Disruption of Oligomerization and Dehydroalanine Formation as Mechanisms for ClpP Protease Inhibition

Over 100 protease inhibitors are currently used in the clinics, and most of them use blockage of the active site for their mode of inhibition. Among the protease drug targets are several enzymes for which the correct multimeric assembly is crucial to their activity, such as the proteasome and the HIV protease. Here, we present a novel mechanism of protease inhibition that relies on active-site-directed small molecules that disassemble the protease complex. We show the applicability of this mechanism within the ClpP protease family, whose members are tetradecameric serine proteases and serve as regulators of several cellular processes, including homeostasis and virulence. Compound binding to ClpP in a substoichiometric fashion triggers the formation of completely inactive heptamers. Moreover, we report the selective β-sultam-induced dehydroalanine formation of the active site serine. This reaction proceeds through sulfonylation and subsequent elimination, thereby obliterating the catalytic charge relay system. The identity of the dehydroalanine was confirmed by mass spectrometry and crystallography. Activity-based protein profiling experiments suggest the formation of a dehydroalanine moiety in living S. aureus cells upon β-sultam treatment. Collectively, these findings extend our view on multicomponent protease inhibition that until now has mainly relied on blockage of the active site or occupation of a regulatory allosteric site

Are Free Radicals Involved in IspH Catalysis? An EPR and Crystallographic Investigation

The [4Fe–4S] protein IspH in the methylerythritol phosphate isoprenoid biosynthesis pathway is an important anti-infective drug target, but its mechanism of action is still the subject of debate. Here, by using electron paramagnetic resonance (EPR) spectroscopy and ²H, ¹⁷O, and ⁵⁷Fe isotopic labeling, we have characterized and assigned two key reaction intermediates in IspH catalysis. The results are consistent with the bioorganometallic mechanism proposed earlier, and the mechanism is proposed to have similarities to that of ferredoxin, thioredoxin reductase, in that one electron is transferred to the [4Fe–4S]²⁺ cluster, which then performs a formal two-electron reduction of its substrate, generating an oxidized high potential iron–sulfur protein (HiPIP)-like intermediate. The two paramagnetic reaction intermediates observed correspond to the two intermediates proposed in the bioorganometallic mechanism: the early π-complex in which the substrate’s 3-CH₂OH group has rotated away from the reduced iron–sulfur cluster, and the next, η³-allyl complex formed after dehydroxylation. No free radical intermediates are observed, and the two paramagnetic intermediates observed do not fit in a Birch reduction-like or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy and X-ray crystallography that two substrate analogues (<b>4</b> and <b>5</b>) follow the same reaction mechanism

Sequential Inactivation of Gliotoxin by the <i>S</i>‑Methyltransferase TmtA

The epipolythiodioxopiperazine (ETP) gliotoxin mediates toxicity via its reactive thiol groups and thereby contributes to virulence of the human pathogenic fungus <i>Aspergillus fumigatus</i>. Self-intoxication of the mold is prevented either by reversible oxidation of reduced gliotoxin or by irreversible conversion to bis­(methylthio)­gliotoxin. The latter is produced by the <i>S</i>-methyltransferase TmtA and attenuates ETP biosynthesis. Here, we report the crystal structure of TmtA in complex with <i>S</i>-(5′-adenosyl)-l-homocysteine. TmtA features one substrate and one cofactor binding pocket per protein, and thus, bis-thiomethylation of gliotoxin occurs sequentially. Molecular docking of substrates and products into the active site of TmtA reveals that gliotoxin forms specific interactions with the protein surroundings, and free energy calculations indicate that methylation of the C10a-SH group precedes alkylation of the C3-SH site. Altogether, TmtA is well suited to selectively convert gliotoxin and to control its biosynthesis, suggesting that homologous enzymes serve to regulate the production of their toxic natural sulfur compounds in a similar manner

Systematic Analyses of Substrate Preferences of 20S Proteasomes Using Peptidic Epoxyketone Inhibitors

Cleavage analyses of 20S proteasomes with natural or synthetic substrates allowed to infer the substrate specificities of the active sites and paved the way for the rational design of high-affinity proteasome inhibitors. However, details of cleavage preferences remained enigmatic due to the lack of appropriate structural data. In a unique approach, we here systematically examined substrate specificities of yeast and human proteasomes using irreversibly acting α′,β′epoxyketone (ep) inhibitors. Biochemical and structural analyses provide unique insights into the substrate preferences of the distinct active sites and highlight differences between proteasome types that may be considered in future inhibitor design efforts. (1) For steric reasons, epoxyketones with Val or Ile at the P1 position are weak inhibitors of all active sites. (2) Identification of the β2c selective compound Ac-LAE-ep represents a promising starting point for the development of compounds that discriminate between β2c and β2i. (3) The compound Ac-LAA-ep was found to favor subunit β5c over β5i by three orders of magnitude. (4) Yeast β1 and human β1c subunits preferentially bind Asp and Leu in their S1 pockets, while Glu and large hydrophobic residues are not accepted. (5) Exceptional structural features in the β1/2 substrate binding channel give rise to the β1 selectivity of compounds featuring Pro at the P3 site. Altogether, 23 different epoxyketone inhibitors, five proteasome mutants, and 43 crystal structures served to delineate a detailed picture of the substrate and ligand specificities of proteasomes and will further guide drug development efforts toward subunit-specific proteasome inhibitors for applications as diverse as cancer and autoimmune disorders

A Predictive Approach for the Optical Control of Carbonic Anhydrase II Activity

Optogenetics and photopharmacology are powerful approaches to investigating biochemical systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is generally a lengthy and cumbersome process. Herein, we describe an <i>in silico</i> screening approach that is designed to propose a small number of optimal combinations. By applying this computational approach to human carbonic anhydrase and a set of three photochromic tethered ligands, the number of potential site-ligand combinations was narrowed from over 750 down to 6, which we then evaluated experimentally. Two of these six combinations resulted in light-responsive human Carbonic Anhydrases (LihCAs), which were characterized with enzymatic activity assays, mass spectrometry, and X-ray crystallography. Our study also provides insights into the reactivity of cysteines toward maleimides and the hydrolytic stability of the adducts obtained

Mechanism of Allosteric Inhibition of the Enzyme IspD by Three Different Classes of Ligands

Enzymes of the nonmevalonate pathway of isoprenoid biosynthesis are attractive targets for the development of herbicides and drugs against infectious diseases. While this pathway is essential for many pathogens and plants, mammals do not depend on it for the synthesis of isoprenoids. IspD, the third enzyme of the nonmevalonate pathway, is unique in that it has an allosteric regulatory site. We elucidated the binding mode of phenylisoxazoles, a new class of allosteric inhibitors. Allosteric inhibition is effected by large conformational changes of a loop region proximal to the active site. We investigated the different roles of residues in this loop by mutation studies and identified repulsive interactions with Asp291 and Asp292 to be responsible for inhibition. Crystallographic data and the response of mutant enzymes to three different classes of allosteric inhibitors provide an in-depth understanding of the allosteric mechanism. The obtained mutant enzymes show selective resistance to allosteric inhibitors and provide conceptually valuable information for future engineering of herbicide-resistant crops. We found that the isoprenoid precursors IPP and DMAPP are natural inhibitors of <i>Arabidopsis thaliana</i> IspD; however, they do not seem to bind to the allosteric site

Mechanism of Allosteric Inhibition of the Enzyme IspD by Three Different Classes of Ligands

Enzymes of the nonmevalonate pathway of isoprenoid biosynthesis are attractive targets for the development of herbicides and drugs against infectious diseases. While this pathway is essential for many pathogens and plants, mammals do not depend on it for the synthesis of isoprenoids. IspD, the third enzyme of the nonmevalonate pathway, is unique in that it has an allosteric regulatory site. We elucidated the binding mode of phenylisoxazoles, a new class of allosteric inhibitors. Allosteric inhibition is effected by large conformational changes of a loop region proximal to the active site. We investigated the different roles of residues in this loop by mutation studies and identified repulsive interactions with Asp291 and Asp292 to be responsible for inhibition. Crystallographic data and the response of mutant enzymes to three different classes of allosteric inhibitors provide an in-depth understanding of the allosteric mechanism. The obtained mutant enzymes show selective resistance to allosteric inhibitors and provide conceptually valuable information for future engineering of herbicide-resistant crops. We found that the isoprenoid precursors IPP and DMAPP are natural inhibitors of <i>Arabidopsis thaliana</i> IspD; however, they do not seem to bind to the allosteric site

Dimerized Linear Mimics of a Natural Cyclopeptide (TMC-95A) Are Potent Noncovalent Inhibitors of the Eukaryotic 20S Proteasome

Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors used in cancer therapy. Starting from a noncovalent linear mimic of TMC-95A, a series of dimerized inhibitors using polyaminohexanoic acid spacers has been designed and optimized to target simultaneously two of the six active sites of the eukaryotic 20S proteasome. The homodimerized compounds actively inhibited chymotrypsin-like (<i>K</i>_i = 6–11 nM) and trypsin-like activities, whereas postacid activity was poorly modified. The noncovalent binding mode was ascertained by X-ray crystallography of the inhibitors complexed with the yeast 20S proteasome. The inhibition of proteasomal activities in human cells was evaluated. The use of the multivalency inhibitor concept has produced highly efficient and selective noncovalent compounds (no inhibition of calpain and cathepsin) that have potential therapeutic advantages compared to covalent binders such as bortezomib and carfilzomib

Potent Proteasome Inhibitors Derived from the Unnatural <i>cis</i>-Cyclopropane Isomer of Belactosin A: Synthesis, Biological Activity, and Mode of Action

The natural product belactosin A (<b>1</b>) with a <i>trans</i>-cyclopropane structure is a useful prototype compound for developing potent proteasome (core particle, CP) inhibitors. To date, <b>1</b> and its analogues are the only CP ligands that bind to both the nonprimed S1 pocket as well as the primed substrate binding channel; however, these molecules harbor a high IC₅₀ value of more than 1 μM. We have performed structure–activity relationship studies, thereby elucidating unnatural <i>cis</i>-cyclopropane derivatives of <b>1</b> that exhibit high potency to primarily block the chymotrypsin-like active site of the human constitutive (cCP) and immunoproteasome (iCP). The most active compound <b>3e</b> reversibly inhibits cCP and iCP similarly with an IC₅₀ of 5.7 nM. X-ray crystallographic analysis of the yeast proteasome in complex with <b>3e</b> revealed that the ligand is accommodated predominantly into the primed substrate binding channel and covalently binds to the active site threonine residue via its β-lactone ring-opening

Gliotoxin Biosynthesis: Structure, Mechanism, and Metal Promiscuity of Carboxypeptidase GliJ

The formation of glutathione (GSH) conjugates, best known from the detoxification of xenobiotics, is a widespread strategy to incorporate sulfur into biomolecules. The biosynthesis of gliotoxin, a virulence factor of the human pathogenic fungus <i>Aspergillus fumigatus</i>, involves attachment of two GSH molecules and their sequential decomposition to yield two reactive thiol groups. The degradation of the GSH moieties requires the activity of the Cys–Gly carboxypeptidase GliJ, for which we describe the X-ray structure here. The enzyme forms a homodimer with each monomer comprising one active site. Two metal ions are present per proteolytic center, thus assigning GliJ to the diverse family of dinuclear metallohydrolases. Depending on availability, Zn²⁺, Fe²⁺, Fe³⁺, Mn²⁺, Cu²⁺, Co²⁺, or Ni²⁺ ions are accepted as cofactors. Despite this high metal promiscuity, a preference for zinc versus iron and manganese was noted. Mutagenesis experiments revealed details of metal coordination, and molecular modeling delivered insights into substrate recognition and processing by GliJ. The latter results suggest a reaction mechanism in which the two scissile peptide bonds of one gliotoxin precursor molecule are hydrolyzed sequentially and in a given order