Jark Böttcher

Targeting the open-flap conformation of HIV-1 protease with pyrrolidine-based inhibitors.

HIV protease is a well‐established drug target in antiviral chemotherapy. Immense research efforts have been made to discover effective inhibitors, thus making the enzyme one of the most studied and best characterized proteins. Although the protease exhibits high flexibility, all approved drugs target virtually the same protein conformation. The development of viral cross‐resistance demands the generation of inhibitors with novel scaffolds and deviating modes of binding. Herein we report the design and the short, high‐yielding stereoselective synthesis of a series of chiral, symmetric pyrrolidine‐based inhibitors targeting the open‐flap conformation of the protease. The obtained co‐crystal structure with one derivative provides a valuable starting point for further inhibitor design.

The Potential of P1 Site Alterations in Peptidomimetic Protease Inhibitors as Suggested by Virtual Screening and Explored by the Use of CC-Coupling Reagents†

A synthetic concept is presented that allows the construction of peptide isostere libraries through polymer‐supported C‐acylation reactions. A phosphorane linker reagent is used as a carbanion equivalent; by employing MSNT as a coupling reagent, the C‐acylation can be conducted without racemization. Diastereoselective reduction was effected with L‐selectride. The reagent linker allows the preparation of a norstatine library with full variation of the isosteric positions including the P1 side chain that addresses the protease S1 pocket. Therefore, the concept was employed to investigate the P1 site specificity of peptide isostere inhibitors systematically. The S1 pocket of several aspartic proteases including plasmepsin II and cathepsin D was modeled and docked with ≈500 amino acid side chains. Inspired by this virtual screen, a P1 site mutation library was designed, synthesized, and screened against three aspartic proteases (plasmepsin II, HIV protease, and cathepsin D). The potency of norstatine inhibitors was found to depend strongly on the P1 substituent. Large, hydrophobic residues such as biphenyl, 4‐bromophenyl, and 4‐nitrophenyl enhanced the inhibitory activity (IC50) by up to 70‐fold against plasmepsin II. In addition, P1 variation introduced significant selectivity, as up to 9‐fold greater activity was found against plasmepsin II relative to human cathepsin D. The active P1 site residues did not fit into the crystal structure; however, molecular dynamics simulation suggested a possible alternative binding mode.

Structural and Kinetic Analysis of Pyrrolidine-Based Inhibitors of the Drug-Resistant Ile84Val Mutant of HIV-1 Protease

Human immunodeficiency virus (HIV) protease is a well-established drug target in HIV chemotherapy. However, continuously increasing resistance towards approved drugs inevitably requires the development of new inhibitors preferably showing no susceptibility against resistant HIV protease strains. Recently, symmetric pyrrolidine-3,4-bis-N-benzyl-sulfonamides have been developed as a new class of HIV-1 protease inhibitors. The most promising candidate exhibited a K(i) of 74 nM towards a wild-type protease. Herein, we report the influence of the active-site mutations Ile50Val and Ile84Val on these inhibitors by structural and kinetic analysis. Although 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 important Ile84Val mutation. By detailed analysis of the crystal structures of two representatives in complex with wild-type and mutant proteases, we were able to elucidate the structural basis of this phenomenon.

Unexpected Novel Binding Mode of Pyrrolidine-Based Aspartyl Protease Inhibitors: Design, Synthesis and Crystal Structure in Complex with HIV Protease

At present nine FDA‐approved HIV protease inhibitors have been launched to market, however rapid drug resistance arising under antiviral therapy calls upon novel concepts. Possible strategies are the development of ligands with less peptide‐like character or the stabilization of a new and unexpected binding‐competent conformation of the protein through a novel ligand‐binding mode. Our rational design of pyrrolidinedimethylene diamines was inspired by the idea to incorporate key structural elements from classical peptidomimetics with a non‐peptidic heterocyclic core comprising an endocyclic amino function to address the catalytic aspartic acid side chains of Asp 25 and 25′. The basic scaffolds were decorated by side chains already optimized for the recognition pockets of HIV protease or cathepsin D. A multistep synthesis has been established to produce the central heterocycle and to give flexible access to side chain decorations. Depending on the substitution pattern of the pyrrolidine moiety, single‐digit micromolar inhibition of HIV‐1 protease and cathepsin D has been achieved. Successful design is suggested in agreement with our modelling concepts. The subsequently determined crystal structure with HIV protease shows that the pyrrolidine moiety binds as expected to the pivotal position between both aspartic acid side chains. However, even though the inhibitors have been equipped symmetrically by polar acceptor groups to address the flap water molecule, it is repelled from the complex, and only one direct hydrogen bond is formed to the flap. A strong distortion of the flap region is detected, leading to a novel hydrogen bond which cross‐links the flap loops. Furthermore, the inhibitor addresses only three of the four available recognition pockets. It achieves only an incomplete desolvation compared with the similarly decorated amprenavir. Taking these considerations into account it is surprising that the produced pyrrolidine derivatives achieve micromolar inhibition and it suggests extraordinary potency of the new compound class. Most likely, the protonated pyrrolidine moiety experiences strong enthalpic interactions with the enzyme through the formation of two salt bridges to the aspartic acid side chains. This might provide challenging opportunities to combat resistance of the rapidly mutating virus.

Achiral oligoamines as versatile tool for the development of aspartic protease inhibitors

Due to the important role that aspartic proteases play in many patho-physiological processes, they have intensively been targeted by modern drug development. However, up to now, only for two family members, renin and HIV protease, approved drugs are available. Inhibitor development, mostly guided by mimicking the natural peptide substrates, resulted in very potent inhibitors for several targets, but the pharmacokinetic properties of these compounds were often not optimal. Herein we report a novel approach for lead structure discovery of non-peptidic aspartic protease inhibitors using easily accessible achiral linear oligoamines as starting point. An initial library comprising 11 inhibitors was developed and screened against six selected aspartic proteases. Several hits could be identified, among them selective as well as rather promiscuous inhibitors. The design concept was confirmed by determination of the crystal structure of two derivatives in complex with the HIV-1 protease, and represents a promising basis for the further inhibitor development.