Aspartic proteases are involved in various physiological and pathophysiological processes. Famous examples highlighting the success of inhibition of aspartic proteases in drug discovery include, among others, e.g. aliskiren which represents a non-peptidic inhibitor of renin, and ten HIV-1 protease (HIV-1 PR) inhibitors which are or have been clinically used in the therapy of AIDS.
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 the 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. Interestingly, indinavir binds at first glance similar to both enzymes. As the H-bond inventory between both proteases is quite conserved, one would not directly expect such a drastic difference in affinity. However, particularly the van der Waals contact inventory established by hydrophobic residues of indinavir seems to be less favorable in the HTLV-1 PR compared to the original HIV-1 PR complex thus presumably accounting for the observed affinity difference.
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
174 8. Summary
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 deter-mined crystal structures of AB84 and AB83, the latter being another representative of this inhibitor series, enabled structure-guided SAR interpretations thus laying the foundation for the deduction of design ideas for the further optimization of this inhibitor scaffold. In addition, with the determined crystal structures in hand the observed affinity differences within the members of this compound class towards HTLV-1 PR and HIV-1 PR could convincingly be rationalized.
The pyrrolidine-based bicyclic compounds exhibit affinities from the three-digit up to the one-digit micromolar range, with NK232, decorated with two benzhydryl moieties, being the most potent inhibitor of this series (Ki-value: 1.4 µM). The crystal structure of NK101 was determined at moderate resolution, nevertheless, this structure provides valuable information about the binding mode of this inhibitor scaffold.
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.
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Figure 8.1. Novel scaffolds for HTLV-1 protease inhibition as promising starting points for structure-based lead optimization identified within this thesis.
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. The presented example highlights the complexity of binding events and their strong dependence on seemingly minor effects of the scaffold decoration. As structure-based drug discovery indispensably relies on the correct deduction and interpretation
series of ten inhibitors:
Ki: >250µM – 15nM
two X-ray structures in
complex with the HTLV-1 PR X-ray structure of NK101 in complex with the HTLV-1 PR series of ten inhibitors:
Ki: 215µM – 1.4µM
NK232 Ki: 1.4µM AB84 Ki: 15nM
AB83 AB84 NK101 indinavir C2-symmetric
3,4-bis-N-alkylsulfonamido-pyrrolidines
pyrrolidine-based bicyclic inhibitors
Ki: 3.5µM X-ray structure (2.4Å)
176 8. Summary
of the underlying SAR, not only the incipient determination of the binding mode of solely a single lead representative or a set of identified hits at the beginning, but also the re-validation of the initially determined binding mode during the optimization process is of utmost importance. Moreover, we could demonstrate that any inconsistent affinity-thermal shift assay (TSA) correlation may serve as an easily available indicator to qualify candidates for structural revalidation, even if an interaction mode is believed to be known.
The detailed analysis presented herein highlights the necessity to continuously monitor binding modes during the hit-to-lead optimization process and questions the commonly accepted hypothesis that similar ligands usually bind in a similar fashion.
Figure 8.2. Multiple binding modes of Gewald reaction-based aspartic protease inhibitors: a representative case study for structure-based lead optimization projects challenging typical design paradigms in medicinal chemistry.
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