Identification of suitable target/E3 ligase pairs for PROTAC development using a rapamycin-induced proximity assay (RiPA).

The development of proteolysis targeting chimeras (PROTACs), which induce the degradation of target proteins by bringing them into proximity with cellular E3 ubiquitin ligases, has revolutionized drug development. While the human genome encodes more than 600 different E3 ligases, current PROTACs use only a handful of them, drastically limiting their full potential. Furthermore, many PROTAC development campaigns fail because the selected E3 ligase candidates are unable to induce degradation of the particular target of interest.

Correlating Predicted Reactivities with Experimental Inhibition Data of Covalent ChlaDUB1 Inhibitors.

Covalent inhibition with electrophiles of suitable reactivity allows for prolonged inactivation of a targeted enzyme, in comparison to noncovalent inhibitors. To fine-tune covalently reacting groups, various computational workflows have been developed for reactivity prediction. Their results are typically evaluated based on assays that use cysteine, glutathione, or other model nucleophiles, but the relevance of the predictions for the situation in a particular enzyme environment remains under-investigated.

Structure-Based Design and Synthesis of Covalent Inhibitors for Deubiquitinase and Acetyltransferase ChlaDUB1 of .

Upon infection by an intracellular pathogen, host cells activate apoptotic pathways to limit pathogen replication. Consequently, efficient proliferation of the obligate intracellular pathogen , a major cause of trachoma and sexually transmitted diseases, depends on the suppression of host cell apoptosis. secretes deubiquitinase ChlaDUB1 into the host cell, leading among other interactions to the stabilization of antiapoptotic proteins and, thus, suppression of host cell apoptosis.

Structural basis for the bi-specificity of USP25 and USP28 inhibitors.

The development of cancer therapeutics is often hindered by the fact that specific oncogenes cannot be directly pharmaceutically addressed. Targeting deubiquitylases that stabilize these oncogenes provides a promising alternative. USP28 and USP25 have been identified as such target deubiquitylases, and several small-molecule inhibitors indiscriminately inhibiting both enzymes have been developed.

Assessment of fragment docking and scoring with the endothiapepsin model system.

Fragment-based screening has become indispensable in drug discovery. Yet, the weak binding affinities of these small molecules still represent a challenge for the reliable detection of fragment hits. The extent of this issue was illustrated in the literature for the aspartic protease endothiapepsin: When seven biochemical and biophysical in vitro screening methods were applied to screen a library of 361 fragments, very poor overlap was observed between the hit fragments identified by the individual approaches, resulting in high levels of false positive and/or false negative results depending on the mutually compared methods.

Combined In-Solution Fragment Screening and Crystallographic Binding-Mode Analysis with a Two-Domain Hsp70 Construct.

Heat shock protein 70 (Hsp70) isoforms are key players in the regulation of protein homeostasis and cell death pathways and are therefore attractive targets in cancer research. Developing nucleotide-competitive inhibitors or allosteric modulators, however, has turned out to be very challenging for this protein family, and no Hsp70-directed therapeutics have so far become available. As the field could profit from alternative starting points for inhibitor development, we present the results of a fragment-based screening approach on a two-domain Hsp70 construct using in-solution NMR methods, together with X-ray-crystallographic investigations and mixed-solvent molecular dynamics simulations.

Molecular Dynamics Simulations for Rationalizing Polymer Bioconjugation Strategies: Challenges, Recent Developments, and Future Opportunities.

The covalent modification of proteins with polymers is a well-established method for improving the pharmacokinetic properties of therapeutically valuable biologics. The conjugated polymer chains of the resulting hybrid represent highly flexible macromolecular structures. As the dynamics of such systems remain rather elusive for established experimental techniques from the field of protein structure elucidation, molecular dynamics simulations have proven as a valuable tool for studying such conjugates at an atomistic level, thereby complementing experimental studies.

MD-Based Assessment of Covalent Inhibitors in Noncovalent Association Complexes: Learning from Cathepsin K as a Test Case.

A sufficiently stable noncovalent association complex between a covalent inhibitor and its protein target is regarded as a prerequisite for the formation of a covalent complex. As this transient form can hardly be assessed experimentally, computational modeling is required to probe the suitability of a given ligand at this particular stage. To investigate which criteria should be fulfilled by suitable candidates in a molecular dynamics (MD) assessment, a systematic study was conducted with 20 complexes of cathepsin K, a papain-like cysteine protease of pharmaceutical relevance.

Unraveling an Alternative Mechanism in Polymer Self-Assemblies: An Order-Order Transition with Unusual Molecular Interactions between Hydrophilic and Hydrophobic Polymer Blocks.

Polymer self-assembly leading to cooling-induced hydrogel formation is relatively rare for synthetic polymers and typically relies on H-bonding between repeat units. Here, we describe a non-H-bonding mechanism for a cooling-induced reversible order-order (sphere-to-worm) transition and related thermogelation of solutions of polymer self-assemblies. A multitude of complementary analytical tools allowed us to reveal that a significant fraction of the hydrophobic and hydrophilic repeat units of the underlying block copolymer is in close proximity in the gel state.

Fragment screening using biolayer interferometry reveals ligands targeting the SHP-motif binding site of the AAA+ ATPase p97.

Biosensor techniques have become increasingly important for fragment-based drug discovery during the last years. The AAA+ ATPase p97 is an essential protein with key roles in protein homeostasis and a possible target for cancer chemotherapy. Currently available p97 inhibitors address its ATPase activity and globally impair p97-mediated processes.

Crystal structure of a covalently linked Aurora-A-MYCN complex.

Formation of the Aurora-A-MYCN complex increases levels of the oncogenic transcription factor MYCN in neuroblastoma cells by abrogating its degradation through the ubiquitin proteasome system. While some small-molecule inhibitors of Aurora-A were shown to destabilize MYCN, clinical trials have not been satisfactory to date. MYCN itself is considered to be `undruggable' due to its large intrinsically disordered regions.

Glycolytic flux control by drugging phosphoglycolate phosphatase.

Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target.

Development of an Indole-Amide-Based Photoswitchable Cannabinoid Receptor Subtype 1 (CBR) "Cis-On" Agonist.

Activation of the human cannabinoid receptor type 1 (CBR) with high spatiotemporal control is useful to study processes involved in different pathologies related to nociception, metabolic alterations, and neurological disorders. To synthesize new agonist ligands for CBR, we have designed different classes of photoswitchable molecules based on an indole core. The modifications made to the central core have allowed us to understand the molecular characteristics necessary to design an agonist with optimal pharmacological properties.

Predicting Bile and Lipid Interaction for Drug Substances.

Predicting biopharmaceutical characteristics and food effects for drug substances may substantially leverage rational formulation outcomes. We established a bile and lipid interaction prediction model for new drug substances and further explored the model for the prediction of bile-related food effects. One hundred and forty-one drugs were categorized as bile and/or lipid interacting and noninteracting drugs using H nuclear magnetic resonance (NMR) spectroscopy.

New peptidomimetic rhodesain inhibitors with improved selectivity towards human cathepsins.

Parasitic cysteine proteases such as rhodesain (TbCatL) from Trypanosoma brucei rhodesiense are relevant targets for developing new potential drugs against parasitic diseases (e. g. Human African Trypanosomiasis).

Photoswitchable Pseudoirreversible Butyrylcholinesterase Inhibitors Allow Optical Control of Inhibition and Enable Restoration of Cognition in an Alzheimer's Disease Mouse Model upon Irradiation.

To develop tools to investigate the biological functions of butyrylcholinesterase (BChE) and the mechanisms by which BChE affects Alzheimer's disease (AD), we synthesized several selective, nanomolar active, pseudoirreversible photoswitchable BChE inhibitors. The compounds were able to specifically influence different kinetic parameters of the inhibition process by light. For one compound, a 10-fold difference in the IC-values (44.

Molecular Insights into Site-Specific Interferon-α2a Bioconjugates Originated from PEG, LPG, and PEtOx.

Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers.

Sesquiterpenes and sesquiterpenoids harbor modulatory allosteric potential and affect inhibitory GABA receptor function in vitro.

Naturally occurring compounds such as sesquiterpenes and sesquiterpenoids (SQTs) have been shown to modulate GABA receptors (GABA Rs). In this study, the modulatory potential of 11 SQTs at GABA Rs was analyzed to characterize their potential neurotropic activity. Transfected HEK293 cells and primary hippocampal neurons were functionally investigated using electrophysiological whole-cell recordings.

Design, Synthesis, and Evaluation of WD-Repeat-Containing Protein 5 (WDR5) Degraders.

Histone H3K4 methylation serves as a post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferase (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat-containing protein 5 (WDR5) that has also been associated with controlling long noncoding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMT complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target.

"Photo-Rimonabant": Synthesis and Biological Evaluation of Novel Photoswitchable Molecules Derived from Rimonabant Lead to a Highly Selective and Nanomolar "-On" CBR Antagonist.

Human cannabinoid receptor type 1 (CBR) plays important roles in the regulation of appetite and development of addictive behaviors. Herein, we describe the design, synthesis, photocharacterization, molecular docking, and characterization of "photo-rimonabant", i.e.

Melatonin- and Ferulic Acid-Based HDAC6 Selective Inhibitors Exhibit Pronounced Immunomodulatory Effects and Neuroprotective Effects in a Pharmacological Alzheimer's Disease Mouse Model.

The structures of melatonin and ferulic acid were merged into tertiary amide-based histone deacetylase 6 (HDAC6) inhibitors to develop multi-target-directed inhibitors for neurodegenerative diseases to incorporate antioxidant effects without losing affinity and selectivity at HDAC6. Structure-activity relationships led to compound as a hybrid molecule showing pronounced and selective inhibition of HDAC6 (IC50 = 30.7 nM, > 25-fold selectivity over other subtypes).

Warhead Reactivity Limits the Speed of Inhibition of the Cysteine Protease Rhodesain.

Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood.

A Long Residence Time Enoyl-Reductase Inhibitor Explores an Extended Binding Region with Isoenzyme-Dependent Tautomer Adaptation and Differential Substrate-Binding Loop Closure.

The enoyl-acyl carrier protein (ACP) reductase (ENR) is a key enzyme within the bacterial fatty-acid synthesis pathway. It has been demonstrated that small-molecule inhibitors carrying the diphenylether (DPE) scaffold bear a great potential for the development of highly specific and effective drugs against this enzyme class. Interestingly, different substitution patterns of the DPE scaffold have been shown to lead to varying effects on the kinetic and thermodynamic behavior toward ENRs from different organisms.

C-2-Linked Dimeric Strychnine Analogues as Bivalent Ligands Targeting Glycine Receptors.

Strychnine is the prototypic antagonist of glycine receptors, a family of pentameric ligand-gated ion channels. Recent high-resolution structures of homomeric glycine receptors have confirmed the presence of five orthosteric binding sites located in the extracellular subunit interfaces of the receptor complex that are targeted by strychnine. Here, we report the synthesis and extensive pharmacological evaluation of bivalent ligands composed of two strychnine pharmacophores connected by appropriate spacers optimized toward simultaneous binding to two adjacent orthosteric sites of homomeric α1 glycine receptors.