Preclinical Characterization of Naturally Occurring Polyketide Cyclophilin Inhibitors from the Sanglifehrin Family

Abstract

Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30-to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC 50 s] of 0.070 M and 0.16 M in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F < 4%) and low solubility (<25 M), although the half-lives (t 1/2 ) of SfA and SfB in mouse blood after intravenous (i.v.) dosing were long (t 1/2 > 5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties.Hepatitis C virus (HCV) is a positive-strand RNA virus and is the major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (39). The World Health Organization (WHO) estimates that there are more than 170 million chronic carriers of HCV, which is approximately 3% of the world population (28,29). In developed countries, 50 to 76% of all cases of liver cancer and two-thirds of all liver transplants are due to chronic HCV infection (37).The current standard of care (SOC) involves subcutaneous injections of pegylated alpha-interferon (peg-IFN-␣) and oral dosing of the nonspecific antiviral drug ribavirin for a period of 24 to 48 weeks (9). Overall, response rates to the SOC depend upon the genotype and the pretreatment HCV RNA levels. Patients with genotypes 2 and 3 are more responsive to the SOC than patients infected with genotype 1 (particularly 1b) (25, 31). A significant number of HCV patients do not respond adequately to the SOC or cannot tolerate it due to side effects, leading to poor compliance and a sustained virological response (SVR) of about 50% (31).Direct-acting antiviral (DAA) drugs are being developed to target viral proteins, such as HCV NS5B polymerase or NS3 protease (35). One concern with the increased use of virustargeted agents is that, as was observed with HIV treatment, drug resistance emerges (47). However, compounds that target human proteins (i.e., cyclophilins), rather than viral targets, are also being developed, which should have higher hurdles for viral breakthrough and incidence of resistance duri...

Multiple Mutations in Hepatitis C Virus NS5A Domain II Are Required To Confer a Significant Level of Resistance to Alisporivir

“…We first found that alisporivir profoundly blocks wild-type Con1 replication (Fig. 2D), confirming that this Cyp inhibitor efficiently inhibits HCV replication in vitro (6,11,18,19,25,26,33,37,39,44,55). Importantly, the D320E or Y321N mutation alone confers only a slight resistance to the Cyp inhibitor, with the Y321N mutation having a more profound resistance impact (Fig.…”

“…We and others showed that CypA, via its isomerase pocket, directly binds NS5A (4,10,22,28,32). We and others also showed that Cyp inhibitors, including CsA, alisporivir, sanglifehrins, and sangamides, totally disrupt NS5A-CypA interactions (5,6,10,14,19,22,54,57). Thus, one possibility explaining how D320E and Y321N mutations render HCV resistant to alisporivir is that the mutations render NS5A-CypA interactions resistant to the drug-mediated dissociation.…”

The Cyclophilin Inhibitor SCY-635 Disrupts Hepatitis C Virus NS5A-Cyclophilin A Complexes

“…These data suggest that the D320E and Y321N mutations confer low-level resistance of HCV (Con1) to Cyp inhibitors, possibly by reducing the need for CypA-dependent isomerization of NS5A. It will be interesting to determine whether the D320E and Y321N mutations confer "universal" cross-resistance among Cyp inhibitors, including SCY-635, NIM811, alisporivir, CsA, sanglifehrins A to D, and sangamides (amide derivatives of sanglifehrin A) (19). It is important to note that the RNA binding of domain II of NS5A containing the D320E mutation was unaffected by CypA (14).…”

Mitochondrial Dysfunction and Permeability Transition in Osteosarcoma Cells Showing the Warburg Effect

“…To further confirm the involvement of the MPT, we studied the effect of the MPT inhibitor SfA on mitochondrial morphology and function in these cells. We preferred SfA to the more commonly used cyclosporin A because SfA was shown to be more specific and less immunosuppressive (32). We treated LM7 and 143B cells with SfA for various times (data not shown) and found that a 7-day incubation produced the most stable effects.…”