Programmed cell death-1 is involved with peripheral blood immune cell profiles in patients with hepatitis C virus antiviral therapy.

Mutations in the non-structural protein regions of hepatitis C virus (HCV) are a cause of a non-sustained virological response (SVR) to treatment with direct-acting antivirals (DAAs) for chronic hepatitis; however, there are non-SVR cases without these mutations. In this study, we examined immune cell profiles in peripheral blood before and after ombitasvir/paritaprevir/ritonavir treatment and screened for genes that could be used to predict the therapeutic effects of DAAs. Fluorescence-activated cell sorting analysis indicated that the median frequencies of programmed cell death-1-positive (PD-1+) effector regulatory T cells (eTregs), PD-1+CD8+ T cells, and PD-1+Helper T cells were decreased significantly in SVR cases, but without significant changes in non-SVR cases. The frequency of PD-1+ naïve Tregs was significantly higher in the SVR group than in the non-SVR group before and after treatment. Similar results were found in patients treated with other DAAs (e.g., daclatasvir plus asunaprevir) and supported an immune response after HCV therapy. RNA-sequencing analysis indicated a significant increase in the expression of genes associated with the immune response in the SVR group, while genes related to intracellular and extracellular signal transduction were highly expressed in the non-SVR group. Therefore, we searched for genes associated with PD-1+ eTregs and CD8+ T cells that were significantly different between the SVR and non-SVR groups and found that T-box transcription factor 21 was associated with the non-SVR state. These results indicate that PD-1-related signaling pathways are associated with a non-SVR mechanism after DAAs treatment separate from mutation-related drug resistance.

Discovery of Selective and Potent Macrocyclic CDK9 Inhibitors for the Treatment of Osimertinib-Resistant Non-Small-Cell Lung Cancer.

Effectiveness of epidermal growth factor receptor (EGFR) inhibitors, including Osimertinib, for treating non-small-cell lung cancer (NSCLC) is limited due to the continuous emergence of drug resistance. Hence, it is urgent to develop new therapeutic approaches. CDK9, a key regulator of RNA transcription, has emerged as a promising target for the development of antitumor drugs due to its crucial role in modulating the levels of antiapoptotic protein Mcl-1. Herein, we present the synthesis, optimization, and evaluation of selective CDK9 inhibitors with a macrocyclic scaffold that effectively suppresses the growth of NSCLC cells. Notably, compound Z11, a potent CDK9 inhibitor (IC50 = 3.20 nM) with good kinase selectivity, significantly inhibits cell proliferation and colony formation and induces apoptosis in Osimertinib-resistant H1975 cells. Furthermore, Z11 demonstrates a significant suppression of tumor growth in six patient-derived organoids, including three organoids resistant to Osimertinib. Overall, Z11 served as a promising macrocycle-based CDK9 inhibitor for treating Osimertinib-resistant NSCLC.

Efficacy and safety of ombitasvir/paritaprevir/ritonavir-based therapy in HCV patients with chronic kidney disease.

BACKGROUND AND STUDY AIMS: Hepatitis C virus (HCV) prevalence inchronic kidney disease (CKD) patients is significantly higher than in the general population. This study evaluated the efficacy and safety of combined ombitasvir/paritaprevir/ritonavir-based therapy in HCV patients with renal impairment. PATIENTS AND METHODS: Our study included 829 patients with normal kidney functions (group 1) and 829 patients with CKD (group 2),which were subdivided into patients not requiring dialysis (group 2a) and those on hemodialysis (group2b). Patients received regimens of ombitasvir/paritaprevir/ritonavir with or without ribavirin or sofosbuvir/ombitasvir/paritaprevir/ritonavir with or without ribavirin for 12 weeks. Clinical and laboratory assessment was done before treatment, and patients were followed up for12 weeks after treatment. RESULTS: The sustained virological response (SVR) at week 12 was significantly higher in group 1 than in the other three groups/subgroups, being 94.2% vs 90.2%, 90%, and 90.7%, respectively. The regimen with the highest SVR was ombitasvir/paritaprevir/ritonavir with ribavirin. The most common adverse event was anemia, which was more common in group 2. CONCLUSION: Ombitasvir/paritaprevir/ritonavir-based therapy in chronic HCV patients with CKD is highly effective, with minimal side effects despite ribavirin-induced anemia.

Synergistic activity and mechanism of cytarabine and MCL-1 inhibitor AZD5991 against acute myeloid leukemia.

The 5-year overall survival rate of acute myeloid leukemia (AML) is less than 30%. Improving clinical outcomes is still a clinical challenge for AML treatment. Simultaneous use of chemotherapeutic drugs and targeting of apoptosis pathways has become a first-line clinical treatment for AML. Myeloid cell leukemia 1 (MCL-1) is a candidate target for AML treatment. In this study, we demonstrated that inhibition of the anti-apoptotic protein MCL-1 by AZD5991 synergistically increased chemotherapeutic agent cytarabine (Ara-C)-induced apoptosis in AML cell lines and primary patient samples. Apoptosis induced by a combination of Ara-C and AZD5991 was partially dependent on caspase activity and Bak/Bax. The downregulation of MCL-1 by Ara-C and the enhancement of Ara-C-induced DNA damage through inhibition of MCL-1 are potential mechanisms underlying the synergistic anti-AML activity between Ara-C and AZD5991. Our data support the application of MCL-1 inhibitor in combination with the conventional chemotherapeutic agent for the clinical treatment of AML.

Cardiac toxicity associated with pharmacokinetic drug-drug interaction between crizotinib and sofosbuvir/velpatasvir: A case report.

This case report describes a pharmacokinetic drug-drug interaction between crizotinib, a tyrosine kinase inhibitor, and sofosbuvir/velpatasvir, a direct-acting antiviral drug, leading to cardiac toxicity. A 75-year-old man, with no cardiovascular history but a diagnosis of metastatic nonsmall cell lung cancer with mesenchymal-epithelial transition exon-14 deletion and hepatitis C virus infection genotype 1A, received both crizotinib and sofosbuvir/velpatasvir. Crizotinib was well tolerated, but 1 week after sofosbuvir/velpatasvir initiation, the patient experienced bilateral lower-limb oedema and class III New York Heart Association dyspnoea. We assumed that increased exposure to crizotinib could account for this cardiac toxicity. Drug causality was probable according to the Naranjo scale. We hypothesized a reciprocal interaction between crizotinib and velpatasvir, mediated by both cytochrome 3A4 (CYP3A4) and P-glycoprotein (P-gp). Clinicians should be aware of the risk of drug-drug interactions between direct-acting antiviral agents that inhibit CYP3A4 (glecaprevir) and/or P-gp (voxilaprevir, velpatasvir) and anticancer tyrosine kinase inhibitors that are mostly CYP3A4 and/or P-gp substrates (gefitinib, afatinib, erlotinib, crizotinib, ceritinib, lorlatinib, brigatinib, capmatinib etc.).

Discovery of D6808, a Highly Selective and Potent Macrocyclic c-Met Inhibitor for Gastric Cancer Harboring MET Gene Alteration Treatment.

MET alterations have been validated as a driven factor in NSCLC and gastric cancers. The c-Met inhibitors, capmatinib, tepotinib, and savolitinib, are only approved for the treatment of NSCLC harboring exon 14 skipping mutant MET. We used a molecular hybridization in conjunction with macrocyclization strategy for structural optimization to obtain a series of 2-(2-(quinolin-6-yl)ethyl)pyridazin-3(2H)-one derivatives as new c-Met inhibitors. One of the macrocyclic compounds, D6808, potently inhibited c-Met kinase and MET-amplified Hs746T gastric cancer cells with IC50 values of 2.9 and 0.7 nM, respectively. It also strongly suppressed Ba/F3-Tpr-Met cells harboring resistance-relevant mutations (F1200L/M1250T/H1094Y/F1200I/L1195V) with IC50 values of 4.2, 3.2, 1.0, 39.0, and 33.4 nM, respectively. Furthermore, D6808 exhibited extraordinary target specificity in a Kinome profiling against 373 wild-type kinases and served as a promising macrocycle-based compound for further anticancer drug development.

c-Myc plays a critical role in the antileukemic activity of the Mcl-1-selective inhibitor AZD5991 in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive disease with a low 5-year overall survival rate of 29.5%. Thus, more effective therapies are in need to prolong survival of AML patients. Mcl-1 is overexpressed in AML and is associated with poor prognosis, representing a promising therapeutic target. The oncoprotein c-Myc is also overexpressed in AML and is a significant prognostic factor. In addition, Mcl-1 is required for c-Myc induced AML, indicating that c-Myc-driven AML harbors a Mcl-1 dependency and co-targeting of Mcl-1 and c-Myc represents a promising strategy to eradicate AML. In this study, we investigated the role of c-Myc in the antileukemic activity of Mcl-1 selective inhibitor AZD5991 and the antileukemic activity of co-targeting of Mcl-1 and c-Myc in preclinical models of AML. We found that c-Myc protein levels negatively correlated with AZD5991 EC50s in AML cell lines and primary patient samples. AZD5991 combined with inhibition of c-Myc synergistically induced apoptosis in AML cell lines and primary patient samples, and cooperatively targeted leukemia progenitor cells. AML cells with acquired resistance to AZD5991 were resensitized to AZD5991 when c-Myc was inhibited. The combination also showed promising and synergistic antileukemic activity in vitro against AML cell lines with acquired resistance to the main chemotherapeutic drug AraC and primary AML cells derived from a patient at relapse post chemotherapy. The oncoprotein c-Myc represents a potential biomarker of AZD5991 sensitivity and inhibition of c-Myc synergistically enhances the antileukemic activity of AZD5991 against AML.

Efficacy and Safety of Ombitasvir/Paritaprevir/ Ritonavir + Dasabuvir ± Ribavirin Combinations in Patients with Genotype 1 Hepatitis C and Inherited Bleeding Disorders.

BACKGROUND: Hepatitis C is one of the leading causes of death in patients with inherited bleeding disorders. Currently, direct-acting antiviral drugs used for the treatment of hepatitis C have become an effective and a reliable option for people with inherited bleeding disorders. The aim of this study is to report the efficacy and safety of ombitasvir + paritaprevir/ritonavir and dasabuvir combination in the treatment of hepatitis C in patients with inherited bleeding disorders. METHODS: In this retrospective study, we evaluated the efficacy and safety of the combination of ombitasvir + paritaprevir/ritonavir and dasabuvir in 10 adult patients with hemophilia A, 4 patients with hemophilia B, and 1 patient with von Willebrand disease who were infected with hepatitis C genotype 1. RESULTS: Five patients had genotype 1a and 10 patients had genotype 1b chronic hepatitis C. One patient had Child A cirrhosis, 14 patients had chronic hepatitis C without cirrhosis. Hepatitis C virus ribonucleic acid was negative in all patients at week 4 and at the end of the treatment. Sustained virologic response was obtained in all patients. Serious side effects were detected in 3 patients, which were intra- muscular bleeding, erosive gastritis-related gastrointestinal bleeding, and pneumonia. CONCLUSION: Ombitasvir + paritaprevir combined with ritonavir and dasabuvir ± ribavirin is an effective treatment for patients infected with genotype 1 hepatitis C who have coagulation disorders. Tolerance and side effects are similar to other treatment options.

hsa-miR-17-5p: A Possible Predictor of Ombitasvir/Paritaprevir/Ritonavir + Dasabuvir ± Ribavirin Therapy Efficacy in Hepatitis C Infection.

Although persistent sustained viral response rates are increased in hepatitis C infection following administration of direct-acting antiviral (DAA) agents, the pre-use predictive parameters of these antivirals and the clinical progression in patients post-treatment remain unknown. To obtain data pertaining to the predictive parameters prior to the use of ombitavir/paritaprevir/ritonavir + dasabuvir and the clinical progression in patients following antiviral treatment. The expression profiles of miR-223-3p, miR-17-5p, miR-24-3p, and TLR2 - 196 to - 174 del/ins polymorphisms from the blood/serum of 34 hepatitis C virus (HCV)-infected patients pre- and post-ombitavir/paritaprevir/ritonavir + dasabuvir treatment were determined by RT-qPCR. The expression levels of miR-17-5p (P < 0.001) and miR-24-3p (P = 0.011) were significantly downregulated post-treatment as compared with those pre-treatment; however, there was no significant difference between these two groups in terms of miR-223-3p expression. In addition, there was no significant difference in TLR2 genotype or allele distribution between pre-and post-treatment (P > 0.05); nevertheless, the TLR2 del allele was decreased post-treatment (16.2%) as compared with that pre-treatment (19.1%), although the difference was not statistically significant. Moreover, a significant difference was found between the mRNA levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and HCV RNA pre-and post-treatment (P < 0.05). Further, miR-17-5p expression correlated with both ALT and AST mRNA levels post-treatment (P.

Glycemic Control in Patients Undergoing Treatment With Paritaprevir/Ombitasvir/Ritonavir and Dasabuvir for Chronic Hepatitis C Infection.

BACKGROUND/AIM: Patients with hepatitis C virus (HCV)-associated cirrhosis are more prone to developing type 2 diabetes mellitus than patients with any other etiology of cirrhosis. The main objective of this study was to evaluate the impact of all oral antiviral treatment with ritonavir-boosted paritaprevir/ombitasvir and dasabuvir (OBV/PTV/r + DSV) in patients with chronic genotype 1b HCV infection. PATIENTS AND METHODS: We retrospectively evaluated 806 patients who underwent antiviral therapy between December 2015 and July 2019. The laboratory data analyzed were liver function tests, kidney function tests, HCV viremia, fasting glucose levels, and glycosylated hemoglobin. RESULTS: Patients with impaired glucose metabolism were predominantly male and of older age compared to patients with normal glucose tolerance, and also had higher levels of transaminases. Proteinuria and higher creatinine levels were found in patients with impaired glucose metabolism. Overall, we found a 98.01% rate of sustained virologic response (SVR), with a non-significant difference between patients with normal and abnormal glucose metabolism. A statistically significant difference in SVR rates in patients with low degrees of fibrosis (F0-F2) versus those with advanced degrees of fibrosis (F3-F4) was found in both groups. Antiviral treatment resulted in significant decreases in fasting glucose levels and glycosylated hemoglobin levels in all patients with impaired glucose metabolism at SVR. CONCLUSION: Patients with pre-diabetes, as well as diabetic patients, achieved a better glycemic control after SVR obtained by ritonavir-boosted paritaprevir/ombitasvir and dasabuvir.

Drug-Drug Interactions With Cyclosporine in the Anti-Hepatitis C Viral PrOD Combination Regimen of Paritaprevir/Ritonavir-Ombitasvir and Dasabuvir in Organ Transplant Recipients With Severe Hepatic Fibrosis or Cirrhosis.

BACKGROUND: The clinical guidelines suggest that the dosing of cyclosporine (CsA), during combination therapy with paritaprevir/ritonavir-ombitasvir and dasabuvir (PrOD), would be only one-fifth of the pre-PrOD total daily dose to be administered once daily. However, this dosing may not be applicable to all patients depending on their clinical condition. This study focuses on the pharmacokinetic dynamics of PrOD with CsA in Asian organ transplant recipients with severe liver fibrosis or cirrhosis who undergo concurrent treatment with PrOD treatment and CsA. The efficacy and safety of PrOD treatment was also evaluated. METHODS: Data from 7 patients obtained between January 2017 and September 2017 were retrospectively analyzed. Determinations of the blood concentrations of CsA were made, whether used as a single treatment or in combination therapy with PrOD. RESULTS: The combination regimen compared with CsA administered alone resulted in a 4.53-fold and 5.52-fold increase in the area under the concentration-time curve from time 0-12 hours (AUC0-12 h) of CsA on days 1 and 15, respectively. In addition, the maximal concentration, time to maximum concentration, and terminal phase elimination half-life (t1/2) of CsA were increased during the combined treatment of PrOD and CsA. The authors proposed reducing the CsA dosage during PrOD treatment to one-seventh of that of the pre-PrOD treatment of the total daily dose to maintain target CsA levels. All patients achieved sustained virologic responses at week 12. There were no episodes of serious adverse events or graft rejections observed. CONCLUSIONS: Although the combination with PrOD significantly affects the pharmacokinetics of CsA, it is effective and safe with regular monitoring of the CsA blood concentrations and appropriate CsA dose adjustment.

Supramolecular Radiosensitizer Based on Hypoxia-Responsive Macrocycle.

Radiotherapy (RT) has been viewed as one of the most effective and extensively applied curatives in clinical cancer therapy. However, the radioresistance of tumor severely discounts the radiotherapy outcomes. Here, an innovative supramolecular radiotherapy strategy, based on the complexation of a hypoxia-responsive macrocycle with small-molecule radiosensitizer, is reported. To exemplify this tactic, a carboxylated azocalix[4]arene (CAC4A) is devised as molecular container to quantitatively package tumor sensitizer banoxantrone dihydrochloride (AQ4N) through reversible host-guest interaction. Benefited from the selective reduction of azo functional groups under hypoxic microenvironment, the supramolecular prodrug CAC4A•AQ4N exhibits high tumor accumulation and efficient cellular internalization, thereby significantly amplifying radiation-mediated tumor destruction without appreciable systemic toxicity. More importantly, this supramolecular radiotherapy strategy achieves an ultrahigh sensitizer enhancement ratio (SER) value of 2.349, which is the supreme among currently reported noncovalent-based radiosensitization approach. Further development by applying different radiosensitizing drugs can make this supramolecular strategy become a general platform for boosting therapeutic effect in cancer radiotherapies, tremendously promising for clinical translation.

Macrocyclic Inhibitors of HGF-Activating Serine Proteases Overcome Resistance to Receptor Tyrosine Kinase Inhibitors and Block Lung Cancer Progression.

Hepatocyte growth factor (HGF), the ligand for the MET receptor tyrosine kinase, is a tumor-promoting factor that is abundant in the tumor microenvironment. Proteolytic activation of inactive pro-HGF by one or more of the serine endopeptidases matriptase, hepsin, and HGF activator is the rate-limiting step in HGF/MET signaling. Herein, we have rationally designed a novel class of side chain cyclized macrocyclic peptide inhibitors. The new series of cyclic tripeptides has superior metabolic stability and significantly improved pharmacokinetics in mice relative to the corresponding linear peptides. We identified the lead compound VD2173 that potently inhibits matriptase and hepsin, which was tested in parallel alongside the acyclic inhibitor ZFH7116 using both in vitro and in vivo models of lung cancer. We demonstrated that both compounds block pro-HGF activation, abrogate HGF-mediated wound healing, and overcome resistance to EGFR- and MET-targeted therapy in lung cancer models. Furthermore, VD2173 inhibited HGF-dependent growth of lung cancer tumors in mice.

Coassembly of hypoxia-sensitive macrocyclic amphiphiles and extracellular vesicles for targeted kidney injury imaging and therapy.

BACKGROUND: Hypoxia is a major contributor to global kidney diseases. Targeting hypoxia is a promising therapeutic option against both acute kidney injury and chronic kidney disease; however, an effective strategy that can achieve simultaneous targeted kidney hypoxia imaging and therapy has yet to be established. Herein, we fabricated a unique nano-sized hypoxia-sensitive coassembly (Pc/C5A@EVs) via molecular recognition and self-assembly, which is composed of the macrocyclic amphiphile C5A, the commercial dye sulfonated aluminum phthalocyanine (Pc) and mesenchymal stem cell-excreted extracellular vesicles (MSC-EVs). RESULTS: In murine models of unilateral or bilateral ischemia/reperfusion injury, MSC-EVs protected the Pc/C5A complex from immune metabolism, prolonged the circulation time of the complex, and specifically led Pc/C5A to hypoxic kidneys via surface integrin receptor α4ß1 and αLß2, where Pc/C5A released the near-infrared fluorescence of Pc and achieved enhanced hypoxia-sensitive imaging. Meanwhile, the coassembly significantly recovered kidney function by attenuating cell apoptosis, inhibiting the progression of renal fibrosis and reducing tubulointerstitial inflammation. Mechanistically, the Pc/C5A coassembly induced M1-to-M2 macrophage transition by inhibiting the HIF-1α expression in hypoxic renal tubular epithelial cells (TECs) and downstream NF-κB signaling pathway to exert their regenerative effects. CONCLUSION: This synergetic nanoscale coassembly with great translational potential provides a novel strategy for precise kidney hypoxia diagnosis and efficient kidney injury treatment. Furthermore, our strategy of coassembling exogenous macrocyclic receptors with endogenous cell-derived membranous structures may offer a functional platform to address multiple clinical needs.

Molecular Characteristics of Repotrectinib That Enable Potent Inhibition of TRK Fusion Proteins and Resistant Mutations.

NTRK chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to TRK inhibitors (larotrectinib and entrectinib) but often mutate, limiting the durability of response for NTRK + patients. Next-generation inhibitors with compact macrocyclic structures (repotrectinib and selitrectinib) were designed to avoid resistance mutations. Head-to-head potency comparisons of TRK inhibitors and molecular characterization of binding interactions are incomplete, obscuring a detailed understanding of how molecular characteristics translate to potency. Larotrectinib, entrectinib, selitrectinib, and repotrectinib were characterized using cellular models of wild-type TRKA/B/C fusions and resistance mutant variants with a subset evaluated in xenograft tumor models. Crystal structures were determined for repotrectinib bound to TRKA (wild-type, solvent-front mutant). TKI-naïve and pretreated case studies are presented. Repotrectinib was the most potent inhibitor of wild-type TRKA/B/C fusions and was more potent than selitrectinib against all tested resistance mutations, underscoring the importance of distinct features of the macrocycle structures. Cocrystal structures of repotrectinib with wild-type TRKA and the TRKAG595R SFM variant elucidated how differences in macrocyclic inhibitor structure, binding orientation, and conformational flexibility affect potency and mutant selectivity. The SFM crystal structure revealed an unexpected intramolecular arginine sidechain interaction. Repotrectinib caused tumor regression in LMNA-NTRK1 xenograft models harboring GKM, SFM, xDFG, and GKM + SFM compound mutations. Durable responses were observed in TKI-naïve and -pretreated patients with NTRK + cancers treated with repotrectinib (NCT03093116). This comprehensive analysis of first- and second-generation TRK inhibitors informs the clinical utility, structural determinants of inhibitor potency, and design of new generations of macrocyclic inhibitors.

Translational Strategies for Repotrectinib in Neuroblastoma.

Limited clinical data are available regarding the utility of multikinase inhibition in neuroblastoma. Repotrectinib (TPX-0005) is a multikinase inhibitor that targets ALK, TRK, JAK2/STAT, and Src/FAK, which have all been implicated in the pathogenesis of neuroblastoma. We evaluated the preclinical activity of repotrectinib monotherapy and in combination with chemotherapy as a potential therapeutic approach for relapsed/refractory neuroblastoma. In vitro sensitivity to repotrectinib, ensartinib, and cytotoxic chemotherapy was evaluated in neuroblastoma cell lines. In vivo antitumor effect of repotrectinib monotherapy, and in combination with chemotherapy, was evaluated using a genotypically diverse cohort of patient-derived xenograft (PDX) models of neuroblastoma. Repotrectinib had comparable cytotoxic activity across cell lines irrespective of ALK mutational status. Combination with chemotherapy demonstrated increased antiproliferative activity across several cell lines. Repotrectinib monotherapy had notable antitumor activity and prolonged event-free survival compared with vehicle and ensartinib in PDX models (P < 0.05). Repotrectinib plus chemotherapy was superior to chemotherapy alone in ALK-mutant and ALK wild-type PDX models. These results demonstrate that repotrectinib has antitumor activity in genotypically diverse neuroblastoma models, and that combination of a multikinase inhibitor with chemotherapy may be a promising treatment paradigm for translation to the clinic.

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.

Prevalence of resistance-associated substitutions to NS3, NS5A and NS5B inhibitors at DAA-failure in hepatitis C virus in Italy from 2015 to 2019.

Despite the high efficacy of direct-acting antivirals (DAAs), the selection of resistance-associated substitutions (RASs) after virological failure of hepatitis C virus (HCV) DAAs can impair the cure of chronic HCV. The aim of the study was to characterize RASs after virological failure of DAAs in Italy over the years. Within the Italian network VIRONET-C, the change in prevalence of NS3/4A-NS5A-NS5B RASs was retrospectively evaluated in patients who failed a DAA regimen over the years 2015-2019. NS3, NS5A and NS5B Sanger sequencing was performed using homemade protocols and the geno2pheno system was used to define HCV-genotype/subtype and predict drug resistance. The changes in the prevalence of RASs over time were evaluated using the chi-square test for trend. Predictors of RASs at failure were analysed by logistic regression. Among 468 HCV-infected patients, HCV genotype 1 was the most prevalent (1b in 154, 33% and 1a in 109, 23%). DAA regimens were: ledipasvir (LDV)/sofosbuvir (SOF) in 131 patients (28%), daclatasvir (DCV)/SOF in 109 (23%), ombitasvir/paritaprevir/ritonavir+dasabuvir (3D) in 89 (19%), elbasvir (EBR)/grazoprevir (GRZ) in 52 (10.5%), velpatasvir (VEL)/SOF in 53 (11%), glecaprevir (GLE)/pibrentasvir (PIB) in 27 (6%) and ombitasvir/paritaprevir/ritonavir (2D) in 7 (1.5%); ribavirin was administered in 133 (28%). The NS5A fasta sequence was available for all patients, NS5B and NS3/4A both for 93%. The prevalence of NS5A and NS3/4A RASs significantly declined from 2015 to 2019; NS5B RAS remained stable. Independent predictors of any RASs included older age and genotype 1a (vs G2 and vs G4). Notably, at least partial susceptibility to all the agents included in the GLE/PIB and VEL/SOF/Voxilaprevir (VOX) combinations was predicted in >95% of cases. As RASs remain common at the failure of DAAs, their identification could play a crucial role in optimizing re-treatment strategies. In Italy RAS prevalence has been decreasing over the years and susceptibility to the latest developed drug combinations is maintained in most cases.

Structure-Enabled Discovery of Novel Macrocyclic Inhibitors Targeting Glutaminase 1 Allosteric Binding Site.

The inhibition of glutaminase 1 (GLS1) represents a potential treatment of malignant tumors. Structural analysis led to the design of a novel series of macrocyclic GLS1 allosteric inhibitors. Through extensive structure-activity relationship studies, a promising candidate molecule 13b (LL202) was identified with robust GLS1 inhibitory activity (IC50 = 6 nM) and high GLS1 binding affinity (SPR, Kd = 24 nM; ITC, Kd = 37 nM). The X-ray crystal structure of the 13b-GLS1 complex was resolved, revealing a unique binding mode and providing a novel structural scaffold for GLS1 allosteric inhibitors. Importantly, 13b clearly adjusted the cellular metabolites and induced an increase in the ROS level by blocking glutamine metabolism. Furthermore, 13b exhibited a similar in vivo antitumor activity as CB839. This study adds to the growing body of evidence that macrocyclization provides an alternative and complementary approach for the design of small-molecule inhibitors, with the potential to improve the binding affinity to the targets.

Treatment failure with DAA therapy: Importance of resistance.

Viral resistance is a major reason for virological failure in patients being treated with direct-acting antivirals (DAAs) for chronic HCV infection. However, the importance of viral resistance mainly depends on the DAA regimen and HCV genotype. For first-line therapy with glecaprevir/pibrentasvir (G/P) or velpatasvir/sofosbuvir (VEL/SOF) no general baseline resistance analysis is required because of the high antiviral activity and high barrier to resistance. If available, resistance testing may help to optimise therapy in certain subgroups of patients with HCV genotype 3 and other rare HCV geno/subtypes. Voxilaprevir/velpatasvir/sofosbuvir (VOX/VEL/SOF) is the first choice for the second-line treatment of patients following a previous DAA failure, with rates of viral eradication above 90% irrespective of the presence of resistance-associated substitutions (RASs). However, in resource-limited settings, only first-generation DAAs may be available for second-line therapy. Here, RASs selected during initial antiviral therapy should be considered if testing is available and rescue treatment should include a switch to a regimen with a new DAA class to optimise treatment response. Patients with HCV genotype 3 are overrepresented in the group who experience DAA treatment failure. Limited data are available for third-line therapies, but promising results have been achieved with G/P plus SOF or VOX/VEL/SOF with or without ribavirin for 12 to 24 weeks; these regimens should be administered irrespective of a patient's RAS profile.