Brain-Permeable Immunoproteasome-Targeting Macrocyclic Peptide Epoxyketones for Alzheimer's Disease.

Previously, we demonstrated that linear peptide epoxyketones targeting the immunoproteasome (iP) could ameliorate cognitive deficits in mouse models of Alzheimer's disease (AD) independently of amyloid deposition. We also reported the first iP-targeting macrocyclic peptide epoxyketones, which exhibit improved metabolic stability compared with their linear counterparts. Here, we prepared additional macrocyclic peptide epoxyketones and compared them with existing macrocyclic iP inhibitors by assessing Caco2 cell-based permeability and microsomal stability, providing the four best macrocyclic iP inhibitors. We then evaluated the four compounds using the Ames test and the potency assays in BV2 cells, selecting compound 5 as our AD drug lead. When 5 was administered intravenously (40 mg/kg) or orally (150 mg/kg) into healthy BALB/c mice, we observed considerable iP inhibition in the mouse brain, indicating good blood-brain barrier permeability and target engagement. Combined results suggest that 5 is a promising AD drug lead that may need further investigation.

Synthesis of calix (4) resorcinarene based amphiphilic macrocycle as an efficient nanocarrier for Amphotericin-B to enhance its oral bioavailability.

The supramolecular-based macrocyclic amphiphiles have fascinating attention and find extensive utilization in the pharmaceutical industry for efficient drug delivery. In this study, we designed and synthesized a new supramolecular amphiphilic macrocycle to serve as an efficient nanocarrier, achieved by treating 4-hydroxybenzaldehyde with 1-bromotetradecane. The derivatized product was subsequently treated with resorcinol to cyclize, resulting in the formation of a calix(4)-resorcinarene-based supramolecular amphiphilic macrocycle. The synthesized macrocycle and intermediate products were characterized using mass spectrometry, IR, and 1H NMR spectroscopic techniques. The amphotericin-B (Amph-B)-loaded and unloaded amphiphiles were screened for biocompatibility studies, vesicle formation, particle shape, size, surface charge, drug entrapment, in-vitro release profile, and stability through atomic force microscopy (AFM), Zetasizer, HPLC, and FT-IR. Amph-B -loaded macrocycle-based niosomal vesicles were investigated for in-vivo bioavailability in rabbits. The synthesized macrocycle exhibited no cytotoxicity against normal mouse fibroblast cells and was found to be hemocompatible and safe in mice following an acute toxicity study. The drug-loaded macrocycle-based vesicles appeared spherical, nano-sized, and homogeneous in size, with a notable negative surface charge. The vesicles remained stable after 30 days of storage. The results of Amph-B oral bioavailability and pharmacokinetics revealed that the newly tailored niosomal formulation enhanced drug solubility, protected drug degradation at gastric pH, facilitated sustained drug release at the specific target site, and delayed plasma drug clearance. Incorporating such advanced niosomal formulations in the field of drug delivery systems has the potential to revolutionize therapeutic outcomes and improve the quality of patient well-being.

Concurrent Optimizations of Efficacy and Blood-Brain Barrier Permeability in New Macrocyclic LRRK2 Inhibitors for Potential Parkinson's Disease Therapeutics.

The elevated activity of leucine-rich repeat kinase 2 (LRRK2) is implicated in the pathogenesis of Parkinson's disease (PD). The quest for effective LRRK2 inhibitors has been impeded by the formidable challenge of crossing the blood-brain barrier (BBB). We leveraged structure-based de novo design and developed robust three-dimensional quantitative structure-activity relationship (3D-QSAR) models to predict BBB permeability, enhancing the likelihood of the inhibitor's brain accessibility. Our strategy involved the synthesis of macrocyclic molecules by linking the two terminal nitrogen atoms of HG-10-102-01 with an alkyl chain ranging from 2 to 4 units, laying the groundwork for innovative LRRK2 inhibitor designs. Through meticulous computational and synthetic optimization of both biochemical efficacy and BBB permeability, 9 out of 14 synthesized candidates demonstrated potent low-nanomolar inhibition and significant BBB penetration. Further assessments of in vitro and in vivo effectiveness, coupled with pharmacological profiling, highlighted 8 as the promising new lead compound for PD therapeutics.

Discovery of Quinoxaline-Based P1-P3 Macrocyclic NS3/4A Protease Inhibitors with Potent Activity against Drug-Resistant Hepatitis C Virus Variants.

The three pan-genotypic HCV NS3/4A protease inhibitors (PIs) currently in clinical use-grazoprevir, glecaprevir, and voxilaprevir-are quinoxaline-based P2-P4 macrocycles and thus exhibit similar resistance profiles. Using our quinoxaline-based P1-P3 macrocyclic lead compounds as an alternative chemical scaffold, we explored structure-activity relationships (SARs) at the P2 and P4 positions to develop pan-genotypic PIs that avoid drug resistance. A structure-guided strategy was used to design and synthesize two series of compounds with different P2 quinoxalines in combination with diverse P4 groups of varying sizes and shapes, with and without fluorine substitutions. Our SAR data and cocrystal structures revealed the interplay between the P2 and P4 groups, which influenced inhibitor binding and the overall resistance profile. Optimizing inhibitor interactions in the S4 pocket led to PIs with excellent antiviral activity against clinically relevant PI-resistant HCV variants and genotype 3, providing potential pan-genotypic inhibitors with improved resistance profiles.

Pharmacodynamic and pharmacokinetic profiles of a neurotensin receptor type 2 (NTS2) analgesic macrocyclic analog.

The current opioid crisis highlights the urgent need to develop safe and effective pain medications. Thus, neurotensin (NT) compounds represent a promising approach, as the antinociceptive effects of NT are mediated by activation of the two G protein-coupled receptor subtypes (i.e., NTS1 and NTS2) and produce potent opioid-independent analgesia. Here, we describe the synthesis and pharmacodynamic and pharmacokinetic properties of the first constrained NTS2 macrocyclic NT(8-13) analog. The Tyr11 residue of NT(8-13) was replaced with a Trp residue to achieve NTS2 selectivity, and a rationally designed side-chain to side-chain macrocyclization reaction was applied between Lys8 and Trp11 to constrain the peptide in an active binding conformation and limit its recognition by proteolytic enzymes. The resulting macrocyclic peptide, CR-01-64, exhibited high-affinity for NTS2 (Ki 7.0 nM), with a more than 125-fold selectivity over NTS1, as well as an improved plasma stability profile (t1/2 > 24 h) compared with NT (t1/2 ~ 2 min). Following intrathecal administration, CR-01-64 exerted dose-dependent and long-lasting analgesic effects in acute (ED50 = 4.6 µg/kg) and tonic (ED50 = 7.1 µg/kg) pain models as well as strong mechanical anti-allodynic effects in the CFA-induced chronic inflammatory pain model. Of particular importance, this constrained NTS2 analog exerted potent nonopioid antinociceptive effects and potentiated opioid-induced analgesia when combined with morphine. At high doses, CR-01-64 did not cause hypothermia or ileum relaxation, although it did induce mild and short-term hypotension, all of which are physiological effects associated with NTS1 activation. Overall, these results demonstrate the strong therapeutic potential of NTS2-selective analogs for the management of pain.

TPX-0131, a Potent CNS-penetrant, Next-generation Inhibitor of Wild-type ALK and ALK-resistant Mutations.

Since 2011, with the approval of crizotinib and subsequent approval of four additional targeted therapies, anaplastic lymphoma kinase (ALK) inhibitors have become important treatments for a subset of patients with lung cancer. Each generation of ALK inhibitor showed improvements in terms of central nervous system (CNS) penetration and potency against wild-type (WT) ALK, yet a key continued limitation is their susceptibility to resistance from ALK active-site mutations. The solvent front mutation (G1202R) and gatekeeper mutation (L1196M) are major resistance mechanisms to the first two generations of inhibitors while patients treated with the third-generation ALK inhibitor lorlatinib often experience progressive disease with multiple mutations on the same allele (mutations in cis, compound mutations). TPX-0131 is a compact macrocyclic molecule designed to fit within the ATP-binding boundary to inhibit ALK fusion proteins. In cellular assays, TPX-0131 was more potent than all five approved ALK inhibitors against WT ALK and many types of ALK resistance mutations, e.g., G1202R, L1196M, and compound mutations. In biochemical assays, TPX-0131 potently inhibited (IC50 <10 nmol/L) WT ALK and 26 ALK mutants (single and compound mutations). TPX-0131, but not lorlatinib, caused complete tumor regression in ALK (G1202R) and ALK compound mutation-dependent xenograft models. Following repeat oral administration of TPX-0131 to rats, brain levels of TPX-0131 were approximately 66% of those observed in plasma. Taken together, preclinical studies show that TPX-0131 is a CNS-penetrant, next-generation ALK inhibitor that has potency against WT ALK and a spectrum of acquired resistance mutations, especially the G1202R solvent front mutation and compound mutations, for which there are currently no effective therapies.

Dinuclear Fe(III) Hydroxypropyl-Appended Macrocyclic Complexes as MRI Probes.

Four high-spin Fe(III) macrocyclic complexes, including three dinuclear and one mononuclear complex, were prepared toward the development of more effective iron-based magnetic resonance imaging (MRI) contrast agents. All four complexes contain a 1,4,7-triazacyclononane macrocyclic backbone with two hydroxypropyl pendant groups, an ancillary aryl or biphenyl group, and a coordination site for a water ligand. The pH potentiometric titrations support one or two deprotonations of the complexes, most likely deprotonation of hydroxypropyl groups at near-neutral pH. Variable-temperature 17O NMR studies suggest that the inner-sphere water ligand is slow to exchange with bulk water on the NMR time scale. Water proton T1 relaxation times measured for solutions of the Fe(III) complexes at pH 7.2 showed that the dinuclear complexes have a 2- to 3-fold increase in r1 relaxivity in comparison to the mononuclear complex per molecule at field strengths ranging from 1.4 T to 9.4 T. The most effective agent, a dinuclear complex with macrocycles linked through para-substitution of an aryl group (Fe2(PARA)), has an r1 of 6.7 mM-1 s-1 at 37 °C and 4.7 T or 3.3 mM-1 s-1 per iron center in the presence of serum albumin and shows enhanced blood pool and kidney contrast in mice MRI studies.

68Ga-Labeled bismacrocyclic methylene phosphonate as potential bone seeking PET radiopharmaceutical.

Phosphonates-based agents are well-known bone-seeking radiopharmaceuticals with application in detection and therapy. With higher sensitivity and resolution offered by Positron Emission Tomography (PET), tracers based on this technique are gaining huge attention. 68Ga-based generator and radiotracers render independence from the on-site cyclotron. We report the development of 68Ga-labeled DOTA-based bismacrocyclic phosphonate derivative, for bone PET imaging. The synthesis and characterization of 68Ga- DO3P-AME-DO3P was carried out in > 95% purity. The radiotracer displayed high stability and low binding affinity (<3%) to blood serum. High in vitro binding affinity were observed for synthetic hydroxyapatite, SAOS-2, osteoclast and osteoblast cells. In vivo pharmacokinetics revealed fast washout with biphasic release pattern. The deposition of radiotracer in osseous tissues was high (Bone/Muscle ratio:18), as studied from the biodistribution studies. In vivo PET/CT and biodistribution analyses revealed the ability of 68Ga-DO3P-AME-DO3P to target and accumulate in bone, thus displaying its potential as a PET bone imaging agent.

Modulating the Properties of Fe(III) Macrocyclic MRI Contrast Agents by Appending Sulfonate or Hydroxyl Groups.

Complexes of Fe(III) that contain a triazacyclononane (TACN) macrocycle, two pendant hydroxyl groups, and a third ancillary pendant show promise as MRI contrast agents. The ancillary group plays an important role in tuning the solution relaxivity of the Fe(III) complex and leads to large changes in MRI contrast enhancement in mice. Two new Fe(III) complexes, one with a third coordinating hydroxypropyl pendant, Fe(L2), and one with an anionic non-coordinating sulfonate group, Fe(L1)(OH2), are compared. Both complexes have a deprotonated hydroxyl group at neutral pH and electrode potentials representative of a stabilized trivalent iron center. The r1 relaxivity of the Fe(L1)(OH2) complex is double that of the saturated complex, Fe(L2), at 4.7 T, 37 °C in buffered solutions. However, variable-temperature 17O-NMR experiments show that the inner-sphere water of Fe(L1)(OH2) does not exchange rapidly with bulk water under these conditions. The pendant sulfonate group in Fe(L1)(OH2) confers high solubility to the complex in comparison to Fe(L2) or previously studied analogues with benzyl groups. Dynamic MRI studies of the two complexes showed major differences in their pharmacokinetics clearance rates compared to an analogue containing a benzyl ancillary group. Rapid blood clearance and poor binding to serum albumin identify Fe(L1)(OH2) for development as an extracellular fluid contrast agent.

Discovery of a High Affinity, Orally Bioavailable Macrocyclic FXIa Inhibitor with Antithrombotic Activity in Preclinical Species.

Oral factor XIa (FXIa) inhibitors may provide a promising new antithrombotic therapy with an improved benefit to bleeding risk profile over existing antithrombotic agents. Herein, we report application of a previously disclosed cyclic carbamate P1 linker which provided improved oral bioavailability in the imidazole-based 13-membered macrocycle to the 12-membered macrocycle. This resulted in identification of compound 4 with desired FXIa inhibitory potency and good oral bioavailability but high in vivo clearance. Further structure-activity relationship (SAR) studies of heterocyclic core modifications to replace the imidazole core as well as various linkers to the P1 group led to the discovery of compound 6f, a potent FXIa inhibitor with selectivity against most of the relevant serine proteases. Compound 6f also demonstrated excellent pharmacokinetics (PK) profile (high oral bioavailability and low clearance) in multiple preclinical species. Compound 6f achieved robust antithrombotic efficacy in a rabbit efficacy model at doses which preserved hemostasis.

Bioanalytical assay for the new-generation ROS1/TRK/ALK inhibitor repotrectinib in mouse plasma and tissue homogenate using liquid chromatography-tandem mass spectrometry.

Repotrectinib, a next-generation ROS1/TRK/ALK tyrosine kinase inhibitor, overcomes resistance due to acquired solvent-front mutations involving ROS1, NTRK1-3, and ALK. A bioanalytical assay for quantification of repotrectinib in mouse plasma and seven tissue-related matrices (brain, liver, spleen, kidney, small intestinal tissue, small intestinal content, and testis homogenates) was developed and validated using liquid chromatography with tandem mass spectrometric detection in a high-throughput 96-well format. Protein precipitation was performed by adding acetonitrile, also containing the internal standard axitinib, to 10-µl samples for all matrices. Chromatographic separation of analytes was done on an ACQUITY UPLC® BEH C18 column by gradient elution using ammonium hydroxide in water and methanol. Compounds were monitored with positive electrospray ionization using a triple quadruple mass spectrometer in selected reaction monitoring mode. The method was successfully validated in the 1-1000 ng/ml calibration range. Precisions (intra- and interday) were in the range of 1.3-8.7% and accuracies were in between 90.5% and 107.3% for all levels in all matrices. The developed method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of repotrectinib in wild-type mice.

Orally bioavailable amine-linked macrocyclic inhibitors of factor XIa.

The discovery of orally bioavailable FXIa inhibitors has been a challenge. Herein, we describe our efforts to address this challenge by optimization of our imidazole-based macrocyclic series. Our optimization strategy focused on modifications to the P2 prime, macrocyclic amide linker, and the imidazole scaffold. Replacing the amide of the macrocyclic linker with amide isosteres led to the discovery of substituted amine linkers which not only maintained FXIa binding affinity but also improved oral exposure in rats. Combining the optimized macrocyclic amine linker with a pyridine scaffold afforded compounds 23 and 24 that were orally bioavailable, single-digit nanomolar FXIa inhibitors with excellent selectivity against relevant blood coagulation enzymes.

Single dose escalation studies with inhaled POL6014, a potent novel selective reversible inhibitor of human neutrophil elastase, in healthy volunteers and subjects with cystic fibrosis.

BACKGROUND: POL6014 is a novel, orally inhaled neutrophil elastase (NE) inhibitor in development for cystic fibrosis (CF). METHODS: Two studies, one in healthy volunteers (HVs, doses 20 to 960 mg) and one in subjects with CF (doses 80 to 320 mg) were conducted to evaluate the safety, tolerability and pharmacokinetics (PK) of single ascending doses of inhaled POL6014 with a Pari eFlow® nebuliser. PK was evaluated over a period of 24 h. In addition, NE activity in CF sputum was measured. RESULTS: After single doses, POL6014 was safe and well tolerated up to 480 mg in HVs and at all doses in subjects with CF. POL6014 showed a dose-linear PK profile in both populations with Cmax between 0.2 and 2.5 µM in HVs and between 0.2 and 0.5 µM in subjects with CF. Tmax was reached at approximately 2-3 h. Mean POL6014 levels in CF sputum rapidly reached 1000 µM and were still above 10 µM at 24 h. >1-log reduction of active NE was observed at 3 h after dosing. CONCLUSION: Inhalation of POL6014 can safely lead to high concentrations within the lung and simultaneously low plasma concentrations, allowing for a clear inhibition of NE in the sputum of subjects with CF after single dosing. TRIAL REGISTRATION: European Medicines Agency EudraCT-Nr. 2015-001618-83 and 2016-000493-38.

QSAR Models for Predicting Five Levels of Cellular Accumulation of Lysosomotropic Macrocycles.

Drugs that accumulate in lysosomes reach very high tissue concentrations, which is evident in the high volume of distribution and often lower clearance of these compounds. Such a pharmacokinetic profile is beneficial for indications where high tissue penetration and a less frequent dosing regime is required. Here, we show how the level of lysosomotropic accumulation in cells can be predicted solely from molecular structure. To develop quantitative structure-activity relationship (QSAR) models, we used cellular accumulation data for 69 lysosomotropic macrocycles, the pharmaceutical class for which this type of prediction model is extremely valuable due to the importance of cellular accumulation for their anti-infective and anti-inflammatory applications as well as due to the fact that they are extremely difficult to model by computational methods because of their large size (Mw > 500). For the first time, we show that five levels of intracellular lysosomotropic accumulation (as measured by liquid chromatography coupled to tandem mass spectrometry-LC-MS/MS), from low/no to extremely high, can be predicted with 60% balanced accuracy solely from the compound's structure. Although largely built on macrocycles, the eight non-macrocyclic compounds that were added to the set were found to be well incorporated by the models, indicating their possible broader application. By uncovering the link between the molecular structure and cellular accumulation as the key process in tissue distribution of lysosomotropic compounds, these models are applicable for directing the drug discovery process and prioritizing the compounds for synthesis with fine-tuned accumulation properties, according to the desired pharmacokinetic profile.

Building upon Nature's Framework: Overview of Key Strategies Toward Increasing Drug-Like Properties of Natural Product Cyclopeptides and Macrocycles.

The pharmaceutical industry has focused mainly in the development of small-molecule entities intended for oral administration for the past decades. As a result, the majority of existing drugs address only a narrow range of biological targets. In the era of post-genomics, transcriptomics, and proteomics, there is an increasing interest on larger modulators of proteins that can span larger surfaces, access new therapeutic mechanisms of action, and provide greater target specificity. Traditional drug-like molecules developed using "rule-of-five" (Ro5) guidelines have been proven ineffective against a variety of challenging targets, such as protein-protein interactions, nucleic acid complexes, and antibacterial modalities. However, natural products are known to be effective at modulating such targets, leading to a renewed focus by medicinal chemists on investigating underrepresented chemical scaffolds associated with natural products. Here we describe recent efforts toward identification of novel natural cyclopeptides and macrocycles as well as selected medicinal chemistry strategies to increase drug-like properties or further exploration of their activity.

In Situ Ring Contraction and Transformation of the Rhizoxin Macrocycle through an Abiotic Pathway.

A Rhizopus sp. culture containing an endosymbiont partner ( Burkholderia sp.) was obtained through a citizen-science-based soil-collection program. An extract prepared from the pair of organisms exhibited strong inhibition of Ewing sarcoma cells and was selected for bioassay-guided fractionation. This led to the purification of rhizoxin (1), a potent antimitotic agent that inhibited microtubule polymerization, along with several new (2-5) and known (6) analogues of 1. The structures of 2-6 were established using a combination of NMR data analysis, while the configurations of the new stereocenters were determined using ROESY spectroscopy and comparison of GIAO-derived and experimental data for NMR chemical shift and 3 JHH coupling values. Whereas compound 1 showed modest selectivity for Ewing sarcoma cell lines carrying the EWSR1/ FLI1 fusion gene, the other compounds were determined to be inactive. Chemically, compound 2 stands out from other rhizoxin analogues because it is the first member of this class that is reported to contain a one-carbon-smaller 15-membered macrolactone system. Through a combination of experimental and computational tests, we determined that 2 is likely formed via an acid-catalyzed Meinwald rearrangement from 1 because of the mild acidic culture environment created by the Rhizopus sp. isolate and its symbiont.

Pharmacokinetic stability of macrocyclic peptide triazole HIV-1 inactivators alone and in liposomes.

Previously, we reported the discovery of macrocyclic peptide triazoles (cPTs) that bind to HIV-1 Env gp120, inhibit virus cell infection with nanomolar potencies, and cause irreversible virion inactivation. Given the appealing virus-killing activity of cPTs and resistance to protease cleavage observed in vitro, we here investigated in vivo pharmacokinetics of the cPT AAR029b. AAR029b was investigated both alone and encapsulated in a PEGylated liposome formulation that was designed to slowly release inhibitor. Pharmacokinetic analysis in rats showed that the half-life of FITC-AAR029b was substantial both alone and liposome-encapsulated, 2.92 and 8.87 hours, respectively. Importantly, liposome-encapsulated FITC-AAR029b exhibited a 15-fold reduced clearance rate from serum compared with the free FITC-cPT. This work thus demonstrated both the in vivo stability of cPT alone and the extent of pharmacokinetic enhancement via liposome encapsulation. The results obtained open the way to further develop cPTs as long-acting HIV-1 inactivators against HIV-1 infection.

Pharmacokinetics of Ombitasvir, Paritaprevir, Ritonavir, and Dasabuvir in Healthy Chinese Subjects and HCV GT1b-Infected Chinese, South Korean and Taiwanese Patients.

BACKGROUND/PURPOSE: The 3 direct-acting antiviral (3D) regimen of ombitasvir/paritaprevir/ritonavir plus dasabuvir has recently been approved in several Asian geographic regions for the treatment of hepatitis C virus (HCV) genotype (GT) 1 infection. The pharmacokinetics of the components of the 3D regimen with or without ribavirin were evaluated in healthy Chinese subjects and HCV GT1b-infected Chinese, South Korean, and Taiwanese patients, with or without cirrhosis, to determine how the drug exposures in Asian populations compare with historical data in Western populations. METHODS: Participants received ombitasvir/paritaprevir/ritonavir 25/150/100 mg once daily plus dasabuvir 250 mg twice daily for 14 days (healthy subjects, n = 36) or 12 weeks (HCV patients, n = 754). Patients with compensated cirrhosis also received ribavirin 1000 or 1200 mg divided twice daily, per the local label. Intensive or sparse pharmacokinetic sampling was performed for assessments of plasma drug concentrations. RESULTS: The exposures [maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC)] of the components of the 3D regimen were comparable (< 20% difference) in healthy Chinese subjects residing in China or the United States. In addition, the trough plasma concentrations (Ctrough) in HCV GT1b-infected Asian patients were either similar to (ombitasvir) or within 75% of (paritaprevir and dasabuvir) those in Western patients without cirrhosis, or similar to (ombitasvir and paritaprevir) or within 100% of (dasabuvir) those in Western patients with cirrhosis, with widely overlapping ranges of individual values. Generally comparable drug exposures were observed among Chinese, South Korean, and Taiwanese ethnicities for noncirrhotic and cirrhotic patients. CONCLUSION: Collectively, the results of these pharmacokinetic analyses support the use of the same dose of the 3D regimen for Asian and Western patients. CLINICALTRIALS.GOV: NCT02534870, NCT02517515, NCT02517528.

GP205, a new hepatitis C virus NS3/4A protease inhibitor, displays higher metabolic stability in vitro and drug exposure in vivo.

NS3/4A serine protease is a prime target for direct-acting antiviral therapies against hepatitis C virus (HCV) infection. Several NS3/4A inhibitors have been widely used in clinic, while new inhibitors with better characteristics are still urgently needed. GP205 is a new macrocyclic inhibitor of NS3/4A with low nanomolar activities against HCV replicons of genotypes 1b, 2a, 4a, and 5a, with EC50 values ranging from 1.5 to 12.8 nmol/L. In resistance selection study in vitro, we found resistance-associated substitutions on D168: The activity of GP205 was significantly attenuated against 1b replicon with D168V or D168A mutation, similar as simeprevir. No cross resistance of GP205 with NS5B or NS5A inhibitor was observed. Combination of GP205 with sofosbuvir or daclatasvir displayed additive or synergistic efficacy. The pharmacokinetic profile of GP205 was characterized in rats and dogs after oral administration, which revealed good drug exposure both in plasma and in liver and long plasma half-life. The in vitro stability test showed ideal microsomal and hepatic cells stability of GP205. The preclinical profiles of GP205 support further research on this NS3/4A inhibitor to expand the existing HCV infection therapies.