Voclosporin and Other Cyclosporin Analogs

Voclosporin, an analog of cyclosporin A (CsA), is a calcineurin inhibitor approved as an immunosuppressant in the treatment of lupus nephritis. Lupus nephritis is a manifestation of a disease called systemic lupus erythematosus. In lupus nephritis, the immune system attacks the kidneys, causing inflammation and kidney damage, leading to chronic renal failure. Therefore, early intervention matters a lot. Voclosporin has an enhanced inhibitory effect on calcineurin and higher metabolic stability. Early intervention with Voclosporin combined with renal response can bring more positive clinical outcomes for lupus nephritis patients. Voclosporin is used with another immunosuppressive drug called mycophenolate mofetil for adults with active lupus nephritis Class III, IV, or V, which are serious forms of lupus nephritis disease.

Voclosporin's chemical structure is very similar to cyclosporin. It replaces the methyl group on the side chain of the amino acid residue 1 of CsA with a vinyl group, and others remain identical.

Fig 1. Chemical structure of cyclosporin and voclosporin¹

Pharmacological properties of cyclosporin analogs

Voclosporin targets calcineurin, which, like CsA, first binds to a receptor called cyclophilin, forming an admixture that acts on calcineurin to inhibit its biological activity and then blocks the production of IL-2 and T-cell-mediated immune responses. Due to calcineurin inhibition, podocytes (cells in the kidney) are stabilized and inflammation is reduced, which can prevent further kidney damage.

• Calcineurin (CaN)

Calcineurin is a calcium and calmodulin dependent serine/threonine protein phosphatase. It activates the immune system's T cells and can be blocked by drugs. Calcineurin activates nuclear factor of activated T cells (NFATc) by dephosphorylation. Activated NFATc is transferred to the nucleus, where it up-regulates the expression of interleukin 2 (IL-2) to stimulate the growth and differentiation of T-cell responses. Calcineurin is the target of a class of drugs called calcineurin inhibitors, including cyclosporin, voclosporin, pimecrolimus, and tacrolimus.

• Cyclophilins (CYPs)

The inhibitory effect of cyclosporine and voclosporin on calcineurin begins with binding to CYP. Cyclophilins are a family of proteins named for their ability to bind to cyclosporine. These proteins exhibit prolyl isomerase activity, which catalyzes the conversion of peptide bonds from trans to cis-form at proline residues and promotes protein folding. Cyclophilin A (CYPA), also known as Peptidylprolyl isomerase A (PPIA), is present in the cytosol and has a β barrel structure with two α-helixes and a β-sheet. Other members of the cyclophilin family have a structure similar to that of cyclophilin A. The cyclosporin-cyclophilin A complex inhibits calcium/calmodulin-dependent calcineurin.

Much experimental evidence also suggests that cyclophilins are of great value to the medical field, not only in cardiovascular disease but also in cancer and viral infections. Overexpression of cyclophilin A has been associated with adverse reactions to inflammatory diseases, progression or metastasis of cancer, and aging. Cyclophilin inhibitors, such as cyclosporin, can be developed to treat neurodegenerative diseases. Cyclophilin inhibition may also be a treatment for liver disease.

During viral infection, cyclophilins support viral replication in cells infected with the hepatitis C virus (HCV), coronavirus (CoV), HIV, and hepatitis B virus (HBV). However, in influenza- and rotavirus-infected cells, cyclophilins play a positive role by disrupting viral proteins or triggering host defense mechanisms in cells.

Pharmacologic potential of cyclosporine analogs

Cyclosporin analogs have been in development for several indications. Similar to cyclosporine, these analogs are potential immunosuppressant drugs that bind to the cyclophilin A (CypA) receptor, then the complex binds to calcineurin to achieve its diverse biological functions. The binding of CsA-CypA to calcineurin inhibits its calcium-dependent phosphatase activity, which plays a critical role in the nuclear translocation of NFAT. Studies have uncovered that the binding of CsA to cyclophilin mainly depends on its 1, 2, 9, and 10 amino acid residues, while other parts contribute relatively little. The immunosuppressive biological activity mainly relies on the combination of CsA and calcineurin, in which amino acid residues at 4, 5, and 6 play an important role.

Given these differences in binding receptors, if the goal of the lead compound is to inhibit the cyclophilin and minimize the immunosuppressive effect, researchers can target the chemical structure of CsA. For example, the introduction of benzyl as a 3-position side chain into CsA to obtain derivative 1 did not affect the ability of CsA to inhibit cyclophilin A but significantly reduced its immunosuppressive effect. This modification produced a non-immunosuppressive cyclophilin inhibitor.

CsA derivative 2, obtained by methyl substitution of 6 isopropyl groups, had 50% of the natural CsA's cyclophilin binding affinity, but reduced immunosuppression to 0.4% of the natural CsA.

Substituting the 4-position amino acid side chain in CsA to produce derivative 3 or 4 preserved or enhanced binding to cyclophilin A while significantly reducing its activity in immunosuppressive assays. Based on these results, it was concluded that calcineurin had a unique P4 pocket that was later confirmed by crystallization experiments.

5 and 6 (NIM811) derived from changes in four amino acids can inhibit HIV replication and significantly reduce immunosuppression in a cell assay. Interestingly, compound 5 was four times more effective than compound 6 in binding analysis but less effective than compound 6 in anti-HIV cell tests. Compound 6 had a stronger affinity for cyclophilin A than natural CsA. The inhibitor had been tested in animal and cellular models for a variety of diseases, as well as in clinical trials as a treatment agent for HCV. After finding its ability to inhibit HCV replication, researchers conducted further tests to see if it could be used as a treatment for HCV infection. It was found that non-immunosuppressive cyclophilin A inhibitors presented a significant genetic barrier to viral evolution, and CsA analogs were expected to be available as host-targeted therapeutic agents in the future.

CsA derivative 7 (debio-025 or alisporivir) was 5-10 times more effective than CsA in inhibiting subgenomic HCV replication. And this inhibitor was more efficient than CsA analog 6. The antiviral activity of debio-025 was higher than that of earlier CsA-like inhibitors, which may be attributed to its altered P3 group, conferring a higher affinity for cyclophilin A. The cyclophilin A inhibitor was very effective in treating HCV genotype 2 and 3 infections. At the same time, debio-25 was also being developed to treat HIV infection. Its anti-HIV effect originated from its ability to prevent cyclophilin A from attaching to the HIV-p24 Gag protein.

The modification of one amino acid residue successfully led to the launch of voclosporin, and interest in the modification of this site continued. By introducing benzimidazole groups into the 1-position side chain, derivative 8 had been developed as an extracellular cyclophilin A inhibitor to inhibit leukocyte transport. Notably, the chemical modification resulted in the cell permeability of derivative 8 is at least 50 times lower than that of CsA. Although its cell permeability was reduced, analog 8 blocked leukocyte migration to cyclophilin A. The inhibitor also reduced white blood cell activation in the murine contact hypersensitivity model.

A recent study showed that the 5-site amino acid-modified CsA analog 9 can act as an NTCP inhibitor and reduce bile acid uptake. This potent-specific inhibitor exhibited extensive antiviral activity against genomic HBV and hepatitis D viruses and demonstrated excellent pharmacokinetics and oral bioavailability. But its immunosuppressive effect was lower than that of CsA.

Conclusion of Cyclosporine and its Analogs

Due to its good biological binding ability to cyclophilins and calcineurin, cyclosporine and its analogs have great potential in a wide range of fields in immunosuppression, antiviral, and treatment of autoimmune diseases. Two drugs, cyclosporine and voclopsorin have been successfully developed to inhibit calcineurin. Cyclosporine has been around since the 1980s, and voclosporin is approved for lupus nephritis nearly 40 years later. But beyond inhibiting calcineurin, the binding of cyclosporin analogs to cyclophilins also plays a key role in many viral diseases. The vision that further research in this area could lead to superior antiviral drugs (hepatitis B, hepatitis C, HIV, Covid-19) is likely to come to reality soon. At the same time, oral delivery of cyclosporine and voclosporin also suggests that these lead compounds have a promising future as oral formulations.

References

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