Jacques Friborg

Identification of I50L as the Signature Atazanavir (ATV)-Resistance Mutation in Treatment-Naive HIV-1-Infected Patients Receiving ATV-Containing Regimens

Atazanavir (ATV) is a once-daily human immunodeficiency virus (HIV) protease inhibitor (PI) shown to be effective and well tolerated. ATV has a distinct resistance profile relative to other PIs, with susceptibility maintained against 86% of isolates resistant to 1-2 PIs. Clinical isolates obtained from PI-naive patients designated as experiencing virologic failure while receiving ATV-containing regimens contained a unique isoleucine-to-leucine substitution at amino acid residue 50 (I50L) of the HIV-1 protease. The I50L substitution, observed in all isolates exhibiting phenotypic resistance to ATV, emerged in a variety of different backgrounds and was most frequently accompanied by A71V, K45R, and/or G73S. Viruses containing an I50L substitution were growth impaired, displayed ATV-specific resistance, and had increased susceptibilities (</=0.4 of reference strain) to other PIs. Comparison of viruses bearing I50L with those bearing I50V revealed specific resistance to ATV and amprenavir, respectively, with no evidence of cross-resistance. The unique I50L substitution is the signature mutation for resistance to ATV.

Combinations of Lambda Interferon with Direct-Acting Antiviral Agents Are Highly Efficient in Suppressing Hepatitis C Virus Replication

ABSTRACT The clinical efficacy of a pegylated form of human lambda 1 interferon (IFN-λ1; also referred to herein as lambda) has been demonstrated in patients chronically infected with hepatitis C virus (HCV) representing genotypes 1 through 4. In these proof-of-concept studies, lambda showed an improved safety profile compared to the pegylated form of alfa interferon (referred to herein as alfa). In the study described in this report, an assessment of the in vitro antiviral activity of type III IFNs toward different HCV replicons revealed that the unpegylated recombinant form of IFN-λ1 (rIFN-λ1) exerted the most robust effect, while rIFN-λ3 exhibited greater activity than rIFN-λ2. More importantly, cross-resistance to rIFN-λ1 was not observed in replicon cell lines known to have reduced susceptibility to investigational direct-acting antiviral (DAA) agents targeting the essential HCV nonstructural protein NS3, NS5A, or NS5B. When combined with either rIFN-α, the NS3 protease inhibitor (NS3 PI) asunaprevir (ASV), the NS5A replication complex inhibitor (NS5A RCI) daclatasvir (DCV), or the NS5B polymerase site I inhibitor (NS5B I) BMS-791325, rIFN-λ1 displayed a mixture of additive and synergistic effects. In three-drug combination studies, inclusion of lambda with ASV and DCV also yielded additive to synergistic effects. In line with these observations, it was demonstrated that a regimen that used a combination of rIFN-λ1 with one or two DAAs was superior to an IFN-free regimen in clearing HCV RNA in genotype 1a cell lines representing wild-type and NS3 protease inhibitor-resistant sequences. Overall, these data support further clinical development of lambda as part of alternative combination treatments with DAAs for patients chronically infected with HCV.

IFN-λ-mediated IL-12 production in macrophages induces IFN-γ production in human NK cells

With increasing interest in alternative options to interferon‐alpha‐based treatments, IFN‐λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN‐λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN‐λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN‐λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon‐alpha, IFN‐λ is unable to directly stimulate NK cells, due to the absence of IFN‐λ receptor chain 1 (IFN‐λR1) on NK cells. However, IFN‐λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL‐12 family of cytokines in monocyte‐derived macrophages. We further show that through macrophage‐mediated IL‐12 production, IFN‐λ is able to indirectly affect NK cells and ultimately induce IFN‐γ production.

Development of a chimeric replicon system for phenotypic analysis of NS3 protease sequences from HCV clinical isolates.

BACKGROUND To support clinical development of HCV non-structural protein (NS) 3 protease inhibitors (PIs), phenotypic monitoring of patient isolates is a prerequisite for understanding the emergence of resistance. HCV isolates typically fail to replicate in cell culture, necessitating the use of alternative phenotyping methods. METHODS An NS3 protease chimeric replicon system was developed to monitor the phenotype of clinical isolates. The transfer of NS3 protease domain sequences from HCV-infected patients to the background of genotype (Gt) 1a-H77c, 1b-Con1 and 2a-JFH-1 lab strain replicons adapted to high-level cell culture replication was investigated. RESULTS NS3 protease sequences derived from HCV Gt 1a or Gt 1b infected patients were transferred into Gt 1a and 1b replicons, respectively. Replication was detected for 20% of Gt 1a and 75% of Gt 1b sequences. Incorporation of known cell culture adaptive change NS3-E176G improved replication of Gt 1b but not of Gt 1a sequences. Transfer of Gt 1a clinical sequences into the Gt 1b background enhanced replication and allowed phenotypic analysis of all sequences. A correlation was observed between clinical isolate sequence polymorphisms and reduced susceptibility to NS3 PI. In mixed populations containing known NS3 PI resistance changes NS3-R155K or D168E/V, sensitivity of resistance detection was ≥ 10%. CONCLUSIONS An HCV replicon capable of supporting phenotypic characterization of patient-derived HCV NS3 protease sequences was developed. Pre-existence of amino acid changes associated with NS3 PI resistance highlights the need for combination therapies in the treatment of HCV.

Impairment of type I but not type III IFN signaling by hepatitis C virus infection influences antiviral responses in primary human hepatocytes.

Peginterferon lambda-1a (Lambda), a type III interferon (IFN), acts through a unique receptor complex with limited cellular expression outside the liver which may result in a differentiated tolerability profile compared to peginterferon alfa (alfa). In Phase 2b clinical studies, Lambda administered in combination with ribavirin (RBV) was efficacious in patients with hepatitis C virus (HCV) infection representing genotypes 1 through 4, and was associated with more rapid declines in HCV RNA compared to alfa plus RBV. To gain insights into potential mechanisms for this finding, we investigated the effects of HCV replication on IFN signaling in primary human hepatocytes (PHH) and in induced hepatocyte-like cells (iHLCs). HCV infection resulted in rapid down-regulation of the type I IFN-α receptor subunit 1 (IFNAR1) transcript in hepatocytes while the transcriptional level of the unique IFN-λ receptor subunit IL28RA was transiently increased. In line with this observation, IFN signaling was selectively impaired in infected cells upon stimulation with alfa but not in response to Lambda. Importantly, in contrast to alfa, Lambda was able to induce IFN-stimulated gene (ISG) expression in HCV-infected hepatocytes, reflecting the onset of innate responses. Moreover, global transcriptome analysis in hepatocytes indicated that Lambda stimulation prolonged the expression of various ISGs that are potentially beneficial to antiviral defense mechanisms. Collectively, these observed effects of HCV infection on IFN receptor expression and signaling within infected hepatocytes provide a possible explanation for the more pronounced early virologic responses observed in patients treated with Lambda compared to alfa.

Structure-activity relationships of 4-hydroxy-4-biaryl-proline acylsulfonamide tripeptides: A series of potent NS3 protease inhibitors for the treatment of hepatitis C virus

The design and synthesis of a series of tripeptide acylsulfonamides as potent inhibitors of the HCV NS3/4A serine protease is described. These analogues house a C4 aryl, C4 hydroxy-proline at the S2 position of the tripeptide scaffold. Information relating to structure-activity relationships as well as the pharmacokinetic and cardiovascular profiles of these analogues is provided.

Isolation and characterization of interferon lambda-resistant hepatitis C virus replicon cell lines.

Pegylated interferon lambda-1a (Lambda) is currently in clinical development for the treatment of chronic hepatitis C virus (HCV) infection. To gain insight into the potential mechanisms of non-responsiveness that may occur in patients treated with Lambda, HCV subgenomic replicon cell-lines with impaired susceptibility to the unpegylated recombinant (r) form of interferon (IFN) lambda-1 (rIFNλ) were isolated and characterized. The selected replicon cell populations showed a defect in the activation of the IFN-dependent JAK-STAT signaling pathway. Reduced phosphorylation of STAT proteins and lower expression levels of the cellular janus kinases Jak1 and Tyk2 were observed in these cell populations, which may account for the impaired JAK-STAT signaling and reduced antiviral responses to rIFNλ. Overall, this in vitro study provides molecular insights into the possible mechanism of viral evasion to rIFNλ in the HCV replicon cell system.

Potent Inhibitors of Hepatitis C Virus NS3 Protease: Employment of a Difluoromethyl Group as a Hydrogen-Bond Donor

The design and synthesis of potent, tripeptidic acylsulfonamide inhibitors of HCV NS3 protease that contain a difluoromethyl cyclopropyl amino acid at P1 are described. A cocrystal structure of 18 with a NS3/4A protease complex suggests the presence of a H-bond between the polarized C-H of the CHF2 moiety and the backbone carbonyl of Leu135 of the enzyme. Structure-activity relationship studies indicate that this H-bond enhances enzyme inhibitory potency by 13- and 17-fold compared to the CH3 and CF3 analogues, respectively, providing insight into the deployment of this unique amino acid.

The discovery and optimization of naphthalene-linked P2-P4 Macrocycles as inhibitors of HCV NS3 protease.

Naphthalene-linked P2-P4 macrocycles within a tri-peptide-based acyl sulfonamide chemotype have been synthesized and found to inhibit HCV NS3 proteases representing genotypes 1a and 1b with single digit nanomolar potency. The pharmacokinetic profile of compounds in this series was optimized through structural modifications along the macrocycle tether as well as the P1 subsite. Ultimately a compound with oral bioavailability of 100% in rat, and a long half-life in plasma was obtained. However, compounds in this macrocyclic series exhibited cardiac effects in an isolated rabbit heart model and for this reason further optimization efforts were discontinued.