Polypeptide Inhibitor Research Articles

Identification of a Polypeptide Inhibitor of O-GlcNAc Transferase with Picomolar Affinity.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that binds thousands of different proteins, including substrates that it glycosylates and nonsubstrate interactors that regulate its biology. OGT also has one proteolytic substrate, the transcriptional coregulator host cell factor 1 (HCF-1), which it cleaves in a process initiated by glutamate side chain glycosylation at a series of central repeats. Although HCF-1 is OGT's most prominent binding partner, its affinity for the enzyme has not been quantified. Here, we report a time-resolved Förster resonance energy transfer assay to measure ligand binding to OGT and show that an HCF-1-derived polypeptide (HCF3R) binds with picomolar affinity to the enzyme (KD ≤ 85 pM). This high affinity is driven in large part by conserved asparagines in OGT's tetratricopeptide repeat domain, which form bidentate contacts to the HCF-1 peptide backbone; replacing any one of these asparagines with alanine reduces binding by more than 5 orders of magnitude. Because the HCF-1 polypeptide binds so tightly to OGT, we tested its ability to inhibit enzymatic function. We found that HCF3R potently inhibits OGT both in vitro and in cells and used this finding to develop a genetically encoded, inducible OGT inhibitor that can be degraded with a small molecule, allowing for reversible and tunable inhibition of OGT.

Abstract 5776: Target identification, selectivity profiling and binding site mapping of small molecule and peptide drugs by LiP-MS

Abstract Target identification in the cellular context is a critical step for both target-based and phenotypic drug discovery. Limited proteolysis coupled with mass spectrometry (LiP-MS) has emerged as a powerful technique for target deconvolution of small molecules or peptides in cell lysate without compound modification or genetic manipulation of cell lines [1,2,3,4]. Utilizing a non-specific protease under well-controlled conditions, LiP-MS exploits drug-induced structural alteration or steric hindrance on protein targets and utilizes quantitative mass spectrometry to probe over 250’000 peptides covering more than 9000 proteins in the proteome. To aid target ID, this workflow incorporates a 7-concentration dose response experiment and a machine-learning framework [1] to compute a LiP-score to rank identified target proteins, as well as predict potential binding site. In this study, we present target deconvolution results on four small molecule compounds and one peptide compound with very distinct pharmacological profile: 1) staurosporine, a broad-specific kinase inhibitor; 2) roniciclib, a failed clinical-stage pan-CDK inhibitor; 3) selumetinib, an allosteric, non-ATP-competitive MEK 1 and 2 inhibitor; 4) NST-628, a newly developed non-degradating molecular glue inhibiting the RAS/MAPK pathway; and 5) Acetyl-calpastatin, a 24 amino-acid polypeptide inhibitor of calpain 1 and 2. Target ID experiment with staurosporine and roniciclib yielded > 150 and 68 kinase targets were identified respectively. For the pan-kinase inhibitor roniciclib, both cell-cycle CDKs (CDK1/cyclin B, CDK2/cyclin E, CDK4 cyclin D) and transcriptional CDK9 were among the targets. For Selumetinib, LiP-score ranking identified MEK1 and MEK2 as the top two targets, and only kinases among a total of 23 targets. Furthermore, mapping the LiP-peptides from this experiment onto MEK1 showed that they are in close proximity to the selumetinib allosteric binding site on co-crystal structure. Last but not least, we assessed the target landscape of NST-628, a novel non-degrading molecular glue and identified MEK1 and MEK2 clearly as the primary targets. This finding is in line with orthogonal data obtained from X-ray crystallography [5]. Collectively, this data demonstrates that LiP-MS can be deployed to effectively identify protein drug targets and predict binding sites in complex cellular millieu independent of the compound’s mechanism of action and without compound modification or labeling. These capabilities make LiP-MS a powerful addition to the target deconvolution toolbox. [1] Piazza and Beaton et al. Nature Comm 2020 [2] Hendricks and Beaton et al. ACS Chem Bio 2021 [3] AACR 2022 [4] AACR 2023 [5] AACR-NCI-EORTC 2023 Citation Format: Fabio Sabino, Martin Soste, Dominique Kamber, Maria Stella Ritorto, Klaus Hoeflich, Michael Hale, Bradley Quade, Xin Huang, Nigel Beaton, Roland Bruderer, Yuehan Feng, Lukas Reiter. Target identification, selectivity profiling and binding site mapping of small molecule and peptide drugs by LiP-MS [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 5776.

Inhibition of GSDMD activation by Z-LLSD-FMK or Z-YVAD-FMK reduces vascular inflammation and atherosclerotic lesion development in ApoE-/- mice.

Pyroptosis is a form of pro-inflammatory cell death that can be mediated by gasdermin D (GSDMD) activation induced by inflammatory caspases such as caspase-1. Emerging evidence suggests that targeting GSDMD activation or pyroptosis may facilitate the reduction of vascular inflammation and atherosclerotic lesion development. The current study investigated the therapeutic effects of inhibition of GSDMD activation by the novel GSDMD inhibitor N-Benzyloxycarbonyl-Leu-Leu-Ser-Asp(OMe)-fluoromethylketone (Z-LLSD-FMK), the specific caspase-1 inhibitor N-Benzyloxycarbonyl-Tyr-Val-Ala-Asp(OMe)-fluoromethylketone (Z-YVAD-FMK), and a combination of both on atherosclerosis in ApoE-/- mice fed a western diet at 5weeks of age, and further determined the efficacy of these polypeptide inhibitors in bone marrow-derived macrophages (BMDMs). In vivo studies there was plaque formation, GSDMD activation, and caspase-1 activation in aortas, which increased gradually from 6 to 18weeks of age, and increased markedly at 14 and 18weeks of age. ApoE-/- mice were administered Z-LLSD-FMK (200µg/day), Z-YVAD-FMK (200µg/day), a combination of both, or vehicle control intraperitoneally from 14 to 18weeks of age. Treatment significantly reduced lesion formation, macrophage infiltration in lesions, protein levels of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and pyroptosis-related proteins such as activated caspase-1, activated GSDMD, cleaved interleukin(IL)-1β, and high mobility group box 1 in aortas. No overt differences in plasma lipid contents were detected. In vitro treatment with these polypeptide inhibitors dramatically decreased the percentage of propidium iodide-positive BMDMs, the release of lactate dehydrogenase and IL-1β, and protein levels of pyroptosis-related proteins both in supernatants and cell lysates elevated by lipopolysaccharide + nigericin. Notably however, there were no significant differences in the above-mentioned results between the Z-LLSD-FMK group and the Z-YVAD-FMK group, and the combination of both did not yield enhanced effects. These findings indicate that suppression of GSDMD activation by Z-LLSD-FMK or Z-YVAD-FMK reduces vascular inflammation and lesion development in ApoE-/- mice.

Sequential delivery of PD-1/PD-L1 blockade peptide and IDO inhibitor for immunosuppressive microenvironment remodeling via an MMP-2 responsive dual-targeting liposome.

Intelligent responsive drug delivery system opens up new avenues for realizing safer and more effective combination immunotherapy. Herein, a kind of tumor cascade-targeted responsive liposome (NLG919@Lip-pep1) is developed by conjugating polypeptide inhibitor of PD-1 signal pathway (AUNP-12), which is also a targeted peptide that conjugated with liposome carrier through matrix metalloproteinase-2 (MMP-2) cleavable peptide (GPLGVRGD). This targeted liposome is prepared through a mature preparation process, and indoleamine-2,3-dioxygenase (IDO) inhibitor NLG919 was encapsulated into it. Moreover, mediated by the enhanced permeability and retention effect (EPR effect) and AUNP-12, NLG919@Lip-pep1 first targets the cells that highly express PD-L1 in tumor tissues. At the same time, the over-expressed MMP-2 in the tumor site triggers the dissociation of AUNP-12, thus realizing the precise block of PD-1 signal pathway, and restoring the activity of T cells. The exposure of secondary targeting module II VRGDC-NLG919@Lip mediated tumor cells targeting, and further relieved the immunosuppressive microenvironment. Overall, this study offers a potentially appealing paradigm of a high efficiency, low toxicity, and simple intelligent responsive drug delivery system for targeted drug delivery in breast cancer, which can effectively rescue and activate the body's anti-tumor immune response and furthermore achieve effective treatment of metastatic breast cancer.

In silico Analysis and Characterization of a Putative Aspartic Proteinase Inhibitor, IA3 from Lachancea kluyveri

Aspartic proteinases play important role in various physiological and biological processes. Understanding catalytic mechanisms of these enzymes could lead to crucial medical and biological applications. Two types of aspartic proteinase inhibitors have been identified i.e. small molecule inhibitor and naturally occurring peptides. Most of aspartic proteinases are highly susceptible to inhibition by a series of small, non-proteinaceous natural products; pepstatin. However, only limited number of naturally-occurring polypeptide inhibitors of aspartic proteinases has so far been discovered. One among these inhibitors is Saccharomyces cerevisiae IA3. The S. cerevisiae Proteinase A (ScPrA) is solely and potently inhibited by this small polypeptide at sub-nanomolar level. It was proven that, not only IA3 has no detectable effect against a wide range of aspartic proteinases, but it was also shown to be cleaved as a substrate of these non-target enzymes. Bioinformatics analysis of the IA3 structure has been undertaken in this study. Database searching for sequence homology from available fungal genome data bank using the Basic Local Alignment Search Tool (BLAST) revealed that 4 structurally related, IA3-like proteins have successfully been identified. The amino acid sequence of IA3-like proteins from Lachancea kluyveri share highest degree of similarity toward wild-type IA3. The Lk-IA3-like gene was synthesized using a PCR-based gene synthesis method. Protein expression in E. coli, protein purification and characterization of Lk-IA3-like by enzyme kinetic assays were performed. The results indicated that Lk-IA3-like protein inhibits ScPrA at the Ki value of 190 ± 0.11 nM and possesses no inhibitory effect toward AfPrA (Aspergillus fumigatus proteinase A) or HuCatD (Human cathepsin D). HIGHLIGHTS Aspartic proteinases enzyme family is crucially involved in various physiological and biological processes including in the pathogenesis of numerous diseases which make them a possible target for drug design Inhibitors of aspartic proteinases are not only applied for human disease treatment but also extended to plant and other economically important animals Aspartic proteinase from cerevisiae (Proteinase A) is solely and potently inhibited by a small peptideinhibitor, IA3 Bioinformatics analysis of the naturally occurring aspartic proteinases inhibitor, IA3 structure has been undertaken in this research and putative IA3-like proteins have successfully been identified IA3-like proteins from Lachancea kluyveri were produced in coli and enzyme inhibition assays revealed that Lk-IA3-like protein inhibits ScPrA at the Ki value of 190 ± 0.11 nM GRAPHICAL ABSTRACT

Taurochotine Drug Design as Sodium Taurocholate Co-Transporting Polypeptide (NTCP) Inhibitor for HBV Treatment, in Silico Approach

Background: Hepatitis B Virus (HBV) is a viral infection that attacks the liver and can cause both acute and chronic disease. The virus is most commonly transmitted from mother to child during birth and delivery. Also, by contact with patient blood or other fluids during sex with an infected partner. The virus enters the cell body by sodium taurocholate cotransporting polypeptide receptor (NTCP), then it replicate and invade other healthy cells. Aim: the aim of this study is to design a drug that can block sodium taurocholate cotransporting polypeptide receptor (NTCP), Using bioinformatics tools and servers. Material and method: Using CHemSketch the drug molecule was drawn and determined and using OpenBabel software the molecule was transferred and designed into MOL 2 format to be applied in the SwissDock server. The target protein from the protein database bank using the PDB ID of NTCP receptor protein was retrieved and then inserted it into SwissDock. The result of docking was then subjected to SwissADME to check the pharmacological effect. Results: A novel drug that can inhibit and block sodium taurocholate cotransporting polypeptide receptor (NTCP) hence can treat the HBV. Conclusion: With these predicted pharmacokinetics and chemical properties of NTCP inhibitor, a new emerging drug to treat hepatitis B virus disease, which affects the majority of people around the world, especially in poor countries, can be developed, and this can be achieved with more research and laboratory procedures.

Hepatitis D virus (HDV) prevalence in Austria is low but causes considerable morbidity due to fast progression to cirrhosis.

BackgroundHepatitis D virus (HDV) coinfection aggravates the course of hepatitis B virus (HBV). The prevalence of HDV in Austria is unknown.ObjectiveThis national study aimed at (i) recording the prevalence of HDV‐infection in Austria and (ii) characterizing the “active” HDV cohort in Austria.MethodsA total of 10 hepatitis treatment centers in Austria participated in this multicenter study and retrospectively collected their HDV patients between Q1/2010 and Q4/2020. Positive anti‐HDV and/or HDV‐RNA‐polymerase chain reaction (PCR) results were retrieved from local database queries. Disease severity was assessed by individual chart review. Viremic HDV patients with clinical visits in/after Q1/2019 were considered as the “active” HDV cohort.ResultsA total of 347 anti‐HDV positive patients were identified. In 202 (58.2%) patients, HDV‐RNA‐PCR test was performed, and 126/202 (62.4%) had confirmed viremia. Hepatocellular carcinoma was diagnosed in 7 (5.6%) patients, 7 (5.6%) patients underwent liver transplantation, and 11 (8.7%) patients died during follow‐up. The “active” Austrian HDV cohort included 74 (58.7%) patients: Evidence for advanced chronic liver disease (ACLD, i.e., histological F3/F4 fibrosis, liver stiffness ≥10 kPa, varices, or hepatic venous pressure gradient ≥6 mmHg) was detected in 38 (51.4%) patients, including 2 (5.3%) with decompensation (ascites/hepatic encephalopathy). About 37 (50.0%) patients of the “active” HDV cohort had previously received interferon treatment. Treatment with the sodium‐taurocholate cotransporting polypeptide inhibitor bulevirtide was initiated in 20 (27.0%) patients.ConclusionThe number of confirmed HDV viremic cases in Austria is low (<1% of HBV patients) but potentially underestimated. Testing all HBV patients will increase the diagnostic yield. More than half of viremic HDV patients had ACLD. Improved HDV testing and workup strategies will facilitate access to novel antiviral therapies.

Modulation of the transport of valproic acid through the blood-brain barrier in rats by the Gastrodia elata extracts

Headings ethnopharmacological relevanceValproic acid (VPA) is primarily used as a medicine for the treatment of seizures. Gastrodia elata (G. elata) extract has been used as an alternative medicine for epilepsy patients. Cotreatment with VPA and G. elata extract is commonly prescribed in Taiwan and mainland China. Nevertheless, the mechanism of the blood-brain barrier (BBB) transportation effect of G. elata extract on VPA has not been characterized. Aim of studyOur hypothesis is that G. elata extract modulates the BBB penetration of VPA through specific transporter transfer. Materials and methodsA validated liquid chromatography-tandem mass spectrometry and multiple microdialysis method was developed to simultaneously monitor VPA in the blood and brain of rats. To investigate the mechanism of BBB modulation by the G. elata extract on VPA in the brain, cyclosporin A, a P-glycoprotein (P-gp) inhibitor and organic anion transporting polypeptide (OATP) inhibitor, was coadministered with the G. elata extract and VPA. ResultsThe pharmacokinetic results demonstrated that the VPA penetration ratio of the BBB, determined by the area under the concentration curve (AUC) ratio of VPA (AUCbrain/AUCblood), was approximately 0.36. After treatment with the G. elata extract (1 and 3 g/kg, p.o. for 5 consecutive days), the VPA penetration ratios were significantly enhanced to 1.47 and 1.02, respectively. However, in the experimental group coadministered cyclosporin A, the G. elata extract was unable to enhance the BBB transportation of VPA. Instead, the VPA penetration ratio in the brain was suppressed back to 0.38. ConclusionsThe present study reveals that the enhancement effect of the transporter mechanism of G. elata extract on VPA transport into the brain occurs through the OATP transporter but not the P-gp transporter.

Design of Dimeric Bile Acid Derivatives as Potent and Selective Human NTCP Inhibitors.

Dimeric bile acid derivatives (DBADs) were developed and tested for their anti-HBV and anti-HDV activities as sodium taurocholate cotransporting polypeptide (NTCP) inhibitors. DBADs exhibited strong and persistent potency of NTCP inhibition, whereas diverse linkers and constitutions showed distinct inhibition features. Motif aa157-165 on NTCP was shown to be a possible binding site of DBADs; therefore, we determined DBADs' selectivity among NTCPs from different species. A cyclized DBAD scaffold DBA-41 exhibited a high affinity to human NTCP (hNTCP). Intraperitoneal administration of DBA-41 to hNTCP-tg mice induced serum total bile acid elevation. DBA-41 may serve as a biological tool to study NTCP physiological function.

Prediction of Transporter-Mediated Drug-Drug Interactions and Phenotyping of Hepatobiliary Transporters Involved in the Clearance of E7766, a Novel Macrocycle-Bridged Dinucleotide.

E7766 represents a novel class of macrocycle-bridged dinucleotides and is under clinical development for immuno-oncology. In this report, we identified mechanism of systemic clearance E7766 and investigated the hepatobiliary transporters involved in the disposition of E7766 and potential drug interactions of E7766 as a victim of organic anion-transporting polypeptide (OATP) inhibitors. In bile-duct cannulated rats and dogs, E7766 was mainly excreted unchanged in bile (>80%) and to a lesser extent in urine (<20%). Sandwich-cultured human hepatocytes (SCHHs), transfected cells, and vesicles were used to phenotype the hepatobiliary transporters involved in the clearance of E7766. SCHH data showed temperature-dependent uptake of E7766 followed by active biliary secretion. In vitro transport assays using transfected cells and membrane vesicles confirmed that E7766 was a substrate of OATP1B1, OATP1B3, and multidrug resistance-associated protein 2. Phenotyping studies suggested predominant contribution of OATP1B3 over OATP1B1 in the hepatic uptake of E7766. Studies in OATP1B1/1B3 humanized mice showed that plasma exposure of E7766 increased 4.5-fold when coadministered with Rifampicin. Physiologically based pharmacokinetic models built upon two independent bottom-up approaches predicted elevation of E7766 plasma exposure when administered with Rifampicin, a clinical OATP inhibitor. In conclusion, we demonstrate that OATP-mediated hepatic uptake is the major contributor to the clearance of E7766, and inhibition of OATP1B may increase its systemic exposure. Predominant contribution of OATP1B3 in the hepatic uptake of E7766 was observed, suggesting polymorphisms in OATP1B1 would be unlikely to cause variability in the exposure of E7766. SIGNIFICANCE STATEMENT: Understanding the clearance mechanisms of new chemical entities is critical to predicting human pharmacokinetics and drug interactions. A physiologically based pharmacokinetic model that incorporated parameters from mechanistic in vitro and in vivo experiments was used to predict pharmacokinetics and drug interactions of E7766, a novel dinucleotide drug. The findings highlighted here may shed a light on the pharmacokinetic profile and transporter-mediated drug interaction propensity of other dinucleotide drugs.

Discovery of a novel sodium taurocholate cotransporting polypeptide (NTCP) inhibitor: Design, synthesis, and anti-proliferative activities

Sodium taurocholate cotransporting polypeptide (NTCP) is identified as the functional receptor for HBV entry, which is responsible for upregulated HBV transcription in the HBV life cycle. Besides, NTCP is also implicated in the progression of HBV-induced hepatocellular carcinoma (HCC). Thereby, NTCP-targeting entry inhibitors are proposed to suppress HBV infection and replication in HBV-induced hepatoma therapy. Herein, we integrated in silico screening and chemical synthesis to obtain a small-molecule NTCP inhibitor B7, which exhibited moderate anti-proliferative activities against HepG2 cells and anti-HBV activity in vitro. Additionally, CETSA assay, molecular docking, and MD simulation validated that B7 could bind to NTCP. Furthermore, western blot analysis demonstrated that B7 induced apoptosis with an increased expression of Bax and caspase 3 cleaving as well as a decreasing expression of Bcl-2 in HepG2 cells. Taken together, our study identified B7 as a novel NTCP inhibitor with anti-proliferation activities which might provide a new opportunity for HCC therapy.

Исследование in silico структуры альфа-амилазы колорадского жука и ее взаимодействия с ингибиторами

To develop effective and environmentally friendly methods for protecting crop plants from pests, it is necessary to know the structure of pests hydrolases and the mechanisms of their interaction with plant inhibitors. The aim of this work was a modeling of the spatial structure of Colorado potato beetle alpha-amylase, analysis of the effect of revealed structural features on interaction with protein inhibitors, as well as modeling of interaction with amylase inhibitors from various organisms.The spatial structure of the Colorado potato beetle alpha-amylase was obtained by the method of computer modeling using the IntFOLD and NOMAD-Ref services. Colorado potato beetle amylase differs in the structure and physicochemical properties of the active site in comparison with flour beetle amylase, but has the same conformation of the main chain. The possible effect of these differences on the interaction of the enzyme with plant inhibitors – a lectin-like bean inhibitor and a RATI inhibitor – was shown. The CABS-dock service was used to model the interaction of Colorado potato beetle amylase with polypeptide inhibitors of amylase from various plant species, bacteria, and actinomycetes. Among the structures obtained, the best values of complex free energy were possessed by the knottin-like inhibitor of amaranth and the purothionin-like inhibitor of oats; the least favorable is the binding with the inhibitors of the actinobacterium Thermopolyspora flexuosa and the aquatic plant Alternanthera sessilis. The model structure of a knottin-like inhibitor in a complex with Colorado potato beetle amylase, in comparison with the known structure of a complex with flour beetle amylase, has a similar chain folding, but significantly different conformation. The data obtained can be used to search for new effective pests’ amylase inhibitors and environmentally friendly methods of protecting potato plants from insect pests, including using the genetic transformation of plants.

Application of the Movable Type Free Energy Method to the Caspase-Inhibitor BindingAffinity Study.

Many studies have provided evidence suggesting that caspases not only contribute to the neurodegeneration associated with Alzheimer’s disease (AD) but also play essential roles in promoting the underlying pathology of this disease. Studies regarding the caspase inhibition draw researchers’ attention through time due to its therapeutic value in the treatment of AD. In this work, we apply the “Movable Type” (MT) free energy method, a Monte Carlo sampling method extrapolating the binding free energy by simulating the partition functions for both free-state and bound-state protein and ligand configurations, to the caspase-inhibitor binding affinity study. Two test benchmarks are introduced to examine the robustness and sensitivity of the MT method concerning the caspase inhibition complexing. The first benchmark employs a large-scale test set including more than a hundred active inhibitors binding to caspase-3. The second benchmark includes several smaller test sets studying the relative binding free energy differences for minor structural changes at the caspase-inhibitor interaction interfaces. Calculation results show that the RMS errors for all test sets are below 1.5 kcal/mol compared to the experimental binding affinity values, demonstrating good performance in simulating the caspase-inhibitor complexing. For better understanding the protein-ligand interaction mechanism, we then take a closer look at the global minimum binding modes and free-state ligand conformations to study two pairs of caspase-inhibitor complexes with (1) different caspase targets binding to the same inhibitor, and (2) different polypeptide inhibitors targeting the same caspase target. By comparing the contact maps at the binding site of different complexes, we revealed how small structural changes affect the caspase-inhibitor interaction energies. Overall, this work provides a new free energy approach for studying the caspase inhibition, with structural insight revealed for both free-state and bound-state molecular configurations.

Prediction of pharmacokinetic drug-drug interactions causing atorvastatin-induced rhabdomyolysis using physiologically based pharmacokinetic modelling

Atorvastatin and its lactone form metabolite are reported to be associated with statin-induced myopathy (SIM) such as myalgia and life-threatening rhabdomyolysis. Though the statin-induced rhabdomyolysis is not common during statin therapy, its incidence will significantly increase due to pharmacokinetic drug-drug interactions (DDIs) with inhibitor drugs which inhibit atorvastatin’s and its lactone’s metabolism and hepatic uptake. Thus, the quantitative analysis of DDIs of atorvastatin and its lactone with cytochrome P450 3A4 (CYP3A4) and organic anion-transporting polypeptide (OATP) inhibitors is of great importance. This study aimed to predict pharmacokinetic DDIs possibly causing atorvastatin-induced rhabdomyolysis using Physiologically Based Pharmacokinetic (PBPK) Modelling. Firstly, we refined the PBPK models of atorvastatin and atorvastatin lactone for predicting the DDIs with CYP3A4 and OATP inhibitors. Thereafter, we predicted the exposure changes of atorvastatin and atorvastatin lactone originating from the case reports of atorvastatin-induced rhabdomyolysis using the refined models. The simulation results show that pharmacokinetic DDIs of atorvastatin and its lactone with fluconazole, palbociclib diltiazem and cyclosporine are significant. Consequently, clinicians should be aware of necessary dose adjustment of atorvastatin being used with these four inhibitor drugs.

PET probes of small molecule polypeptide immunosuppressive agents guiding tumor immunotherapy

Immunosuppressant targeting programmed death protein-1 and its ligand (PD-1/PD-L1) is a hot topic in solid tumor immunotherapy. Detection of PD-1/PD-L1 expression in solid tumor patients by molecular imaging can predict whether tumor patients can benefit from immunotherapy. Small molecular polypeptide inhibitors have the advantages of low molecular weight, fast diffusion in tumor microenvironment, uniform distribution and easy access to the deep part of solid tumor. Non-invasive, real-time and quantitative detection of PD-1/PD-L1 expression in tumor patients can be performed by radionuclide labeled small molecular polypeptide inhibitor molecular probe PET imaging. It is expected to provide new detection methods for patient screening, efficacy monitoring, treatment plan optimization and prognosis evaluation. Key words: Immunosuppressive agents; Positron-emission tomography; Immunotherapy

Towards Improved Pharmacokinetic Models for the Analysis of Transporter-Mediated Hepatic Disposition of Drug Molecules with Positron Emission Tomography

Positron emission tomography (PET) imaging with radiolabeled drugs holds great promise to assess the influence of membrane transporters on hepatobiliary clearance of drugs. To exploit the full potential of PET, quantitative pharmacokinetic models are required. In this study, we evaluated the suitability of different compartment models to describe the hepatic disposition of [11C]erlotinib as a small-molecule model drug which undergoes transporter-mediated hepatobiliary excretion. We analyzed two different, previously published data sets in healthy volunteers, in which a baseline [11C]erlotinib PET scan was followed by a second PET scan either after oral intake of unlabeled erlotinib (300 mg) or after intravenous infusion of the prototypical organic anion-transporting polypeptide inhibitor rifampicin (600 mg). We assessed a three-compartment (3C) and a four-compartment (4C) model, in which either a sampled arterial blood input function or a mathematically derived dual input function (DIF), which takes the contribution of the portal vein to the liver blood supply into account, was used. Both models provided acceptable fits of the observed PET data in the liver and extrahepatic bile duct and gall bladder. Changes in model outcome parameters between scans were consistent with the involvement of basolateral hepatocyte uptake and canalicular efflux transporters in the hepatobiliary clearance of [11C]erlotinib. Our results demonstrated that inclusion of a DIF did not lead to substantial improvements in model fits. The models developed in this work represent a step forward in applying PET as a tool to assess the impact of hepatic transporters on drug disposition and their involvement in drug-drug interactions.

Modeling Gadoxetate Liver Uptake and Efflux Using Dynamic Contrast-Enhanced Magnetic Resonance Imaging Enables Preclinical Quantification of Transporter Drug-Drug Interactions.

The aim of this study was to model the in vivo transporter-mediated uptake and efflux of the hepatobiliary contrast agent gadoxetate in the liver. The efficacy of the proposed technique was assessed for its ability to provide quantitative insights into drug-drug interactions (DDIs), using rifampicin as inhibitor. Three groups of C57 mice were scanned twice with a dynamic gadoxetate-enhanced magnetic resonance imaging protocol, using a 3-dimensional spoiled gradient-echo sequence for approximately 72 minutes. Before the second magnetic resonance imaging session, 2 of the groups received a rifampicin dose of 20 (n = 7) or 40 (n = 7) mg/kg, respectively. Data from regions of interest in the liver were analyzed using 2 simplifications of a 2-compartment uptake and efflux model to provide estimates for the gadoxetate uptake rate (ki) into the hepatocytes and its efflux rate (kef) into the bile. Both models were assessed for goodness-of-fit in the group without rifampicin (n = 9), and the appropriate model was selected for assessing the ability to monitor DDIs in vivo. Seven of 9 mice from the group without rifampicin were assessed for model implementation and reproducibility. A simple 3 parameter model (ki, kef, and extracellular space, vecs) adequately described the observed liver concentration time series with mean ki = 0.47 ± 0.11 min and mean kef = 0.039 ± 0.016 min. Visually, the area under the liver concentration time profile was reduced for the groups receiving rifampicin. Furthermore, tracer kinetic modeling demonstrated a significant dose-dependent decrease in the uptake (5.9- and 17.3-fold decrease for 20 mg/kg and 40 mg/kg, respectively) and efflux rates (2.2- and 7.9-fold decrease) compared with the first scan for each group. This study presents the first in vivo implementation of a 2-compartment uptake and efflux model to monitor DDIs at the transporter-protein level, using the clinically relevant organic anion transporting polypeptide inhibitor rifampicin. The technique has the potential to be a novel alternative to other methods, allowing real-time changes in transporter DDIs to be measured directly in vivo.

A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering

Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry-based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPIM17L,I18F,S19F,F34V (APPI-4M), an APPI variant with a KLK6 inhibition constant (Ki ) of 160 pm and a turnover time of 10 days. To the best of our knowledge, APPI-4M is the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPIWT). We further demonstrate that APPI-4M acts as a functional inhibitor in a cell-based model of KLK6-dependent breast cancer invasion. Finally, the crystal structures of the APPIWT/KLK6 and APPI-4M/KLK6 complexes revealed the structural and mechanistic bases for the improved KLK6 binding and proteolytic resistance of APPI-4M. We anticipate that APPI-4M will have substantial translational potential as both imaging agent and therapeutic.

In vitro evaluation of the hepatic disposition of colistin

ObjectiveColistin (polymyxin E) is a polycation antibiotic which is increasingly used (administered as Colistin Methane Sulfonate ‐ CMS) as a salvage therapy in (critically ill) patients with multidrug resistant gram‐negative infection[1]. Because colistin was introduced into clinical practice over 50 years ago, detailed trials on efficacy, safety and pharmacokinetics including underlying mechanisms are lacking. For instance, the metabolic fate of colistin is still largely unknown. Colistin is handled by glomerular filtration in the kidney, but almost completely reabsorbed in the renal tubuli. Less than 1% is eventually cleared by the kidney[2]. The purpose of the present study was to characterize the hepatic disposition of colistin and the role of transporters.MethodsTo characterize the metabolism of colistin, initial incubations were performed using colistin sulphate with rat liver microsomes (up to 1 mg protein/ml). Subsequently, rat and human hepatocytes in suspension (8 million/ml) were used as more complete in vitro drug metabolism tools. To measure hepatic uptake rates of colistin, the oil‐spin method was also applied to suspended hepatocytes (1 million cells/ml). Organic Anion Transporting Polypeptide (OATP) inhibitors (atazanavir and rifampicin) and Multidrug Resistance‐associated Protein (MRP) inhibitors (MK571 and benzbromarone) were used to evaluate the role of these uptake and efflux transporters in the disposition of colistin in rat and human hepatocytes. All samples were analysed by a validated LC‐MSMS method.ResultsColistin, when incubated at 2 mg/L, which is the PKPD target correlating with a high probability of clinical efficacy, was stable up to 4 hr during the incubation with rat liver microsomes. However, a slow time‐dependent decrease in colistin concentration (C0= 2 μg/mL) was found in suspended rat and human hepatocytes, with half‐life values of 9.9 h and 18 h, respectively. Based on these in vitro metabolism data, the predicted human in vivo blood clearance, assuming the well‐stirred model (fu=0.5), was 0.10 ml/min.kg body weight, explaining only a minor part of the total blood clearance based on clinical data [0.67 ml/min.kg body weight]. Both a saturable (transporter‐mediated) and non‐saturable (passive diffusion) component were found to contribute to colistin uptake by hepatocytes. When incubated at the clinically relevant concentration, the intracellular accumulation of colistin in rat and human hepatocytes was 1.9 and 6.2‐fold higher, respectively, in the presence of the MRP inhibitor benzbromarone (30 μM).ConclusionColistin was slowly metabolised in (rat &gt; human) hepatocytes, apparently without involvement of CYP450 enzymes. MRP‐mediated efflux dominates the hepatic disposition of colistin, while one or more uptake transporters may also be involved. Transporter mediated uptake will be explored using Chinese hamster ovary (CHO) cell lines. These in vitro results will be used to simulate the in vivo disposition of colistin using Physiology‐based Pharmacokinetic modelling in Simcyp®.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Organic Anion Transporter 2 Mediates Hepatic Uptake of Tolbutamide, a CYP2C9 Probe Drug.

Tolbutamide is primarily metabolized by CYP2C9, and, thus, is frequently applied as a clinical probe substrate for CYP2C9 activity. However, there is a marked discrepancy in the in vitro-in vivo extrapolation of its metabolic clearance, implying a potential for additional clearance mechanisms. The goal of this study was to evaluate the role of hepatic uptake transport in the pharmacokinetics of tolbutamide and to identify the molecular mechanism thereof. Transport studies using singly transfected cells expressing six major hepatic uptake transporters showed that tolbutamide is a substrate to organic anion transporter 2 (OAT2) alone with transporter affinity [Michaelis-Menten constant (Km)] of 19.5 ± 4.3 µM. Additionally, OAT2-specific transport was inhibited by ketoprofen (an OAT2 inhibitor) and 1 mM rifamycin SV (pan inhibitor), but not by cyclosporine and rifampicin (OAT polypeptides/Na+-taurocholate cotransporting polypeptide inhibitors). Uptake studies in primary human hepatocytes confirmed the predominant role of OAT2 in the active uptake with significant inhibition by rifamycin SV and ketoprofen, but not by the other inhibitors. Concentration-dependent uptake was noted in human hepatocytes with active transport characterized by Km and Vmax values of 39.3 ± 6.6 µM and 426 ± 30 pmol/min per milligram protein, respectively. Bottom-up physiologically based pharmacokinetic modeling was employed to verify the proposed role of OAT2-mediated hepatic uptake. In contrast to the rapid equilibrium (CYP2C9-only) model, the permeability-limited (OAT2-CYP2C9 interplay) model better described the plasma concentration-time profiles of tolbutamide. Additionally, the latter well described tolbutamide pharmacokinetics in carriers of CYP2C9 genetic variants and quantitatively rationalized its known drug-drug interactions. Our results provide first-line evidence for the role of OAT2-mediated hepatic uptake in the pharmacokinetics of tolbutamide, and imply the need for additional clinical studies in this direction.