This thesis focuses on the identification and synthesis as well as kinetic and structural characterization of non-peptidic small molecule inhibitors of the two aspartic proteases HTLV-1 protease (HTLV-1 PR) and endothiapepsin.
The HTLV-1 PR, a promising target for the treatment of viral infections caused by the human T-cell leukemia virus type-1, is related to the well-known HIV-1 PR, however, it exhibits a substantially different substrate specificity and inhibition profile than the latter. However, due to the similarity in the active site, HIV-1 PR inhibitors provide a promising starting point for the identification and further optimization of novel small molecule inhibitors against HTLV-1 PR.
First, after successful establishment of an in-house HTLV-1 protease technology platform, the well-known HIV-1 PR inhibitor indinavir, which displays a Ki-value in the one-digit micromolar range (3.5 µM) against the HTLV-1 PR, was chosen as auspicious starting point although in comparison to the HIV-1 PR (540 pM) its affinity is strongly reduced. However, other highly potent HIV-1 protease inhibitors (saquinavir, ritonavir, nelfinavir, and amprenavir) do not show any relevant affinity against HTLV-1 protease (Ki > 20 μM). Within this thesis the X-ray structure of indinavir in complex with the HTLV-1 PR was determined at 2.40 Å resolution, representing, to the best of our knowledge, the first HTLV-1 PR crystal structure with a non-peptidic inhibitor. This structural information laid the foundation for rationalizing the rather moderate affinity of indinavir against the HTLV-1 PR and thus provided the basis for further structure-guided optimization strategies.
As a second approach for lead identification, the privileged structure concept was exploited as tool to identify novel small molecule scaffolds for HTLV-1 PR inhibition. A screening of our in-house aspartic protease inhibitor library was performed and resulted in the identification of C2-symmetric 3,4-bis-N-alkylsulfonamido-pyrrolidines and pyrrolidine-based bicyclic HIV-1 PR inhibitors as promising candidates for HTLV-1 PR inhibition. Both inhibitor classes were characterized in more detail regarding their kinetic as well as structural properties.
Out of a series of ten 3,4-bis-N-alkylsulfonamido-pyrrolidine inhibitors, AB84 exhibits an affinity of 15 nM (Ki-value) and represents, to the best of our knowledge, the most potent non-peptidic inhibitor of HTLV-1 PR described so far. The successfully determined crystal structures of AB84 and another representative of this inhibitor series, enabled structure-guided SAR interpretations thus laying the foundation for the deduction of design ideas for further optimization of this inhibitor scaffold. The pyrrolidine-based bicyclic compounds exhibit affinities from the three-digit up to the one-digit micromolar range, the most potent inhibitor of this series (Ki-value: 1.4 µM) is substituted with two benzhydryl moieties. A crystal structure of this inhibitor series was determined with NK101.
Based on these fundamental insights and the deduced SAR described in this thesis, both scaffolds represent promising starting points for the further inhibitor optimization utilizing structure-based drug design.
The second part of this thesis deals with the aspartic protease endothiapepsin that serves as a model system for aspartic proteases in general. Various 2-aminothiophene compounds were synthesized as inhibitors of endothiapepsin utilizing the Gewald reaction. Surprisingly, the binding mode analysis of eight similar 2-aminothiophene inhibitors resulted in four completely different binding modes, hence, explaining retrospectively, why the initial deduction of the SAR based on the obtained affinity data had failed. Moreover, we could demonstrate that discrepancies between affinity data based on enzyme kinetics and TSA results within one inhibitor series may be exploited as a hint for putative changes in the adopted binding geometry. The presented example highlights the complexity of binding events, their strong dependence on seemingly minor effects of the scaffold decoration and the necessity to continuously monitor binding modes during the hit-to-lead optimization process.