors with up to 30‐fold improved binding affinity. Interactions with the protein were explained by molecular modeling studies, which indicate that the new fragment combinations interact with the catalytic aspartic acid dyad, as well as with the adjacent binding sites required for potency.     

Design,Synthesis, and Characterization of Macrocyclic Inhibitors of the Proprotein Convertase Furin

The activation of viral glycoproteins by the host protease furin is an essential step in the replication of numerous pathogenic viruses. Thus, effective inhibitors of furin could serve as broad-spectrum antiviral drugs. A crystal structure of an inhibitory hexapeptide derivative in complex with furin served as template for the rational design of various types of new cyclic inhibitors. Most of the prepared derivatives are relatively potent furin inhibitors with inhibition constants in the low nanomolar or even sub-nanomolar range. For seven derivatives the crystal structures in complex with furin could be determined. In three complexes, electron density was found for the entire inhibitor. In the other cases the structures could be determined only for the P6/P5-P1 segments, which directly interact with furin. The cyclic derivatives together with two non-cyclic reference compounds were tested as inhibitors of the proteolytic activation and replication of respiratory syncytial virus in cells. Significant antiviral activity was found for both linear reference inhibitors, whereas a negligible efficacy was determined for the cyclic derivatives.     

Potent HIV-1 Protease Inhibitors Containing Carboxylic and Boronic Acids: Effect on Enzyme Inhibition and Antiviral Activity and Protein-Ligand X-ray Structural Studies

We report the synthesis and biological evaluation of phenylcarboxylic acid and phenylboronic acid containing HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. Inhibitors bearing bis-THF ligand as P2 ligand and phenylcarboxylic acids and carboxamide as the P2′ ligands, showed very potent HIV-1 protease inhibitory activity. However, carboxylic acid containing inhibitors showed very poor antiviral activity relative to carboxamide-derived inhibitors which showed good antiviral IC₅₀ value. Boronic acid derived inhibitor with bis-THF as the P2 ligand showed very potent enzyme inhibitory activity, but it showed lower antiviral activity than darunavir in the same assay. Boronic acid containing inhibitor with a P2-Crn-THF ligand also showed potent enzyme K_i but significantly decreased antiviral activity. We have evaluated antiviral activity against a panel of highly drug-resistant HIV-1 variants. One of the inhibitors maintained good antiviral activity against HIV_DRV^R_P20 and HIV_DRV^R_P30 viruses. We have determined high resolution X-ray structures of two synthetic inhibitors bound to HIV-1 protease and obtained molecular insight into the ligand-binding site interactions.     

E‐64c‐Hydrazide: A Lead Structure for the Development of Irreversible Cathepsin C Inhibitors

Cathepsin C is a papain‐like cysteine protease with dipeptidyl aminopeptidase activity that is thought to activate various granule‐associated serine proteases. Its exopeptidase activity is structurally explained by the so‐called exclusion domain, which blocks the active‐site cleft beyond the S2 site and, with its Asp 1 residue, provides an anchoring point for the N terminus of peptide and protein substrates. Here, the hydrazide of (2S,3S)‐trans‐epoxysuccinyl‐L ‐leucylamido‐3‐methylbutane (E‐64c) (k₂/K_i=140±5 M ^?1 s^?1) is demonstrated to be a lead structure for the development of irreversible cathepsin C inhibitors. The distal amino group of the hydrazide moiety addresses the acidic Asp 1 residue at the entrance of the S2 pocket by hydrogen bonding while also occupying the flat hydrophobic S1′–S2′ area with its leucine‐isoamylamide moiety. Furthermore, structure–activity relationship studies revealed that functionalization of this distal amino group with alkyl residues can be used to occupy the conserved hydrophobic S2 pocket. In particular, the n‐butyl derivative was identified as the most potent inhibitor of the series (k₂/K_i=56 000±1700 M ^?1 s^?1).     

Identification of Kukoamine A,Zeaxanthin, and Clexane as New Furin Inhibitors

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.     

Fluorogenic peptide-based substrates for monitoring thrombin activity

The synthesis of a series of peptides containing C‐terminal 7‐amino‐4‐methylcoumarin (AMC) for use in the thrombin generation test (TGT) is described. The lead structure in this project was H‐Gly‐Gly‐Arg‐AMC, of which the water solubility and kinetic parameters (K_M and k_cat) are greatly improved over those of the substrate in current use in the TGT: Cbz‐Gly‐Gly‐Arg‐AMC. A series of N‐terminally substituted Gly‐Gly‐Arg‐AMC derivatives were synthesized, as well as implementation of structural changes at either the P₂ or P₃ position of the peptide backbone. Furthermore, two substrates were synthesized that have structural similarities to the chromogenic thrombin substrate SQ68 or that contain a 1,2,3‐triazole moiety in the peptide chain, mimicking an amide bond. To determine the applicability of newly synthesized fluorogenic substrates for monitoring continuous thrombin generation, the K_M and k_cat values of the conversion of these fluorogenic substrates by thrombin (FIIa) and factor Xa (FXa) were quantified. An initial selection was made on basis of these data, and suitable substrates were further evaluated as substrates in the thrombin generation assay. Assessment of the acquired data showed that several substrates, including the SQ68 derivative Et‐malonate‐Gly‐Arg‐AMC and N‐functionalized Gly‐Gly‐Arg‐AMC derivatives, are suitable candidates for replacement of the substrate currently in use.     

Ala226 to Gly and Ser189 to Asp mutations convert rat chymotrypsin B to a trypsin-like protease

In a previous successful attempt to convert trypsin to a chymotrypsin-like protease, 15 residues of trypsin were replaced with the corresponding ones in chymotrypsin. This suggests a complex mechanism of substrate recognition instead of a relatively simple one that only involves three sites, residues 189, 216 and 226. However, both trypsin-->elastase and chymotrypsin-->trypsin conversion experiments carried out according to the complex model resulted in non-specific proteases with low catalytic activity. Chymotrypsin used in the latter studies was of B-type, containing an Ala residue at position 226. Trypsins, however, contain a conserved Gly at this site. The substantially decreased trypsin-like activity of the G226A trypsin mutant also suggests a specific role for this site in substrate binding. Here we investigate the role of site 226 by introducing the A226G substitution into chymotrypsin-->trypsin mutants which were constructed according to both the simple (S189D mutant) and the complex model (S(1) mutant) of specificity determination. The kinetic parameters show that the A226G substitution in the S(1) mutant increased the chymotrypsin-like activity, while the trypsin-like activity did not change. In contrast, this substitution in the S189D chymotrypsin mutant resulted in a 100-fold increase in trypsin-like activity and a trypsin-like specificity profile as tested on a competing oligopeptide substrate library. Additionally, the S189D+A226G mutant is the first trypsin-like chymotrypsin that undergoes autoactivation, an exclusive property of trypsinogen among pancreatic serine proteases.     

Synergistic Inhibitor Binding to the Papain‐Like Protease of Human SARS Coronavirus: Mechanistic and Inhibitor Design Implications

We previously developed two potent chemical classes that inhibit the essential papain‐like protease (PLpro) of severe acute respiratory syndrome coronavirus. In this study, we applied a novel approach to identify small fragments that act synergistically with these inhibitors. A fragment library was screened in combination with four previously developed lead inhibitors by fluorescence‐based enzymatic assays. Several fragment compounds synergistically enhanced the inhibitory activity of the lead inhibitors by approximately an order of magnitude. Surface plasmon resonance measurements showed that three fragments bind specifically to the PLpro enzyme. Mode of inhibition, computational solvent mapping, and molecular docking studies suggest that these fragments bind adjacent to the binding site of the lead inhibitors and further stabilize the inhibitor‐bound state. We propose potential next‐generation compounds based on a computational fragment‐merging approach. This approach provides an alternative strategy for lead optimization for cases in which direct co‐crystallization is difficult.     

Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity.     

Protease Inhibitors from Plants with Antimicrobial Activity

Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C₁₈ reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents.     

An Endoperoxide‐Based Hybrid Approach to Deliver Falcipain Inhibitors Inside Malaria Parasites

The emergence of artemisinin‐resistant Plasmodium falciparum malaria in Southeast Asia has reinforced the urgent need to discover novel chemotherapeutic strategies to treat and control malaria. To address this problem, we prepared a set of dual‐acting tetraoxane‐based hybrid molecules designed to deliver a falcipain‐2 (FP‐2) inhibitor upon activation by iron(II) in the parasite digestive vacuole. These hybrids are active in the low nanomolar range against chloroquine‐sensitive and chloroquine‐resistant P. falciparum strains. We also demonstrate that in the presence of FeBr₂ or within infected red blood cells, these molecules fragment to release falcipain inhibitors with nanomolar protease inhibitory activity. Molecular docking studies were performed to better understand the molecular interactions established between the tetraoxane‐based hybrids and the cysteine protease binding pocket residues. Our results further indicate that the intrinsic activity of the tetraoxane partner compound can be masked, suggesting that a tetraoxane‐based delivery system offers the potential to attenuate the off‐target effects of known drugs.     

Functional Investigations of Thromboxane Synthase (CYP5A1) in Lipid Bilayers of Nanodiscs

CYP5A1 is a membrane‐associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH₂) into thromboxane A₂ (TXA₂), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER‐bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug‐metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small‐molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.