Philipp Beck

Covalent and non-covalent reversible proteasome inhibition.

Abstract 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.

Hydroxyureas as Noncovalent Proteasome Inhibitors

Inhibitors with a new mechanism of action are needed for 20S proteasome (CP) inhibition owing to the ineffectiveness of current market drugs against some types of solid tumors. A novel class of nonpeptidic CP inhibitors has been developed, which display reversible and noncovalent binding. The structure-based design of these highly active and site-specific inhibitors revealed unexplored binding subpockets.

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.

One-shot NMR analysis of microbial secretions identifies highly potent proteasome inhibitor.

Natural products represent valuable lead structures for drug discovery. However, for most bioactive compounds no cellular target is yet identified and many substances predicted from genome analysis are inaccessible due to their life stage-dependent biosynthesis, which is not reflected in common isolation procedures. In response to these issues, an NMR-based and target-directed protease assay for inhibitor detection of the proteasome was developed. The methodology is suitable for one-shot identification of inhibitors in conglomerates and crude culture broths. The technique was applied for analysis of the different life stages of the bacterium Photorhabdus luminescens, which resulted in the isolation and characterization of cepafungin I (CepI), the strongest proteasome inhibitor described to date. Its biosynthesis is strictly regulated and solely induced by the specific environmental conditions determined by our methodology. The transferability of the developed technique to other drug targets may disclose an abundance of novel compounds applicable for drug development.

A Single Methylene Group in Oligoalkylamine-Based Cationic Polymers and Lipids Promotes Enhanced mRNA Delivery

The development of chemically modified mRNA holds great promise as a new class of biologic therapeutics. However, the intracellular delivery and endosomal escape of mRNA encapsulated in nanoparticles has not been systematically investigated. Here, we synthesized a diverse set of cationic polymers and lipids from a series of oligoalkylamines and subsequently characterized their mRNA delivery capability. Notably, a structure with an alternating alkyl chain length between amines showed the highest transfection efficiency, which was linked to a high buffering capacity in a narrow range of pH 6.2 to 6.5. Variation in only one methylene group resulted in enhanced mRNA delivery to both the murine liver as well as porcine lungs after systemic or aerosol administration, respectively. These findings reveal a novel fundamental structure-activity relationship for the delivery of mRNA that is independent of the class of mRNA carrier and define a promising new path of exploration in the field of mRNA therapeutics.

Indolo-Phakellins as β5-Specific Noncovalent Proteasome Inhibitors†

The proteasome represents an invaluable target for the treatment of cancer and autoimmune disorders. The application of proteasome inhibitors, however, remains limited to blood cancers because their reactive headgroups and peptidic scaffolds convey unfavorable pharmacodynamic properties. Thus, the discovery of more drug-like lead structures is indispensable. In this study, we present the first structure of the proteasome in complex with an indolo-phakellin that exhibits a unique noncovalent binding mode unparalleled by all hitherto reported inhibitors. The natural product inspired pentacyclic alkaloid binds solely and specificially into the spacious S3 subpocket of the proteasomal β5 substrate binding channel, gaining major stabilization through halogen bonding with the protein backbone. The presented compound provides an ideal scaffold for the structure-based design of subunit-specific nonpeptidic proteasome-blockers.

Structure and Reaction Mechanism of Pyrrolysine Synthase (PylD).

Pyrrolysine (Pyl, 4), the recently discovered 22nd genetically encoded amino acid, has almost instantaneously become a hotspot of protein biochemistry, although its natural occurrence appears to be limited to just three proteins that are involved in the breakdown of methylamines in a small subgroup of archaea and bacteria. The novel amino acid is incorporated by a cognate pair of a tRNA and its synthetase (specified as pylT and pylS, respectively) through recognition of an amber stop codon (UAG). Biosynthesis of 4 is accomplished from two molecules of lysine (1) by sequential action of PylB, PylC, and PylD (Scheme 1). Specifically, the iron–sulfur S-adenosylmethionine protein PylB catalyzes the conversion of 1 to (3R)-3methyl-d-ornithine (3MO, 2), which is subsequently hooked up ATP-dependently to the e amino group of a second lysine molecule by PylC resulting in 3. Dehydrogenation at the C5 position of the methylornithine moiety of the isopeptide and subsequent ring closure catalyzed by PylD completes the biosynthesis of the unusual amino acid pyrrolysine. The present report on PylD describes the structural investigation and implementation on the reaction mechanism of the enzymes required for the biosynthesis of pyrrolysine and may open novel opportunities for the harnessing of the system for biotechnology purposes. We expressed the pylD gene of Methanosarcina barkeri Fusaro in an Escherichia coli strain. The recombinant protein was purified by metal-affinity chromatography and showed in vitro catalytic activity with Km = 3.6 mm 0.5 mm (Figure S1 in the Supporting Information) and kcat = 0.76 s 1 0.04 s 1 using the surrogate l-lysine-N-d-ornithine (LysN-d-Orn, 3a) as substrate. PylD was crystallized together with 3a and/ or a pyridine nucleotide cofactor (NADH or NAD). Crystals diffracted to a maximum resolution of 1.8 and starting phases were obtained by a combination of single-wavelength anomalous diffraction (SAD) methods using a selenomethionine derivative and twofold noncrystallographic symmetry (NCS) averaging. Real-space electron density map averaging was performed with MAIN in combination with CCP4 routines. Model building was carried out with MAIN and refinement was completed with REFMAC5 (see Table S1). After structural elucidation of the selenomethionine-labeled PylD holoenzyme (PylD:holo (peak), PDB ID: 4JK3), we crystallized and determined the structure of native PylD in the presence of NAD (PylD:holo, PDB ID: 4J43) to 2.2 resolution (Rfree = 20.1%, Table S1). Its molecular architecture is shown schematically in Figure 1a: the C-terminal segment (residues 139–259) resembles a Rossmann motif of five parallel b strands (S6–S10) with 21345 topology, which is Nand C-terminally flanked by helices H5 and H10, respectively (secondary-structure nomenclature: see Figure S3), yielding in the DALI search a highest Z-score of 15.6 for the Rhodospirillum rubrum Transhydrogenase Domain I (PDB ID: 1L7D). The N-terminal segment (residues 1–138) comprises a b sheet of five strands (S1–S5) whose overall orientation is orthogonal to that of the Rossmann motif of the C-terminal part. Interestingly, the C-terminal helix (H10) of PylD is wedged between the two b sheets supporting the correct fold and orientation of the N-terminal half of the dehydrogenase. In contrast to the Rossmann fold, the DALI search for proteins resembling the topology of the N-terminal segment resulted in only some similarities with the tRNA binding domain of certain tRNA synthetases (Zscore< 8). The nicotinamide adenine dinucleotide coenzyme NAD is bound to PylD in an extended conformation, inside a groove at the C-terminal pole of the Rossmann b sheet; both furanose rings have C2’-endo conformation. A typical VXGXGXXGXXXA motif (residues 146–157) is part of the coenzyme binding site and the backbone elements are predominantly involved in a network of hydrogen bonds with Scheme 1. Biosynthesis of pyrrolysine. Note: PylB generates only 3MO (2); PylC and PylD also catalyze the reactions of 2a and 3a, respectively.

Identification of a β1/β2-Specific Sulfonamide Proteasome Ligand by Crystallographic Screening

The proteasome represents a validated drug target for the treatment of cancer, however, new types of inhibitors are required to tackle the development of resistant tumors. Current fluorescence-based screening methods suffer from low sensitivity and are limited to the detection of ligands with conventional binding profiles. In response to these drawbacks, a crystallographic screening procedure for the discovery of agents with a novel mode of action was utilized. The optimized workflow was applied to the screening of a focused set of compounds, resulting in the discovery of a β1/β2-specific sulfonamide derivative that noncovalently binds between subunits β1 and β2. The binding pocket displays significant differences in size and polarity between the immuno- and constitutive proteasome. The identified ligand thus provides valuable insights for the future structure-based design of subtype-specific proteasome inhibitors.

Interactions of the natural product kendomycin and the 20S proteasome.

Natural products are a valuable source for novel lead structures in drug discovery, but for the majority of isolated bioactive compounds, the cellular targets are unknown. The structurally unique ansa-polyketide kendomycin (KM) was reported to exert its potent cytotoxic effects via impairment of the ubiquitin proteasome system, but the exact mode of action remained unclear. Here, we present a systematic biochemical characterization of KM-proteasome interactions in vitro and in vivo, including complex structures of wild type and mutant yeast 20S proteasome with KM. Our results provide evidence for a polypharmacological mode of action for KMs cytotoxic effect on cancer cells.

α‐Keto Phenylamides as P1′‐Extended Proteasome Inhibitors

The major challenge for proteasome inhibitor design lies in achieving high selectivity for, and activity against, the target, which requires specific interactions with the active site. Novel ligands aim to overcome off‐target‐related side effects such as peripheral neuropathy, which is frequently observed in cancer patients treated with the FDA‐approved proteasome inhibitors bortezomib (1) or carfilzomib (2). A systematic comparison of electrophilic headgroups recently identified the class of α‐keto amides as promising for next generation drug development. On the basis of crystallographic knowledge, we were able to develop a structure–activity relationship (SAR)‐based approach for rational ligand design using an electronic parameter (Hammett’s σ) and in silico molecular modeling. This resulted in the tripeptidic α‐keto phenylamide BSc4999 [(S)‐3‐(benzyloxycarbonyl‐(S)‐leucyl‐(S)‐leucylamino)‐5‐methyl‐2‐oxo‐N‐(2,4‐dimethylphenyl)hexanamide, 6 a], a highly potent (IC50=38 nM), cell‐permeable, and slowly reversible covalent inhibitor which targets both the primed and non‐primed sites of the proteasome’s substrate binding channel as a special criterion for selectivity. The improved inhibition potency and selectivity of this new α‐keto phenylamide makes it a promising candidate for targeting a wider range of tumor subtypes than commercially available proteasome inhibitors and presents a new candidate for future studies.