Structure-based design of small molecule inhibitors of the cagT4SS ATPase Cagα of Helicobacter pylori.

We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori. The secretion system is encoded by the cag pathogenicity island, and we chose Cagα, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cagα in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cagα subunits and mutagenesis of the binding site identified Cagα residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889, and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cagα, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect.

Discovery and Characterization of Selective, First-in-Class Inhibitors of Citron Kinase.

Citron kinase (CITK) is an AGC-family serine/threonine kinase that regulates cytokinesis. Despite knockdown experiments implicating CITK as an anticancer target, no selective CITK inhibitors exist. We transformed a previously reported kinase inhibitor with weak off-target CITK activity into a first-in-class CITK chemical probe, C3TD879. C3TD879 is a Type I kinase inhibitor which potently inhibits CITK catalytic activity (biochemical IC50 = 12 nM), binds directly to full-length human CITK in cells (NanoBRET Kd < 10 nM), and demonstrates favorable DMPK properties for in vivo evaluation. We engineered exquisite selectivity for CITK (>17-fold versus 373 other human kinases), making C3TD879 the first chemical probe suitable for interrogating the complex biology of CITK. Our small-molecule CITK inhibitors could not phenocopy the effects of CITK knockdown in cell proliferation, cell cycle progression, or cytokinesis assays, providing preliminary evidence that the structural roles of CITK may be more important than its kinase activity.

Structure-Based Optimization of ML300-Derived, Noncovalent Inhibitors Targeting the Severe Acute Respiratory Syndrome Coronavirus 3CL Protease (SARS-CoV-2 3CLpro).

Starting from the MLPCN probe compound ML300, a structure-based optimization campaign was initiated against the recent severe acute respiratory syndrome coronavirus (SARS-CoV-2) main protease (3CLpro). X-ray structures of SARS-CoV-1 and SARS-CoV-2 3CLpro enzymes in complex with multiple ML300-based inhibitors, including the original probe ML300, were obtained and proved instrumental in guiding chemistry toward probe compound 41 (CCF0058981). The disclosed inhibitors utilize a noncovalent mode of action and complex in a noncanonical binding mode not observed by peptidic 3CLpro inhibitors. In vitro DMPK profiling highlights key areas where further optimization in the series is required to obtain useful in vivo probes. Antiviral activity was established using a SARS-CoV-2-infected Vero E6 cell viability assay and a plaque formation assay. Compound 41 demonstrates nanomolar activity in these respective assays, comparable in potency to remdesivir. These findings have implications for antiviral development to combat current and future SARS-like zoonotic coronavirus outbreaks.

Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system.

Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system encoded by the cag pathogenicity island encodes 27 proteins and most are essential for virulence. We here present the identification and characterization of inhibitors of Cagα, a hexameric ATPase and member of the family of VirB11-like proteins that is essential for translocation of the CagA cytotoxin into mammalian cells. We conducted fragment-based screening using a differential scanning fluorimetry assay and identified 16 molecules that stabilize the protein suggesting that they bind Cagα. Several molecules affect binding of ADP and four of them inhibit the ATPase activity. Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the active site. Cross-linking suggests that the active molecules change protein conformation and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. Addition of the molecule 1G2 inhibits the induction of interleukin-8 production in gastric cancer cells after co-incubation with H. pylori suggesting that it inhibits Cagα in vivo. Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis.

Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

Fragment-based screening identifies novel targets for inhibitors of conjugative transfer of antimicrobial resistance by plasmid pKM101.

The increasing frequency of antimicrobial resistance is a problem of global importance. Novel strategies are urgently needed to understand and inhibit antimicrobial resistance gene transmission that is mechanistically related to bacterial virulence functions. The conjugative transfer of plasmids by type IV secretion systems is a major contributor to antimicrobial resistance gene transfer. Here, we present a structure-based strategy to identify inhibitors of type IV secretion system-mediated bacterial conjugation. Using differential scanning fluorimetry we screened a fragment library and identified molecules that bind the essential TraE protein of the plasmid pKM101 conjugation machinery. Co-crystallization revealed that fragments bind two alternative sites of the protein and one of them is a novel inhibitor binding site. Based on the structural information on fragment binding we designed novel small molecules that have improved binding affinity. These molecules inhibit the dimerization of TraE, bind to both inhibitor binding sites on TraE and inhibit the conjugative transfer of plasmid pKM101. The strategy presented here is generally applicable for the structure-based design of inhibitors of antimicrobial resistance gene transfer and of bacterial virulence.

Monomer-to-dimer transition of Brucella suis type IV secretion system component VirB8 induces conformational changes.

Secretion systems are protein complexes essential for bacterial virulence and potential targets for antivirulence drugs. In the intracellular pathogen Brucella suis, a type IV secretion system mediates the translocation of virulence factors into host cells and it is essential for pathogenicity. VirB8 is a core component of the secretion system and dimerization is important for functionality of the protein complex. We set out to study dimerization and possible conformational changes of VirB8 from B. suis (VirB8s) using nuclear magnetic resonance, X-ray crystallography, and differential scanning fluorimetry. We identified changes of the protein induced by a concentration-dependent monomer-to-dimer transition of the periplasmic domain (VirB8sp). We also show that the presence of the detergent CHAPS alters several signals in the heteronuclear single quantum coherence (HSQC) spectra and some of these chemical shift changes correspond to those observed during monomer-dimer transition. X-ray analysis of a monomeric variant (VirB8spM102R ) demonstrates that significant structural changes occur in the protein's α-helical regions (α2 and α4). We localized chemical shift changes of residues at the dimer interface as well as to the α1 helix that links this interface to a surface groove that binds dimerization inhibitors. Fragment-based screening identified small molecules that bind to VirB8sp and two of them have differential binding affinity for wild-type and the VirB8spM102R variant underlining their different conformations. The observed chemical shift changes suggest conformational changes of VirB8s during monomer-dimer transition that may play a role during secretion system assembly or function and they provide insights into the mechanism of inhibitor action. DATABASE: BMRB accession no. 26852 and PDB 5JBS.

Chemical shift assignments of zinc finger domain of methionine aminopeptidase 1 (MetAP1) from Homo sapiens.

Methionine aminopeptidase Type I (MetAP1) cleaves the initiator methionine from about 70 % of all newly synthesized proteins in almost every living cell. Human MetAP1 is a two domain protein with a zinc finger on the N-terminus and a catalytic domain on the C-terminus. Here, we report the chemical shift assignments of the amino terminal zinc binding domain (ZBD) (1-83 residues) of the human MetAP1 derived by using advanced NMR spectroscopic methods. We were able to assign the chemical shifts of ZBD of MetAP1 nearly complete, which reveal two helical fragments involving residues P44-L49 (α1) and Q59-K82 (α2). The protein structure unfolds upon complex formation with the addition of 2 M excess EDTA, indicated by the appearance of amide resonances in the random coil chemical shift region of (15)NHSQC spectrum.

Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: Crystal structure in complex with E. coli aminopeptidase N.

Actinonin is a pseudotripeptide that displays a high affinity towards metalloproteases including peptide deformylases (PDFs) and M1 family aminopeptidases. PDF and M1 family aminopeptidases belong to thermolysin-metzincin superfamily. One of the major differences in terms of substrate binding pockets between these families is presence (in M1 aminopeptidases) or absence (in PDFs) of an S1 substrate pocket. The binding mode of actinonin to PDFs has been established previously; however, it is not clear how the actinonin, without a P1 residue, would bind to the M1 aminopeptidases. Here we describe the crystal structure of Escherichia coli aminopeptidase N (ePepN), a model protein of the M1 family aminopeptidases in complex with actinonin. For comparison we have also determined the structure of ePepN in complex with a well-known tetrapeptide inhibitor, amastatin. From the comparison of the actinonin and amastatin ePepN complexes, it is clear that the P1 residue is not critical as long as strong metal chelating head groups, like hydroxamic acid or α-hydroxy ketone, are present. Results from this study will be useful for the design of selective and efficient hydroxamate inhibitors against M1 family aminopeptidases.

Identification of the molecular basis of inhibitor selectivity between the human and streptococcal type I methionine aminopeptidases.

The methionine aminopeptidase (MetAP) family is responsible for the cleavage of the initiator methionine from newly synthesized proteins. Currently, there are no small molecule inhibitors that show selectivity toward the bacterial MetAPs compared to the human enzyme. In our current study, we have screened 20 α-aminophosphonate derivatives and identified a molecule (compound 15) that selectively inhibits the S. pneumonia MetAP in low micromolar range but not the human enzyme. Further bioinformatics, biochemical, and structural analyses suggested that phenylalanine (F309) in the human enzyme and methionine (M205) in the S. pneumonia MetAP at the analogous position render them with different susceptibilities against the identified inhibitor. X-ray crystal structures of various inhibitors in complex with wild type and F309M enzyme further established the molecular basis for the inhibitor selectivity.

Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents.

Methionine aminopeptidases (MetAPs) cleave initiator methionine from ~ 70% of the newly synthesized proteins in every living cell, and specific inhibition or knockdown of this function is detrimental. MetAPs are metalloenzymes, and are broadly classified into two subtypes, type I and type II. Bacteria contain only type I MetAPs, and the active site of these enzymes contains a conserved cysteine. By contrast, in type II enzymes the analogous position is occupied by a conserved glycine. Here, we report the reactivity of the active site cysteine in a type I MetAP, MetAP1c, of Mycobacterium tuberculosis (MtMetAP1c) towards highly selective cysteine-specific reagents. The authenticity of selective modification of Cys105 of MtMetAP1c was established by using site-directed mutagenesis and crystal structure determination of covalent and noncovalent complexes. On the basis of these observations, we propose that metal ions in the active site assist in the covalent modification of Cys105 by orienting the reagents appropriately for a successful reaction. These studies establish, for the first time, that the conserved cysteine of type I MetAPs can be targeted for selective inhibition, and we believe that this chemistry can be exploited for further drug discovery efforts regarding microbial MetAPs.

Discovery of a new genetic variant of methionine aminopeptidase from Streptococci with possible post-translational modifications: biochemical and structural characterization.

Protein N-terminal methionine excision is an essential co-translational process that occurs in the cytoplasm of all organisms. About 60-70% of the newly synthesized proteins undergo this modification. Enzyme responsible for the removal of initiator methionine is methionine aminopeptidase (MetAP), which is a dinuclear metalloprotease. This protein is conserved through all forms of life from bacteria to human except viruses. MetAP is classified into two isoforms, Type I and II. Removal of the map gene or chemical inhibition is lethal to bacteria and to human cell lines, suggesting that MetAP could be a good drug target. In the present study we describe the discovery of a new genetic variant of the Type I MetAP that is present predominantly in the streptococci bacteria. There are two inserts (insert one: 27 amino acids and insert two: four residues) within the catalytic domain. Possible glycosylation and phosphorylation posttranslational modification sites are identified in the 'insert one'. Biochemical characterization suggests that this enzyme behaves similar to other MetAPs in terms of substrate specificity. Crystal structure Type Ia MetAP from Streptococcus pneumoniae (SpMetAP1a) revealed that it contains two molecules in the asymmetric unit and well ordered inserts with structural features that corroborate the possible posttranslational modification. Both the new inserts found in the SpMetAP1a structurally align with the P-X-X-P motif found in the M. tuberculosis and human Type I MetAPs as well as the 60 amino acid insert in the human Type II enzyme suggesting possible common function. In addition, one of the ß-hairpins within in the catalytic domain undergoes a flip placing a residue which is essential for enzyme activity away from the active site and the ß-hairpin loop of this secondary structure in the active site obstructing substrate binding. This is the first example of a MetAP crystallizing in the inactive form.

Identification, biochemical and structural evaluation of species-specific inhibitors against type I methionine aminopeptidases.

Methionine aminopeptidases (MetAPs) are essential enzymes that make them good drug targets in cancer and microbial infections. MetAPs remove the initiator methionine from newly synthesized peptides in every living cell. MetAPs are broadly divided into type I and type II classes. Both prokaryotes and eukaryotes contain type I MetAPs, while eukaryotes have additional type II MetAP enzyme. Although several inhibitors have been reported against type I enzymes, subclass specificity is scarce. Here, using the fine differences in the entrance of the active sites of MetAPs from Mycobacterium tuberculosis , Enterococcus faecalis , and human, three hotspots have been identified and pyridinylpyrimidine-based molecules were selected from a commercial source to target these hotspots. In the biochemical evaluation, many of the 38 compounds displayed differential behavior against these three enzymes. Crystal structures of four selected inhibitors in complex with human MetAP1b and molecular modeling studies provided the basis for the binding specificity.