Fibroblast activation protein (FAP) has been extensively studied as a cancer biomarker for decades. Recently, small-molecule FAP inhibitors have been widely adopted as a targeting moiety of experimental theranostic radiotracers. Here we present a fast qPCR-based analytical method allowing FAP inhibition screening in a high-throughput regime. To identify clinically relevant compounds that might interfere with FAP-targeted approaches, we focused on a library of FDA-approved drugs. Using the DNA-linked Inhibitor Antibody Assay (DIANA), we tested a library of 2667 compounds within just a few hours and identified numerous FDA-approved drugs as novel FAP inhibitors. Among these, prodrugs of cephalosporin antibiotics and reverse transcriptase inhibitors, along with one elastase inhibitor, were the most potent FAP inhibitors in our dataset. In addition, by employing FAP DIANA in the quantification mode, we were able to determine FAP concentrations in human plasma samples. Together, our work expands the repertoire of FAP inhibitors, analyzes the potential interference of co-administered drugs with FAP-targeting strategies, and presents a sensitive and low-consumption ELISA alternative for FAP quantification with a detection limit of 50 pg/ml.
- MeSH
- cefalosporiny chemie farmakologie MeSH
- endopeptidasy * metabolismus MeSH
- knihovny malých molekul farmakologie chemie MeSH
- lidé MeSH
- membránové proteiny * antagonisté a inhibitory metabolismus MeSH
- molekulární struktura MeSH
- rychlé screeningové testy * MeSH
- schvalování léčiv MeSH
- serinové endopeptidasy * metabolismus MeSH
- Úřad Spojených států pro potraviny a léky MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- želatinasy * antagonisté a inhibitory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Spojené státy americké MeSH
Immune checkpoint blockade (ICB) using monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) is the treatment of choice for cancer immunotherapy. However, low tissue permeability, immunogenicity, immune-related adverse effects, and high cost could be possibly improved using alternative approaches. On the other hand, synthetic low-molecular-weight (LMW) PD-1/PD-L1 blockers have failed to progress beyond in vitro studies, mostly due to low binding affinity or poor pharmacological characteristics resulting from their limited solubility and/or stability. Here, we report the development of polymer-based anti-human PD-L1 antibody mimetics (α-hPD-L1 iBodies) by attaching the macrocyclic peptide WL12 to a N-(2-hydroxypropyl)methacrylamide copolymer. We characterized the binding properties of iBodies using surface plasmon resonance, enzyme-linked immunosorbent assay, flow cytometry, confocal microscopy, and a cellular ICB model. We found that the α-hPD-L1 iBodies specifically target human PD-L1 (hPD-L1) and block the PD-1/PD-L1 interaction in vitro, comparable to the atezolizumab, durvalumab, and avelumab licensed monoclonal antibodies targeting PD-L1. Our findings suggest that iBodies can be used as experimental tools to target hPD-L1 and could serve as a platform to potentiate the therapeutic effect of hPD-L1-targeting small molecules by improving their affinity and pharmacokinetic properties.
Glutamate carboxypeptidase II (GCPII, also known as PSMA or FOLH1) is responsible for the cleavage of N-acetyl-aspartyl-glutamate (NAAG) to N-acetyl-aspartate and glutamate in the central nervous system and facilitates the intestinal absorption of folate by processing dietary folyl-poly-γ-glutamate in the small intestine. The physiological function of GCPII in other organs like kidneys is still not known. GCPII inhibitors are neuroprotective in various conditions (e.g., ischemic brain injury) in vivo; however, their utilization as potential drug candidates has not been investigated in regard to not yet known GCPII activities. To explore the GCPII role and possible side effects of GCPII inhibitors, we performed parallel metabolomic and lipidomic analysis of the cerebrospinal fluid (CSF), urine, plasma, and brain tissue of mice with varying degrees of GCPII deficiency (fully deficient in Folh1, -/-; one allele deficient in Folh1, +/-; and wild type, +/+). Multivariate analysis of metabolites showed no significant differences between wild-type and GCPII-deficient mice (except for NAAG), although changes were observed between the sex and age. NAAG levels were statistically significantly increased in the CSF, urine, and plasma of GCPII-deficient mice. However, no difference in NAAG concentrations was found in the whole brain lysate likely because GCPII, as an extracellular enzyme, can affect only extracellular and not intracellular NAAG concentrations. Regarding the lipidome, the most pronounced genotype-linked changes were found in the brain tissue. In brains of GCPII-deficient mice, we observed statistically significant enrichment in phosphatidylcholine-based lipids and reduction of sphingolipids and phosphatidylethanolamine plasmalogens. We hypothesize that the alteration of the NAA-NAAG axis by absent GCPII activity affected myelin composition. In summary, the absence of GCPII and thus similarly its inhibition do not have detrimental effects on metabolism, with just minor changes in the brain lipidome.
- MeSH
- dipeptidy metabolismus MeSH
- glutamátkarboxypeptidasa II * genetika metabolismus MeSH
- kyselina glutamová MeSH
- lipidomika * MeSH
- lipidy chemie MeSH
- metabolomika * MeSH
- mozek metabolismus MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cocaine- and amphetamine-regulated transcript peptide (CARTp) is an anorexigenic neuropeptide whose receptor is undisclosed. Previously, we reported the specific binding of CART(61-102) to pheochromocytoma PC12 cells, where CART(61-102) affinity and the number of binding sites per cell corresponded to ligand-receptor binding. Recently, Yosten et al. designated orphan GPR160 as the CARTp receptor, because the GPR160 antibody abolished neuropathic pain and anorexigenic effects induced by CART(55-102) and exogenous CART(55-102) coimmunoprecipitated with GPR160 in KATOIII cells. As no direct evidence that CARTp is a ligand for GPR160 has been described, we decided to verify this hypothesis by testing CARTp affinity to the GPR160 receptor. We investigated the GPR160 expression in PC12 cells since it is cell line known to specifically bind CARTp. Moreover, we examined the specific CARTp binding in THP1 cells, with high endogenous GPR160 expression and GPR160-transfected cell lines U2OS and U-251 MG. In PC12 cells, the GPR160 antibody did not compete for specific binding with 125I-CART(61-102) or with 125I-CART(55-102), and GPR160 mRNA expression and GPR160 immunoreactivity were not detected. Moreover, THP1 cells did not show any 125I-CART(61-102) or 125I-CART(55-102) specific binding despite GPR160 detection by fluorescent immunocytochemistry (ICC). Finally, no 125I-CART(61-102) or 125I-CART(55-102) specific binding in the GPR160-transfected cell lines U2OS and U-251 MG, selected due to their negligible endogenous expression of GPR160, was detected, despite the detection of GPR160 by fluorescent ICC. Our binding studies clearly demonstrated that GPR160 cannot be a receptor for CARTp. Further studies are needed to identify true CARTp receptors.
- MeSH
- kokain * MeSH
- krysa rodu rattus MeSH
- ligandy MeSH
- proteiny nervové tkáně * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
Peptide display methods are a powerful tool for discovering new ligands of pharmacologically relevant targets. However, the selected ligands often suffer from low affinity. Using phage display, we identified a new bicyclic peptide binder of prostate-specific membrane antigen (PSMA), a metalloprotease frequently overexpressed in prostate cancer. We show that linking multiple copies of a selected low-affinity peptide to a biocompatible water-soluble N-(2-hydroxypropyl)methacrylamide copolymer carrier (iBody) improved binding of the conjugate by several orders of magnitude. Furthermore, using ELISA, enzyme kinetics, confocal microscopy, and other approaches, we demonstrate that the resulting iBody can distinguish between different conformations of the target protein. The possibility to develop stable, fully synthetic, conformation-selective antibody mimetics has potential applications for molecular recognition, diagnosis and treatment of many pathologies. This strategy could significantly contribute to more effective drug discovery and design.
- MeSH
- biomimetické materiály chemie MeSH
- kalikreiny chemie MeSH
- lidé MeSH
- nosiče léků chemie MeSH
- peptidová knihovna * MeSH
- prostatický specifický antigen chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Peptidomimetic inhibitors of fibroblast activation protein (FAP) are regarded as promising tools for tumor targeting in vivo. Even though several peptidomimetic compounds with nanomolar potency have been described, broad chemical space for further modification remained unexplored. Therefore, we set to analyze the structure-activity relationship (SAR) of pseudopeptide compound series with α-ketoamide warheads in order to explore the contributions of the P1' and P2' moieties to the inhibitory potency. A series of novel inhibitors bearing varied P1' and/or P2' moieties was synthesized by combining a Passerini reaction-Amine Deprotection-Acyl Migration (PADAM) approach with peptide coupling and subsequent oxidation. The resulting compounds inhibited FAP and the related prolyl endopeptidase (PREP) with potencies in the nanomolar to sub-nanomolar range. The most potent FAP inhibitor IOCB22-AP446 (6d, IC50 = 89 pM) had about 36-fold higher inhibition potency than the most potent inhibitor published to date. The compounds were selective over FAP's closest homolog DPP-IV, were stable in human and mouse plasma and in mouse microsomes, and displayed minimal cytotoxicity in tissue cultures.
- MeSH
- fibroblasty metabolismus MeSH
- lidé MeSH
- molekulární struktura MeSH
- myši MeSH
- prolyloligopeptidasy metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.
- MeSH
- antifungální látky chemie metabolismus farmakologie MeSH
- aspartátové proteasy antagonisté a inhibitory genetika metabolismus MeSH
- Cryptococcus neoformans enzymologie MeSH
- fungální proteiny antagonisté a inhibitory genetika metabolismus MeSH
- HIV-proteasa chemie metabolismus MeSH
- HIV enzymologie MeSH
- houby účinky léků MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- preklinické hodnocení léčiv MeSH
- rekombinantní proteiny biosyntéza chemie izolace a purifikace MeSH
- simulace molekulární dynamiky MeSH
- substrátová specifita MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Publikační typ
- abstrakt z konference MeSH
