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. 2024 Mar 28;67(6):4833-4854.
doi: 10.1021/acs.jmedchem.3c02388. Epub 2024 Mar 13.

Mechanism-Based Macrocyclic Inhibitors of Serine Proteases

Vishnu C Damalanka  1 , Victoria Banas  1 , Paolo De Bona  1 , Maithri M Kashipathy  2 , Kevin Battaile  3 , Scott Lovell  2 , James W Janetka  1
Affiliations

Mechanism-Based Macrocyclic Inhibitors of Serine Proteases

Vishnu C Damalanka et al. J Med Chem. .

Abstract

Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, and rapid clearance of peptides. Here, we describe the rational design, synthesis, and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead. We have identified potent and selective inhibitors of TMPRSS2, matriptase, hepsin, and HGFA and demonstrated their improved protease selectivity, metabolic stability, and pharmacokinetic (PK) properties. We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD4162 (8b) bound to matriptase, revealing an unexpected binding conformation. Cyclic biphenyl ether VD5123 (11) displayed the best PK properties in mice with a half-life of 4.5 h and compound exposure beyond 24 h. These new cyclic tripeptide scaffolds can be used as easily modifiable templates providing a new strategy to overcoming the obstacles presented by linear acyclic peptides in protease inhibitor drug discovery.

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Figures

Figure 1.
Figure 1.
Examples of natural products and protease inhibitors containing side-chain-cyclized peptides.
Figure 2.
Figure 2.
Rational design of side-chain-cyclized serine protease inhibitors targeting HGFA. (A) X-ray structure of Ac-KQLR-cmk bound to HGFA (PDB: 2WUC) indicates a 6.1 Å distance from the Lys and Leu side chains. (B) Connecting the side chains into different macrocyclic ring systems like a cycloamide from a P4 Lys and P2 Asp (green) or phenyl ether from a P4 Tyr and P2 AllylGly (magenta) yields conformationally restricted B-sheet mimetics (shown with aldehyde warheads). These were docked to HGFA and shown to fit nicely with the cyclic linker branching the S4─S2 pockets, whereas the P3 side chain binds near the S3 pocket.
Figure 3.
Figure 3.
(A) Fo-Fc omit map (green mesh) contoured at 3σ showing 8b covalently bound to S805 (light green cylinders). (B) Hydrogen-bond interactions (dashed lines) between 8b and matriptase (magenta).
Figure 4.
Figure 4.
Binding mode of the second 8b molecule (gold) above the matriptase active site. (A) Fo-Fc omit map (green mesh) contoured at 3σ. (B) Hydrogen-bond interactions (dashed lines) between 8y and matriptase (magenta).
Figure 5.
Figure 5.
Electrostatic surface representation showing the 8b molecule (A) covalently bound (gray) to matriptase and (B) the second binding site (gold). The electrostatic surface spans from −0.5 (red) to 0.5 V (blue).
Figure 6.
Figure 6.
Pharmacokinetics of compounds 4, 5, 8b, and 11a in mice. Shown is the plasma concentration over time following a 13–20 mg/kg IP dose of compound. Concentrations from individual mice are shown in different colored curves. The terminal half-life of biphenyl ether macrocycle 11a was the best (t1/2 = 4.5 h) with compound coverage out to 24 h.
Scheme 1.
Scheme 1.
Synthesis of P2─P4 Alkenyl-Phenyl Ether Macrocyclic Analog
Scheme 2.
Scheme 2.
Synthesis of P2─P4 Phenyl Alkyl Ether Macrocyclic Analogs
Scheme 3.
Scheme 3.
Synthesis of P2─P4 Alkenyl Amide-Phenyl Ether Linker Macrocyclic Compounds
Scheme 4.
Scheme 4.
Synthesis of P2─P4 Triazole Linkers
Scheme 5.
Scheme 5.
Synthesis of P2─P4 Para–Meta Biphenyl Ether Macrocyclic Analog
Scheme 6.
Scheme 6.
Synthesis of P2─P4 Para–Para Biphenyl Ether Macrocyclic Analog
Scheme 7.
Scheme 7.
Synthesis of P2─P4 Meta–Para Biphenyl Ether Macrocyclic Analog
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References

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