Structure-based Development of Secondary Amines as Aspartic Protease Inhibitors

As novel promising scaffold for HIV protease inhibition pyrrolidine-derived inhibitors have recently been reported. In this thesis the stepwise improvement of this compound class to potent inhibitors of wildtype as well as selected mutant proteases utilizing rational drug discovery methods is reported. Based on the crystal structure of a (rac)-3,4-dimethyleneamino-pyrrolidine in complex with HIV-1 protease symmetric pyrrolidine-diesters possessing the same stereochemistry were synthesized following a chiral-pool approach. The most potent compounds of the series achieve one-digit micromolar inhibition towards wild type as well as two mutant proteases (Ile50Val and Ile84Val). The cocrystal structure of one derivative in complex with the Ile84Val HIV protease revealed that two inhibitor molecules are bound in the large active site cavity comprising an area encompassed by the catalytic dyad and the flaps in the open conformation. This is the first HIV protease cocrystal structure in which the open-flap conformation of the enzyme is stabilized by an inhibitor that concomitantly addresses the catalytic dyad. As an alternative approach towards HIV protease inhibitors, the development of symmetric 3,4-bis N-alkyl sulfonamide-pyrrolidines is described. The initial lead structure possessing benzene sulfonamide groups and benzyl substituents exhibited a Ki of 2.2 µM. The X-ray structure in complex with the HIV protease enabled the rational design of a second series of inhibitors and revealed three promising symmetric substitution patterns for further lead optimization: (A) Elongation of the P1/P1’-benzyl moieties with hydrophobic substituents in para-position, (B) ortho-substitution at the P2/P2’-phenyl ring systems, and (C) para-substitution at the P2/P2’-phenyl moieties. All three strategies were pursued and resulted in inhibitors with improved affinities up to 260 nM. To elucidate the underlying factors accounting for the SAR, the crystal structures of four representatives, at least one of each modification type, in complex with HIV protease were determined. These structures provided deeper insights into the protein–ligand interactions and the underlying principles of the SAR thus enabling to choose the most promising combination of substituents in the next design cycle. The combination of these substituents rendered a final inhibitor showing a significantly improved affinity of Ki = 74 nM and the cocrystal structure in complex with the HIV protease confirmed the successful application of the pursued optimization strategy. Subsequently the influence of the active site mutations Ile50Val and Ile84Val on these inhibitors is investigated by structural and kinetic analysis. Whereas the Ile50Val mutation leads to a significant decrease in affinity for all compounds in this series, they retain or even show increased affinity towards the crucial Ile84Val mutation. By detailed analysis of the crystal structures of two representatives in complex with wild-type and mutant proteases the structural basis of this phenomenon was elucidated. Inhibitors bearing smaller N-alkyl substituents revealed a selectivity profile not being explicable with the initial SAR. By cocrystallization of the most potent derivative of a small series with HIV-1 protease, astonishingly two different crystal forms, P2(1)2(1)2(1) and P6(1)22, were obtained. Structural analysis revealed two completely different binding modes, the interaction of the pyrrolidine nitrogen atom to the catalytic aspartates being the only similarity. Encouraged by the successful utilization of cyclic secondary amines as anchoring group in the development of HIV protease inhibitors, this strategy was expanded into a general approach for lead structure identification for aspartic proteases. An initial library comprising eleven inhibitors based on easily accessible achiral linear oligoamines was developed and screened against six selected aspartic proteases (HIV-1 protease, plasmepsin II, plasmepsin IV, renin, BACE-1, and pepsin). Several hits could be identified, among them selective as well as rather promiscuous inhibitors. The design concept was consecutively confirmed by determination of the crystal structure of two derivatives in complex with HIV-1 protease. The binding modes exhibit high similarity to the binding orientation of substrates as well as to that of peptidomimetic inhibitors. Using this information, a generalization of this binding situation to other aspartic proteases appears reasonable, thus providing a first insight into the observed structure-activity relationships

Structure-based Development of Secondary Amines as Aspartic Protease Inhibitors

As novel promising scaffold for HIV protease inhibition pyrrolidine-derived inhibitors have recently been reported. In this thesis the stepwise improvement of this compound class to potent inhibitors of wildtype as well as selected mutant proteases utilizing rational drug discovery methods is reported. Based on the crystal structure of a (rac)-3,4-dimethyleneamino-pyrrolidine in complex with HIV-1 protease symmetric pyrrolidine-diesters possessing the same stereochemistry were synthesized following a chiral-pool approach. The most potent compounds of the series achieve one-digit micromolar inhibition towards wild type as well as two mutant proteases (Ile50Val and Ile84Val). The cocrystal structure of one derivative in complex with the Ile84Val HIV protease revealed that two inhibitor molecules are bound in the large active site cavity comprising an area encompassed by the catalytic dyad and the flaps in the open conformation. This is the first HIV protease cocrystal structure in which the open-flap conformation of the enzyme is stabilized by an inhibitor that concomitantly addresses the catalytic dyad. As an alternative approach towards HIV protease inhibitors, the development of symmetric 3,4-bis N-alkyl sulfonamide-pyrrolidines is described. The initial lead structure possessing benzene sulfonamide groups and benzyl substituents exhibited a Ki of 2.2 µM. The X-ray structure in complex with the HIV protease enabled the rational design of a second series of inhibitors and revealed three promising symmetric substitution patterns for further lead optimization: (A) Elongation of the P1/P1’-benzyl moieties with hydrophobic substituents in para-position, (B) ortho-substitution at the P2/P2’-phenyl ring systems, and (C) para-substitution at the P2/P2’-phenyl moieties. All three strategies were pursued and resulted in inhibitors with improved affinities up to 260 nM. To elucidate the underlying factors accounting for the SAR, the crystal structures of four representatives, at least one of each modification type, in complex with HIV protease were determined. These structures provided deeper insights into the protein–ligand interactions and the underlying principles of the SAR thus enabling to choose the most promising combination of substituents in the next design cycle. The combination of these substituents rendered a final inhibitor showing a significantly improved affinity of Ki = 74 nM and the cocrystal structure in complex with the HIV protease confirmed the successful application of the pursued optimization strategy. Subsequently the influence of the active site mutations Ile50Val and Ile84Val on these inhibitors is investigated by structural and kinetic analysis. Whereas the Ile50Val mutation leads to a significant decrease in affinity for all compounds in this series, they retain or even show increased affinity towards the crucial Ile84Val mutation. By detailed analysis of the crystal structures of two representatives in complex with wild-type and mutant proteases the structural basis of this phenomenon was elucidated. Inhibitors bearing smaller N-alkyl substituents revealed a selectivity profile not being explicable with the initial SAR. By cocrystallization of the most potent derivative of a small series with HIV-1 protease, astonishingly two different crystal forms, P2(1)2(1)2(1) and P6(1)22, were obtained. Structural analysis revealed two completely different binding modes, the interaction of the pyrrolidine nitrogen atom to the catalytic aspartates being the only similarity. Encouraged by the successful utilization of cyclic secondary amines as anchoring group in the development of HIV protease inhibitors, this strategy was expanded into a general approach for lead structure identification for aspartic proteases. An initial library comprising eleven inhibitors based on easily accessible achiral linear oligoamines was developed and screened against six selected aspartic proteases (HIV-1 protease, plasmepsin II, plasmepsin IV, renin, BACE-1, and pepsin). Several hits could be identified, among them selective as well as rather promiscuous inhibitors. The design concept was consecutively confirmed by determination of the crystal structure of two derivatives in complex with HIV-1 protease. The binding modes exhibit high similarity to the binding orientation of substrates as well as to that of peptidomimetic inhibitors. Using this information, a generalization of this binding situation to other aspartic proteases appears reasonable, thus providing a first insight into the observed structure-activity relationships

Discovery of a chemical probe to study implications of BPTF bromodomain inhibition in cellular and in vivo experiments

The bromodomain and PHD-finger containing transcription factor (BPTF) is part of the nucleosome remodeling factor (NURF) complex and has been implicated in multiple cancer types. Here, we report the discovery of a potent and selective chemical probe targeting the bromodomain of BPTF with an attractive pharmacokinetic profile enabling cellular and in vivo experiments in mice. Microarray-based transcriptomics in presence of the probe in two lung cancer cell lines revealed only minor effects on the transcriptome. Profiling against a panel of cancer cell lines revealed that the antiproliferative effect does not correlate with BPTF dependency score in depletion screens. Both observations and the multi-domain architecture of BPTF suggest that depleting the protein by proteolysis targeting chimeras (PROTACs) could be a promising strategy to target cancer cell proliferation. We envision that the presented chemical probe and the related negative control will enable the research community to further explore scientific hypotheses with respect to BPTF bromodomain inhibition

HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin–proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein–protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins

Drugit: Crowd-sourcing molecular design of non-peptidic VHL binders

Given the role of human intuition in current drug design efforts, crowd-sourced \u27citizen scientist\u27 games have the potential to greatly expand the pool of potential drug designers. Here, we introduce ‘Drugit\u27, the small molecule design mode of the online ‘citizen science’ game Foldit. We demonstrate its utility for design with a use case to identify novel binders to the von Hippel Lindau E3 ligase. Several thousand molecule suggestions were obtained from players in a series of 10 puzzle rounds. The proposed molecules were then evaluated by in silico methods and by an expert panel and selected candidates were synthesized and tested. One of these molecules, designed by a player, showed dose-dependent shift perturbations in protein-observed NMR experiments. The co-crystal structure in complex with the E3 ligase revealed that the observed binding mode matched in major parts the player’s original idea. The completion of one full design cycle is a proof of concept for the Drugit approach and highlights the potential of involving citizen scientists in early drug discovery