Surface plasmon resonance biosensor assay for the analysis of small-molecule inhibitor binding to human and parasitic phosphodiesterases.

In the past decade, surface plasmon resonance (SPR) biosensor-based technology has been exploited more and more to characterize the interaction between drug targets and small-molecule modulators. Here, we report the successful application of SPR methodology for the analysis of small-molecule binding to two therapeutically relevant cAMP phosphodiesterases (PDEs), Trypanosoma brucei PDEB1 which is implicated in African sleeping sickness and human PDE4D which is implicated in a plethora of disease conditions including inflammatory pulmonary disorders such as asthma, chronic obstructive pulmonary disease and central nervous system (CNS) disorders. A protocol combining the use of directed capture using His-tagged PDE_CDs with covalent attachment to the SPR surface was developed. This methodology allows the determination of the binding kinetics of small-molecule PDE inhibitors and also allows testing their specificity for the two PDEs. The SPR-based assay could serve as a technology platform for the development of highly specific and high-affinity PDE inhibitors, accelerating drug discovery processes.

Fragment-based screening in tandem with phenotypic screening provides novel antiparasitic hits.

Methods to discover biologically active small molecules include target-based and phenotypic screening approaches. One of the main difficulties in drug discovery is elucidating and exploiting the relationship between drug activity at the protein target and disease modification, a phenotypic endpoint. Fragment-based drug discovery is a target-based approach that typically involves the screening of a relatively small number of fragment-like (molecular weight <300) molecules that efficiently cover chemical space. Here, we report a fragment screening on TbrPDEB1, an essential cyclic nucleotide phosphodiesterase (PDE) from Trypanosoma brucei, and human PDE4D, an off-target, in a workflow in which fragment hits and a series of close analogs are subsequently screened for antiparasitic activity in a phenotypic panel. The phenotypic panel contained T. brucei, Trypanosoma cruzi, Leishmania infantum, and Plasmodium falciparum, the causative agents of human African trypanosomiasis (sleeping sickness), Chagas disease, leishmaniasis, and malaria, respectively, as well as MRC-5 human lung cells. This hybrid screening workflow has resulted in the discovery of various benzhydryl ethers with antiprotozoal activity and low toxicity, representing interesting starting points for further antiparasitic optimization.

Catechol pyrazolinones as trypanocidals: fragment-based design, synthesis, and pharmacological evaluation of nanomolar inhibitors of trypanosomal phosphodiesterase B1.

Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of Trypanosoma brucei, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. We used homology modeling and docking studies to guide fragment growing into the parasite-specific P-pocket in the enzyme binding site. The resulting catechol pyrazolinones act as potent TbrPDEB1 inhibitors with IC50 values down to 49 nM. The compounds also block parasite proliferation (e.g., VUF13525 (20b): T. brucei rhodesiense IC50 = 60 nM, T. brucei brucei IC50 = 520 nM, T. cruzi = 7.6 µM), inducing a typical multiple nuclei and kinetoplast phenotype without being generally cytotoxic. The mode of action of 20b was investigated with recombinantly engineered trypanosomes expressing a cAMP-sensitive FRET sensor, confirming a dose-response related increase of intracellular cAMP levels in trypanosomes. Our findings further validate the TbrPDEB family as antitrypanosomal target.

The hydrophobic core of twin-arginine signal sequences orchestrates specific binding to Tat-pathway related chaperones.

Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA) and TorD (specific for trimethylamine N-oxide reductase, TorA). Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function.

Mechanism of USP7/HAUSP activation by its C-terminal ubiquitin-like domain and allosteric regulation by GMP-synthetase.

The ubiquitin-specific protease USP7/HAUSP regulates p53 and MDM2 levels, and cellular localization of FOXO4 and PTEN, and hence is critically important for their role in cellular processes. Here we show how the 64 kDa C-terminal region of USP7 can positively regulate deubiquitinating activity. We present the crystal structure of this USP7/HAUSP ubiquitin-like domain (HUBL) comprised of five ubiquitin-like (Ubl) domains organized in 2-1-2 Ubl units. The last di-Ubl unit, HUBL-45, is sufficient to activate USP7, through binding to a "switching" loop in the catalytic domain, which promotes ubiquitin binding and increases activity 100-fold. This activation can be enhanced allosterically by the metabolic enzyme GMPS. It binds to the first three Ubl domains (HUBL-123) and hyperactivates USP7 by stabilization of the HUBL-45-dependent active state.

Nonhydrolyzable ubiquitin-isopeptide isosteres as deubiquitinating enzyme probes.

We demonstrate that oxime ligation is an efficient, straightforward, and generally applicable strategy for generating nonhydrolyzable ubiquitin (Ub)-isopeptide isosteres. We synthesized nonhydrolyzable K48- and K63-linked Ub-isopeptide isosteres to investigate the selectivity of deubiquitinating enzymes for specific linkages employing surface plasmon resonance spectroscopy. The results indicate that deubiquitinating enzymes specifically recognize the local peptide sequence flanking Ub-branched lysine residues in target proteins. The described strategy allows the systematic investigation of sequence requirements for substrate selectivity of deubiquitinating enzymes.

DUBs and disease: activity assays for inhibitor development.

Deubiquitinating enzymes (DUBs) cleave ubiquitin from substrate proteins and so influence the biochemical properties of these proteins, such as their half-life, by a reduction in proteasomal degradation. Several DUBs have been demonstrated to deubiquitinate oncogenic proteins and tumor suppressors, and their activities have been implicated in numerous processes related to disease, including cancer and neurodegeneration. The importance of various DUBs is well established, but the understanding of both their selectivity and reactivity in the ubiquitin-proteasome system is comparatively poor. In this review, the published methods that are available to study DUB action in vitro and their application in finding inhibitors are discussed.

Membrane binding of twin arginine preproteins as an early step in translocation.

The twin arginine transport (Tat) system translocates folded proteins across the bacterial inner membrane. Transport substrates are recognized by means of evolutionarily well-conserved N-terminal signal peptides. The precise role of signal peptides in the actual transport process is not yet fully understood. Potentially, much insight into the molecular details of the transport process could be gained from step-by-step in vitro experiments under controlled conditions. Here, we employ purified preproteins to study their interaction with the phospholipid membrane by using surface plasmon resonance spectroscopy. It turns out that preproteins interact tightly with a model membrane consisting of only phospholipids. This interaction, which is stabilized by both electrostatic and hydrophobic contributions, appears to constitute an early step in protein translocation by the Tat system.