Vadim S. Korotkov

Oligosubstituted Pyrroles Directly from Substituted Methyl Isocyanides and Acetylenes

The formal cycloaddition of alpha-metallated methyl isocyanides 1 onto the triple bond of electron-deficient acetylenes 2 represents a direct and convenient approach to oligosubstituted pyrroles 3. The scope and limitations of this reaction (24 examples, 25-97% yield) are reported along with an optimization of the reaction conditions and a rationalization of the mechanism. In addition, a related newly developed Cu(I)-mediated synthesis of 2,3-disubstituted pyrroles by the reaction of copper acetylides derived from unactivated terminal alkynes with substituted methyl isocyanides is described (11 examples, 5-88% yield).

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA+ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.

The mechanism of caseinolytic protease (ClpP) inhibition.

Maintaining homeostasis at the protein level is an important prerequisite for cellular viability for which prokaryotes exhibit several proteolytic machineries, including ClpXP. In 2008, we reported the first small-molecule inhibitor for the proteolytic subunit ClpP and demonstrated that the inhibition of the enzyme in living bacteria significantly attenuates their capability to produce virulence factors, such as life-threatening toxins. Although ClpP has been extensively studied by biochemical and structural methods, the mechanism of small-molecule inhibition of this enzyme is currently poorly understood. Because chemical inhibition may lead to a novel antibacterial therapy, it is important to systematically analyze the binding site, the mechanism of inhibition, the stereogenic preference of the enzyme for inhibitors, the chemical space of putative inhibitors, and how other members of the ClpP family can be inhibited. One major step towards these aims was accomplished by the recently solved crystal structure of homotetradecameric ClpP from Staphylococcus aureus (SaClpP) in its active conformation. With the structural data at hand, we herein report an in-depth mechanistic analysis of S. aureus ClpP inhibition by b-lactones. A screen of a focused library of enantiopure b-lactones revealed the S,Sstereopreference of the protease, which was rationalized by molecular docking. Docking experiments also gave insight into a hitherto unnoted deep hydrophobic pocket next to the active site that accommodates b-lactone substituents in the aposition to the carbonyl group. The binding hypothesis was verified by binding studies with model compounds, detailed kinetic analysis, and protein mutagenesis studies. Furthermore, the replacement of the b-lactone core by other scaffolds resulted in the loss of inhibitory potency, thereby highlighting the importance of a b-lactone moiety for mechanism-based ClpP inhibition. Taken together, these results open intriguing perspectives in the mechanistic understanding of ClpP inhibition and provide direction for the design of potent and pharmacologically optimized inhibitors. We started by testing 22 enantiopure trans-substituted blactones 1–22 for ClpP inhibition (Supporting Information, Figure S1 A). These molecules share a high structural similarity with our previous b-lactone candidates. They feature a decyl chain as R substituent and structural variations in chain lengths as well as in functional groups at the R position (Figure 1A). For all of the compounds, both trans-configured enantiomers (that is, R,R and S,S) were tested for inhibition of recombinantly expressed SaClpP in an assay monitoring the cleavage of a fluorogenic substrate. Almost all of the compounds inhibited SaClpP at 100 mm concentration (100-fold excess over enzyme) after 15 min incubation at 32 8C (Supporting Information, Figure S1 A). By lowering the inhibitor concentration to 10 mm, we were able to differentiate the compounds tested. While most S,Sconfigured lactones lead to inhibition below 10% residual activity, R,R-configured lactones showed essentially no inhibition (Figure 1 B). Incubation of SaClpP with 1.3-fold molar excess of the most potent compound, 2, led to modification of all 14 subunits as revealed by intact-protein mass spectrometry (Figure 1 C). To investigate if the potent in vitro inhibition correlates with ClpP binding in living cells we applied the structurally related alkynylated probe 23 with S,S-configuration for an

A β‐Lactone‐Based Antivirulence Drug Ameliorates Staphylococcus aureus Skin Infections in Mice

Skin infections caused by Staphylococcus aureus are a major clinical concern, especially if they are caused by multi‐resistant strains. In these cases, a spread into deeper soft tissues or the bloodstream results in life‐threatening conditions that are difficult to treat by conventional antibiotics. Previous in vitro experiments with a small β‐lactone‐based molecule demonstrated that antibiotic‐sensitive and ‐resistant S. aureus strains are effectively disarmed in their virulence and corresponding pathogenicity. In this work, in vivo mouse studies show that this methodology is effective for the treatment of skin abscesses in mice. A single dose of the β‐lactone significantly decreased abscess size even when applied 6 h post‐infection. Although the molecule requires pharmacological optimization (improved stability, for example), this study emphasizes the potential value of antivirulence therapies.

Development and characterization of improved β-lactone-based anti-virulence drugs targeting ClpP.

Here, we report the synthesis and in depth characterization of a second generation β-lactone derived virulence inhibitors. Based on initial results that emphasized the intriguing possibility to disarm bacteria in their virulence the present study develops this concept further and analyses the potential of this strategy for drug development. We were able to expand the collection of bioactive compounds via an efficient synthetic route. Testing of all compounds revealed several hits with anti-virulence activity. Moreover, we demonstrated that these molecules act solely by reducing virulence but not killing bacteria which is an important prerequisite for preserving the useful microbiome. Finally, incubation of lactones with eukaryotic cell lines indicated a tolerable cytotoxicity which is essential for entering animal studies.

A Whole Proteome Inventory of Background Photocrosslinker Binding

Affinity-based protein profiling (AfBPP) is a widely applied method for the target identification of bioactive molecules. Probes containing photocrosslinkers, such as benzophenones, diazirines, and aryl azides, irreversibly link the molecule of interest to its target protein upon irradiation with UV light. Despite their prevalent application, little is known about photocrosslinker-specific off-targets, affecting the reliability of results. Herein, we investigated background protein labeling by gel-free quantitative proteomics. Characteristic off-targets were identified for each photoreactive group and compiled in a comprehensive inventory. In a proof-of-principle study, H8, a protein kinase A inhibitor, was equipped with a diazirine moiety. Application of this photoprobe revealed, by alignment with the diazirine background, unprecedented insight into its in situ proteome targets. Taken together, our findings guide the identification of biologically relevant binders in photoprobe experiments.

Synthesis and biological activity of optimized belactosin C congeners

Successful biochemical studies of the natural products belactosin A and C as well as their more stable acylated derivatives have proved them to be powerful proteasome inhibitors and thereby potential candidates as pharmacologically relevant active compounds. In order to understand their structure-biological activity relations in detail and to find ways of improving their biological activity, four new modified belactosin congeners have been synthesized and tested. One of them (compound 6) turned out to be a more potent inhibitor against HeLa cells than the known proteasome inhibitor MG132.

Reversible Inhibitors Arrest ClpP in a Defined Conformational State that Can Be Revoked by ClpX Association

Caseinolytic protease P (ClpP) is an important regulator of Staphylococcus aureus pathogenesis. A high-throughput screening for inhibitors of ClpP peptidase activity led to the identification of the first non-covalent binder for this enzyme class. Co-crystallization of the small molecule with S. aureus ClpP revealed a novel binding mode: Because of the rotation of the conserved residue proline 125, ClpP is locked in a defined conformational state, which results in distortion of the catalytic triad and inhibition of the peptidase activity. Based on these structural insights, the molecule was optimized by rational design and virtual screening, resulting in derivatives exceeding the potency of previous ClpP inhibitors. Strikingly, the conformational lock is overturned by binding of ClpX, an associated chaperone that enables proteolysis by substrate unfolding in the ClpXP complex. Thus, regulation of inhibitor binding by associated chaperones is an unexpected mechanism important for ClpP drug development.

Synthesis and biological activity of simplified belactosin C analogues

Successful biochemical studies of the natural products belactosin A and C and their acylated congeners have shown a β-lactonecarboxamide moiety to be a possible core structure of powerful proteasome inhibitors. As a part of further investigations, variously decorated simplified β-lactonecarboxamides have been synthesized in order to understand structure-biological activity relations in detail, to find ways of improving their biological activity and stability and to reduce the complexity of their preparation. Biological tests showed that the best compounds possess a high potential against phytopathogenic fungi in the greenhouse.

A Minimal β‐Lactone Fragment for Selective β5c or β5i Proteasome Inhibitors

Broad-spectrum proteasome inhibitors are applied as anticancer drugs, whereas selective blockage of the immunoproteasome represents a promising therapeutic rationale for autoimmune diseases. We here aimed at identifying minimal structural elements that confer β5c or β5i selectivity on proteasome inhibitors. Based on the natural product belactosin C, we synthesized two β-lactones featuring a dimethoxybenzyl moiety and either a methylpropyl (pseudo-isoleucin) or an isopropyl (pseudo-valine) P1 side chain. Although the two compounds differ only by one methyl group, the isoleucine analogue is six times more potent for β5i (IC50=14 nM) than the valine counterpart. Cell culture experiments demonstrate the cell-permeability of the compounds and X-ray crystallography data highlight them as minimal fragments that occupy primed and non-primed pockets of the active sites of the proteasome. Together, these results qualify β-lactones as a promising lead-structure motif for potent nonpeptidic proteasome inhibitors with diverse pharmaceutical applications.