Optimization of Covalent MKK7 Inhibitors via Crude Nanomole-Scale Libraries.

High-throughput nanomole-scale synthesis allows for late-stage functionalization (LSF) of compounds in an efficient and economical manner. Here, we demonstrated that copper-catalyzed azide-alkyne cycloaddition could be used for the LSF of covalent kinase inhibitors at the nanoscale, enabling the synthesis of hundreds of compounds that did not require purification for biological assay screening, thus reducing experimental time drastically. We generated crude libraries of inhibitors for the kinase MKK7, derived from two different parental precursors, and analyzed them via the high-throughput In-Cell Western assay. Select inhibitors were resynthesized, validated via conventional biological and biochemical methods such as western blots and liquid chromatography-mass spectrometry (LC-MS) labeling, and successfully co-crystallized. Two of these compounds showed over 20-fold increased inhibitory activity compared to the parental compound. This study demonstrates that high-throughput LSF of covalent inhibitors at the nanomole-scale level can be an auspicious approach in improving the properties of lead chemical matter.

Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

Structural Elucidation of a Nonpeptidic Inhibitor Specific for the Human Immunoproteasome.

Selective inhibition of the immunoproteasome is a promising approach towards the development of immunomodulatory drugs. Recently, a class of substituted thiazole compounds that combine a nonpeptidic scaffold with the absence of an electrophile was reported in a patent. Here, we investigated the mode of action of the lead compound by using a sophisticated chimeric yeast model of the human immunoproteasome for structural studies. The inhibitor adopts a unique orientation perpendicular to the ß5i substrate-binding channel. Distinct interactions between the inhibitor and the subpockets of the human immunoproteasome account for its isotype selectivity.

Tunable Probes with Direct Fluorescence Signals for the Constitutive and Immunoproteasome.

Electrophiles are commonly used for the inhibition of proteases. Notably, inhibitors of the proteasome, a central determinant of cellular survival and a target of several FDA-approved drugs, are mainly characterized by the reactivity of their electrophilic head groups. We aimed to tune the inhibitory strength of peptidic sulfonate esters by varying the leaving groups. Indeed, proteasome inhibition correlated well with the pKa of the leaving group. The use of fluorophores as leaving groups enabled us to design probes that release a stoichiometric fluorescence signal upon reaction, thereby directly linking proteasome inactivation to the readout. This principle could be applicable to other sulfonyl fluoride based inhibitors and allows the design of sensitive probes for enzymatic studies.

Selective Inhibition of the Immunoproteasome by Structure-Based Targeting of a Non-catalytic Cysteine.

Clinically applied proteasome inhibitors induce cell death by concomitant blockage of constitutive and immunoproteasomes. In contrast, selective immunoproteasome inhibition is less cytotoxic and has the potential to modulate chronic inflammation and autoimmune diseases. In this study, we rationally designed decarboxylated peptides that covalently target a non-catalytic cysteine of the immunoproteasome subunit ß5i with α-chloroacetamide-containing sidechains. The enhanced isoform specificity decreased cytotoxic effects and the compound suppressed the production of inflammatory cytokines. Structure-based optimization led to over 150-fold selectivity for subunit ß5i over ß5c. This new compound class provides a promising starting point for the development of selective immunoproteasome inhibitors as potential anti-inflammatory agents.

Macyranones: Structure, Biosynthesis, and Binding Mode of an Unprecedented Epoxyketone that Targets the 20S Proteasome.

In our screening efforts to identify unique scaffolds from myxobacteria for the drug discovery process, we used LC-SPE-NMR-MS techniques to isolate six linear peptides, termed macyranone A-F, from Cystobacter fuscus MCy9118. The macyranones are characterized by a rare 2-methylmalonamide moiety and an α-amino ketone fragment including an α',ß'-epoxyketone in macyranone A. Gene disruption experiments confirmed the biosynthetic gene cluster of the macyranones as PKS/NRPS hybrid. Detailed in silico and phylogenetic analysis unraveled that the biosynthesis involves two conspicuous amide bond formations accomplished by an amidotransferase and a unique condensation domain. The gene cluster provides further insights into the formation of the powerful epoxyketone residue involving an acyl-CoA dehydrogenase and an unconventional free-standing thioesterase. Macyranone A was found to inhibit the chymotrypsin-like activity of the yeast 20S proteasome with an IC50 of 5.9 nM and the human constitutive proteasome and immunoproteasome with IC50 values of 21 and 15 nM, respectively. The ß5 subunit of the 20S proteasome was characterized as target by X-ray crystallography revealing an irreversible binding mode similar to the natural product epoxomicin. The presence of the methylmalonamide residue facilitates the stabilization of macyranone A with the active ß5 subunit of the proteasome. Macyranone A exhibits a potent inhibitory effect against the parasites Trypanosoma brucei rhodesiense and Leishmania donovani with IC50 values of 1.55 and 0.22 µM, respectively.

A mass spectrometry platform for a streamlined investigation of proteasome integrity, posttranslational modifications, and inhibitor binding.

The proteasome is responsible for the majority of protein degradation within eukaryotic cells and proteasome inhibitors have gained blockbuster status as anticancer drugs. Here, we introduce an analytical platform comprising reverse phase chromatography, intact protein mass spectrometry, and customized data analysis that allows a streamlined investigation of proteasome integrity and posttranslational modifications. We report the complete mass spectrometric assignment of all subunits of the yeast core particle, as well as of the human constitutive 20S proteasome and the human immunoproteasome, including phosphorylated isoforms of α7. Importantly, we found several batches of commercially available immunoproteasome to also contain constitutive catalytic subunits. Moreover, we applied the method to study the binding mechanisms of proteasome inhibitors, both validating the approach and providing a direct readout of subunit preferences complementary to biochemical methods. Collectively, our platform facilitates an easy, reliable and comprehensive detection of different types of covalent modifications on multisubunit protein complexes with high accuracy.

Selective inhibition of the immunoproteasome by ligand-induced crosslinking of the active site.

The concept of proteasome inhibition ranks among the latest achievements in the treatment of blood cancer and represents a promising strategy for modulating autoimmune diseases. In this study, we describe peptidic sulfonyl fluoride inhibitors that selectively block the catalytic ß5 subunit of the immunoproteasome by inducing only marginal cytotoxic effects. Structural and mass spectrometric analyses revealed a novel reaction mechanism involving polarity inversion and irreversible crosslinking of the proteasomal active site. We thus identified the sulfonyl fluoride headgroup for the development and optimization of immunoproteasome selective compounds and their possible application in autoimmune disorders.

Systematic comparison of peptidic proteasome inhibitors highlights the α-ketoamide electrophile as an auspicious reversible lead motif.

The ubiquitin-proteasome system (UPS) has been successfully targeted by both academia and the pharmaceutical industry for oncological and immunological applications. Typical proteasome inhibitors are based on a peptidic backbone endowed with an electrophilic C-terminus by which they react with the active proteolytic sites. Although the peptide moiety has attracted much attention in terms of subunit selectivity, the target specificity and biological stability of the compounds are largely determined by the reactive warheads. In this study, we have carried out a systematic investigation of described electrophiles by a combination of in vitro, in vivo, and structural methods in order to disclose the implications of altered functionality and chemical reactivity. Thereby, we were able to introduce and characterize the class of α-ketoamides as the most potent reversible inhibitors with possible applications for the therapy of solid tumors as well as autoimmune disorders.

Covalent and non-covalent reversible proteasome inhibition.

The 20S proteasome core particle (CP) is the proteolytically active key element of the ubiquitin proteasome system that directs the majority of intracellular protein degradation in eukaryotic cells. Over the past decade, the CP has emerged as an anticancer therapy target after approval of the first-in-class drug bortezomib (Velcade(®)) by the US Food and Drug Administration. However, bortezomib and all second-generation CP inhibitors that are currently explored in clinical phase studies react covalently and most often irreversibly with the proteolytic sites of the CP, hereby causing permanent CP blockage. Furthermore, reactive head groups result in unspecific binding to proteasomal active centers and in substantial enzymatic off-target activities that translate to severe side effects. Thus, reversible proteasome inhibitors might be a promising alternative, overcoming these drawbacks, but are challenging with respect to their urge for thorough enthalpic and entropic optimization. This review describes developments in the hitherto neglected field of reversible proteasome inhibitors focusing on insights gained from crystal structures, which provide valuable knowledge and strategies for future directions in drug development.