Synthesis of an Antimicrobial Enterobactin-Muraymycin Conjugate for Improved Activity Against Gram-Negative Bacteria.

Overcoming increasing antibiotic resistance requires the development of novel antibacterial agents that address new targets in bacterial cells. Naturally occurring nucleoside antibiotics (such as muraymycins) inhibit the bacterial membrane protein MraY, a clinically unexploited essential enzyme in peptidoglycan (cell wall) biosynthesis. Even though a range of synthetic muraymycin analogues has already been reported, they generally suffer from limited cellular uptake and a lack of activity against Gram-negative bacteria. We herein report an approach to overcome these hurdles: a synthetic muraymycin analogue has been conjugated to a siderophore, i. e. the enterobactin derivative EntKL , to increase the cellular uptake into Gram-negative bacteria. The resultant conjugate showed significantly improved antibacterial activity against an efflux-deficient E. coli strain, thus providing a proof-of-concept of this novel approach and a starting point for the future optimisation of such conjugates towards potent agents against Gram-negative pathogens.

Targeting the binding pocket of the fluorophore 8-anilinonaphthalene-1-sulfonic acid in the bacterial enzyme MurA.

8-Anilinonaphthalene-1-sulfonic acid (ANS) has been extensively used as a fluorescent probe to detect conformational changes of proteins. It has been cocrystallized with several of the proteins it is used to monitor, including the bacterial cell wall synthesis enzyme MurA. MurA catalyzes the first committed step of peptidoglycan biosynthesis, converting UDP-N-acetylglucosamine (UDP-GlcNAc) into enolpyruvyl UDP-GlcNAc. It has been reported before that ANS binds to MurA from Enterobacter cloacae without inhibiting the enzyme's activity up to a concentration of 1 mM ANS. In this study, we present evidence that ANS inhibits the activity of several isoforms of MurA with IC50 values of 18, 22, and 31 µM against wild-type Escherichia coli, C115D E. coli, and E. cloacae MurA, respectively. This prompted us to test a larger series of structural analogs of ANS for the inhibition of these MurA enzymes, which led to the discovery of compound 26. This ANS analog showed enhanced inhibition of MurA (WT and C115D MurA from E. coli, and E. cloacae MurA) with IC50 values of 2.7, 10, and 14 µM, respectively. Based on our results, the ANS binding pocket was identified as a novel target site for the development of potential antibiotics.

Pyridoxal-5'-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis.

Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5'S,6'S)-5'-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR-GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-L-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5'-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.

Enzymatic Cß-H Functionalization of l-Arg and l-Leu in Nonribosomally Derived Peptidyl Natural Products: A Tale of Two Oxidoreductases.

Muraymycins are peptidyl nucleoside antibiotics that contain two Cß-modified amino acids, (2S,3S)-capreomycidine and (2S,3S)-ß-OH-Leu. The former is also a component of chymostatins, which are aldehyde-containing peptidic protease inhibitors that─like muraymycin─are derived from nonribosomal peptide synthetases (NRPSs). Using feeding experiments and in vitro characterization of 12 recombinant proteins, the biosynthetic mechanism for both nonproteinogenic amino acids is now defined. The formation of (2S,3S)-capreomycidine is shown to involve an FAD-dependent dehydrogenase:cyclase that requires an NRPS-bound pathway intermediate as a substrate. This cryptic dehydrogenation strategy is both temporally and mechanistically distinct in comparison to the biosynthesis of other capreomycidine diastereomers, which has previously been shown to proceed by Cß-hydroxylation of free l-Arg catalyzed by a member of the nonheme Fe2+- and α-ketoglutarate (αKG)-dependent dioxygenase family and (eventually) a dehydration-mediated cyclization process catalyzed by a distinct enzyme(s). Contrary to our initial expectation, the sole nonheme Fe2+- and αKG-dependent dioxygenase candidate Mur15 encoded within the muraymycin gene cluster is instead demonstrated to catalyze specific Cß hydroxylation of the Leu residue to generate (2S,3S)-ß-OH-Leu that is found in most muraymycin congeners. Importantly, and in contrast to known l-Arg-Cß-hydroxylases, the Mur15-catalyzed reaction occurs after the NRPS-mediated assembly of the peptide scaffold. This late-stage functionalization affords the opportunity to exploit Mur15 as a biocatalyst, proof of concept of which is provided.

Pyridoxal-5'-phosphate as an oxygenase cofactor: Discovery of a carboxamide-forming, α-amino acid monooxygenase-decarboxylase.

Capuramycins are antimycobacterial antibiotics that consist of a modified nucleoside named uridine-5'-carboxamide (CarU). Previous biochemical studies have revealed that CarU is derived from UMP, which is first converted to uridine-5'-aldehyde in a reaction catalyzed by the dioxygenase CapA and subsequently to 5'-C-glycyluridine (GlyU), an unusual ß-hydroxy-α-amino acid, in a reaction catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent transaldolase CapH. The remaining steps that are necessary to furnish CarU include decarboxylation, O atom insertion, and oxidation. We demonstrate that Cap15, which has sequence similarity to proteins annotated as bacterial, PLP-dependent l-seryl-tRNA(Sec) selenium transferases, is the sole catalyst responsible for complete conversion of GlyU to CarU. Using a complementary panel of in vitro assays, Cap15 is shown to be dependent upon substrates O2 and (5'S,6'R)-GlyU, the latter of which was unexpected given that (5'S,6'S)-GlyU is the isomeric product of the transaldolase CapH. The two products of Cap15 are identified as the carboxamide-containing CarU and CO2 While known enzymes that catalyze this type of chemistry, namely α-amino acid 2-monooxygenase, utilize flavin adenine dinucleotide as the redox cofactor, Cap15 remarkably requires only PLP. Furthermore, Cap15 does not produce hydrogen peroxide and is shown to directly incorporate a single O atom from O2 into the product CarU and thus is an authentic PLP-dependent monooxygenase. In addition to these unusual discoveries, Cap15 activity is revealed to be dependent upon the inclusion of phosphate. The biochemical characteristics along with initiatory mechanistic studies of Cap15 are reported, which has allowed us to assign Cap15 as a PLP-dependent (5'S,6'R)-GlyU:O2 monooxygenase-decarboxylase.

Towards Zwitterionic Oligonucleotides with Improved Properties: the NAA/LNA-Gapmer Approach.

Oligonucleotides (ON) are promising therapeutic candidates, for instance by blocking endogenous mRNA (antisense mechanism). However, ON usually require structural modifications of the native nucleic acid backbone to ensure satisfying pharmacokinetic properties. One such strategy to design novel antisense oligonucleotides is to replace native phosphate diester units by positively charged artificial linkages, thus leading to (partially) zwitterionic backbone structures. Herein, we report a "gapmer" architecture comprised of one zwitterionic central segment ("gap") containing nucleosyl amino acid (NAA) modifications and two outer segments of locked nucleic acid (LNA). This NAA/LNA-gapmer approach furnished a partially zwitterionic ON with optimised properties: i) the formation of stable ON-RNA duplexes with base-pairing fidelity and superior target selectivity at 37 °C; and ii) excellent stability in complex biological media. Overall, the NAA/LNA-gapmer approach is thus established as a strategy to design partially zwitterionic ON for the future development of novel antisense agents.

Specific, Sensitive, and Quantitative Detection of HER-2 mRNA Breast Cancer Marker by Fluorescent Light-Up Hybridization Probes.

Currently, there is demand for fluorescent oligonucleotide probes for diagnostic purposes. To address this necessity, we developed nucleosides containing a flexible spacer with an intercalating moiety at its end (NIC molecules). The intercalator is based on 4-hydroxybenzylidene imidazolinone (HBI), found in the Green Fluorescent Protein. We synthesized 20-mer oligonucleotides, ON1-ON4, incorporating the DMTr phosphorodiamidite monomer of dUHBI, 2, and the corresponding dUDFHBI, 5b, monomer. ON1-ON4 target the HER-2 mRNA breast cancer marker for the diagnostics of breast cancer subtype. Hybridization of ON1/ON2 and ON3/ON4 with complementary 2'-OMe-RNA resulted in emission at 462 and 481 nm, respectively, and up to 46-fold increase in fluorescence intensity. CD and 19F-NMR data indicated that HBI and DFHBI fluorophores bind as intercalators and stabilize the duplexes (up to ΔTm 6 °C). Furthermore, addition of ON1-ON4 to total RNA extracted from cancer cells that overexpress HER-2 mRNA, resulted in a significant fluorescence enhancement of ON3 and ON4. The latter sensitively detected low concentrations of the target mRNA (at total RNA 30 ng/µL). These probes were photostable for 200 min. Using a dilution curve, we quantified the number of HER-2 transcripts in a cell. In conclusion, ON3 and ON4 are promising diagnostic probes for an easy, instantaneous, specific, and sensitive detection of levels of oncogenes. Importantly, the NIC concept, demonstrated here for diagnostics of breast cancer, is universal and may be applied not only in a clinical setting but also for the detection of any RNA.

Merging Natural Products: Muraymycin-Sansanmycin Hybrid Structures as Novel Scaffolds for Potential Antibacterial Agents.

To overcome bacterial resistances, the need for novel antimicrobial agents is urgent. The class of so-called nucleoside antibiotics furnishes promising candidates for the development of new antibiotics, as these compounds block a clinically unexploited bacterial target: the integral membrane protein MraY, a key enzyme in cell wall (peptidoglycan) biosynthesis. Nucleoside antibiotics exhibit remarkable structural diversity besides their uridine-derived core motifs. Some sub-classes also show specific selectivities towards different Gram-positive and Gram-negative bacteria, which are poorly understood so far. Herein, the synthesis of a novel hybrid structure is reported, derived from the 5'-defunctionalized uridine core moiety of muraymycins and the peptide chain of sansanmycin B, as a new scaffold for the development of antimicrobial agents. The reported muraymycin-sansanmycin hybrid scaffold showed nanomolar activity against the bacterial target enzyme MraY, but displayed no significant antibacterial activity against S. aureus, E. coli, and P. aeruginosa.

Cellular Targeting of Oligonucleotides by Conjugation with Small Molecules.

Drug candidates derived from oligonucleotides (ON) are receiving increased attention that is supported by the clinical approval of several ON drugs. Such therapeutic ON are designed to alter the expression levels of specific disease-related proteins, e.g., by displaying antigene, antisense, and RNA interference mechanisms. However, the high polarity of the polyanionic ON and their relatively rapid nuclease-mediated cleavage represent two major pharmacokinetic hurdles for their application in vivo. This has led to a range of non-natural modifications of ON structures that are routinely applied in the design of therapeutic ON. The polyanionic architecture of ON often hampers their penetration of target cells or tissues, and ON usually show no inherent specificity for certain cell types. These limitations can be overcome by conjugation of ON with molecular entities mediating cellular 'targeting', i.e., enhanced accumulation at and/or penetration of a specific cell type. In this context, the use of small molecules as targeting units appears particularly attractive and promising. This review provides an overview of advances in the emerging field of cellular targeting of ON via their conjugation with small-molecule targeting structures.

Muraymycin Nucleoside Antibiotics: Structure-Activity Relationship for Variations in the Nucleoside Unit.

Muraymycins are a subclass of naturally occurring nucleoside antibiotics with promising antibacterial activity. They inhibit the bacterial enzyme translocase I (MraY), a clinically yet unexploited target mediating an essential intracellular step of bacterial peptidoglycan biosynthesis. Several structurally simplified muraymycin analogues have already been synthesized for structure-activity relationship (SAR) studies. We now report on novel derivatives with unprecedented variations in the nucleoside unit. For the synthesis of these new muraymycin analogues, we employed a bipartite approach facilitating the introduction of different nucleosyl amino acid motifs. This also included thymidine- and 5-fluorouridine-derived nucleoside core structures. Using an in vitro assay for MraY activity, it was found that the introduction of substituents in the 5-position of the pyrimidine nucleobase led to a significant loss of inhibitory activity towards MraY. The loss of nucleobase aromaticity (by reduction of the uracil C5-C6 double bond) resulted in a ca. tenfold decrease in inhibitory potency. In contrast, removal of the 2'-hydroxy group furnished retained activity, thus demonstrating that modifications of the ribose moiety might be well-tolerated. Overall, these new SAR insights will guide the future design of novel muraymycin analogues for their potential development towards antibacterial drug candidates.

Self-Resistance during Muraymycin Biosynthesis: a Complementary Nucleotidyltransferase and Phosphotransferase with Identical Modification Sites and Distinct Temporal Order.

Muraymycins are antibacterial natural products from Streptomyces spp. that inhibit translocase I (MraY), which is involved in cell wall biosynthesis. Structurally, muraymycins consist of a 5'-C-glycyluridine (GlyU) appended to a 5″-amino-5″-deoxyribose (ADR), forming a disaccharide core that is found in several peptidyl nucleoside inhibitors of MraY. For muraymycins, the GlyU-ADR disaccharide is further modified with an aminopropyl-linked peptide to generate the simplest structures, annotated as the muraymycin D series. Two enzymes encoded in the muraymycin biosynthetic gene cluster, Mur29 and Mur28, were functionally assigned in vitro as a Mg·ATP-dependent nucleotidyltransferase and a Mg·ATP-dependent phosphotransferase, respectively, both modifying the 3″-OH of the disaccharide. Biochemical characterization revealed that both enzymes can utilize several nucleotide donors as cosubstrates and the acceptor substrate muraymycin also behaves as an inhibitor. Single-substrate kinetic analyses revealed that Mur28 preferentially phosphorylates a synthetic GlyU-ADR disaccharide, a hypothetical biosynthetic precursor of muraymycins, while Mur29 preferentially adenylates the D series of muraymycins. The adenylated or phosphorylated products have significantly reduced (170-fold and 51-fold, respectively) MraY inhibitory activities and reduced antibacterial activities, compared with the respective unmodified muraymycins. The results are consistent with Mur29-catalyzed adenylation and Mur28-catalyzed phosphorylation serving as complementary self-resistance mechanisms, with a distinct temporal order during muraymycin biosynthesis.

Oligonucleotides with Cationic Backbone and Their Hybridization with DNA: Interplay of Base Pairing and Electrostatic Attraction.

Non-natural oligonucleotides represent important (bio)chemical tools and potential therapeutic agents. Backbone modifications altering hybridization properties and biostability can provide useful analogues. Here, we employ an artificial nucleosyl amino acid (NAA) motif for the synthesis of oligonucleotides containing a backbone decorated with primary amines. An oligo-T sequence of this cationic DNA analogue shows significantly increased affinity for complementary DNA. Notably, hybridization with DNA is still governed by Watson-Crick base pairing. However, single base pair mismatches are tolerated and some degree of sequence-independent interactions between the cationic NAA backbone and fully mismatched DNA are observed. These findings demonstrate that a high density of positive charges directly connected to the oligonucleotide backbone can affect Watson-Crick base pairing. This provides a paradigm for the design of therapeutic oligonucleotides with altered backbone charge patterns.

Enzymatic Synthesis of the Ribosylated Glycyl-Uridine Disaccharide Core of Peptidyl Nucleoside Antibiotics.

Muraymycins belong to a family of nucleoside antibiotics that have a distinctive disaccharide core consisting of 5-amino-5-deoxyribofuranose (ADR) attached to 6'- N-alkyl-5'- C-glycyluridine (GlyU). Here, we functionally assign and characterize six enzymes from the muraymycin biosynthetic pathway involved in the core assembly that starts from uridine monophosphate (UMP). The biosynthesis is initiated by Mur16, a nonheme Fe(II)- and α-ketoglutarate-dependent dioxygenase, followed by four transferase enzymes: Mur17, a pyridoxal-5'-phosphate (PLP)-dependent transaldolase; Mur20, an aminotransferase; Mur26, a pyrimidine phosphorylase; and Mur18, a nucleotidylyltransferase. The pathway culminates in glycosidic bond formation in a reaction catalyzed by an additional transferase enzyme, Mur19, a ribosyltransferase. Analysis of the biochemical properties revealed several noteworthy discoveries including that (i) Mur16 and downstream enzymes can also process 2'-deoxy-UMP to generate a 2-deoxy-ADR, which is consistent with the structure of some muraymycin congeners; (ii) Mur20 prefers l-Tyr as the amino donor source; (iii) Mur18 activity absolutely depends on the amine functionality of the ADR precursor consistent with the nucleotidyltransfer reaction occurring after the Mur20-catalyzed aminotransfer reaction; and (iv) the bona fide sugar acceptor for Mur19 is (5' S,6' S)-GlyU, suggesting that ribosyltransfer occurs prior to N-alkylation of GlyU. Finally, a one-pot, six-enzyme reaction was utilized to generate the ADR-GlyU disaccharide core starting from UMP.

Antibacterial Muraymycins from Mutant Strains of Streptomyces sp. NRRL 30471.

Muraymycins are nucleoside antibiotics isolated from Streptomyces sp. NRRL 30471 and several mutant strains thereof that were generated by random, chemical mutagenesis. Reinvestigation of two mutant strains using new media conditions led to the isolation of three new muraymycin congeners, named B8, B9, and C6 (1-3), as well as a known muraymycin, C1. Structures of the compounds were elucidated by HRMS and 1D and 2D NMR spectroscopic analyses. Complete 2D NMR assignments for the known muraymycin C1 are also provided for the first time. Compounds 1 and 2, which differ from other muraymycins by having an elongated, terminally branched fatty acid side chain, had picomolar IC50 values against Staphylococcus aureus and Aquifex aeolicus MraY and showed good antibacterial activity against S. aureus (MIC = 2 and 6 µg/mL, respectively) and Escherichia coli Δ tolC (MIC = 4 and 2 µg/mL, respectively). Compound 3, which is characterized by an N-acetyl modification of the primary amine of the dissacharide core that is shared among nearly all of the reported muraymycin congeners, greatly reduced its inhibitory and antibacterial activity compared to nonacylated muraymycin C1, which possibly indicates this modification is used for self-resistance.

Design and synthesis of novel annulated thienopyrimidines as phosphodiesterase 5 (PDE5) inhibitors.

Novel cycloalkene-fused thienopyrimidine analogues with enhanced phosphodiesterase 5 (PDE5) inhibitory properties are presented. The structure of the reported scaffold was modulated through variation of the terminal cycloalkene ring size, as well as by varying the substituents at position 4 through the attachment of different groups including aniline, benzylamine, cyclohexylethylamine, methyl/acetyl/aryl piperazines, and aryl hydrazones. Compound 15Y with a benzylamine substituent and cycloheptene as terminal ring showed the highest PDE5 inhibitory activity with an IC50 value as low as 190 nM and with good selectivity versus PDE7 and PDE9.

Aminoribosylated Analogues of Muraymycin Nucleoside Antibiotics.

Nucleoside antibiotics are uridine-derived natural products that inhibit the bacterial membrane protein MraY. MraY is a key enzyme in the membrane-associated intracellular stages of peptidoglycan biosynthesis and therefore considered to be a promising, yet unexploited target for novel antibacterial agents. Muraymycins are one subclass of such naturally occurring MraY inhibitors. As part of structure-activity relationship (SAR) studies on muraymycins and their analogues, we now report on novel derivatives with different attachment of one characteristic structural motif, i.e., the aminoribose moiety normally linked to the muraymycin glycyluridine core unit. Based on considerations derived from an X-ray co-crystal structure, we designed and synthesised muraymycin analogues having the aminoribose attached (via a linker) to either the glycyluridine amino group or to the uracil nucleobase. Reference compounds bearing the non-aminoribosylated linker units were also prepared. It was found that the novel aminoribosylated analogues were inactive as MraY inhibitors in vitro, but that the glycyluridine-modified reference compound retained most of the inhibitory potency relative to the unmodified parent muraymycin analogue. These results point to 6'-N-alkylated muraymycin analogues as a potential novel variation of the muraymycin scaffold for future SAR optimisation.

Enhanced Stability of DNA Oligonucleotides with Partially Zwitterionic Backbone Structures in Biological Media.

Deficient stability towards nuclease-mediated degradation is one of the most relevant tasks in the development of oligonucleotide-derived biomedical agents. This hurdle can be overcome through modifications to the native oligonucleotide backbone structure, with the goal of simultaneously retaining the unique hybridization properties of nucleic acids. The nucleosyl amino acid (NAA)-modification is a recently introduced artificial cationic backbone linkage. Partially zwitterionic NAA-modified oligonucleotides had previously shown hybridization with DNA strands with retained base-pairing fidelity. In this study, we report the significantly enhanced stability of NAA-modified oligonucleotides towards 3'- and 5'-exonuclease-mediated degradation as well as in complex biological media such as human plasma and whole cell lysate. This demonstrates the potential versatility of the NAA-motif as a backbone modification for the development of biomedically active oligonucleotide analogues.

Analogues of Muraymycin Nucleoside Antibiotics with Epimeric Uridine-Derived Core Structures.

Nucleoside analogues have found widespread application as antiviral and antitumor agents, but not yet as antibacterials. Naturally occurring uridine-derived 'nucleoside antibiotics' target the bacterial membrane protein MraY, an enzyme involved in peptidoglycan biosynthesis and a promising target for the development of novel antibacterial agents. Muraymycins represent a nucleoside-peptide subgroup of such MraY-inhibiting natural products. As part of detailed structure-activity relationship (SAR) studies on muraymycins and their analogues, we now report novel insights into the effects of stereochemical variations in the nucleoside core structure. Using a simplified version of the muraymycin scaffold, it was shown that some formal inversions of stereochemistry led to about one order of magnitude loss in inhibitory potency towards the target enzyme MraY. In contrast, epimers of the core motif with retained inhibitory activity were also identified. These 5',6'-anti-configured analogues might serve as novel chemically tractable variations of the muraymycin scaffold for the future development of uridine-derived drug candidates.

Oligonucleotide analogues with cationic backbone linkages.

Their unique ability to selectively bind specific nucleic acid sequences makes oligonucleotides promising bioactive agents. However, modifications of the nucleic acid structure are an essential prerequisite for their application in vivo or even in cellulo. The oligoanionic backbone structure of oligonucleotides mainly hampers their ability to penetrate biological barriers such as cellular membranes. Hence, particular attention has been given to structural modifications of oligonucleotides which reduce their overall number of negative charges. One such approach is the site-specific replacement of the negatively charged phosphate diester linkage with alternative structural motifs which are positively charged at physiological pH, thus resulting in zwitterionic or even oligocationic backbone structures. This review provides a general overview of this concept and summarizes research on four according artificial backbone linkages: aminoalkylated phosphoramidates (and related systems), guanidinium groups, S-methylthiourea motifs, and nucleosyl amino acid (NAA)-derived modifications. The synthesis and properties of the corresponding oligonucleotide analogues are described.

Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases.

Secreted virulence factors like bacterial collagenases are conceptually attractive targets for fighting microbial infections. However, previous attempts to develop potent compounds against these metalloproteases failed to achieve selectivity against human matrix metalloproteinases (MMPs). Using a surface plasmon resonance-based screening complemented with enzyme inhibition assays, we discovered an N-aryl mercaptoacetamide-based inhibitor scaffold that showed sub-micromolar affinities toward collagenase H (ColH) from the human pathogen Clostridium histolyticum. Moreover, these inhibitors also efficiently blocked the homologous bacterial collagenases, ColG from C. histolyticum, ColT from C. tetani, and ColQ1 from the Bacillus cereus strain Q1, while showing negligible activity toward human MMPs-1, -2, -3, -7, -8, and -14. The most active compound displayed a more than 1000-fold selectivity over human MMPs. This selectivity can be rationalized by the crystal structure of ColH with this compound, revealing a distinct non-primed binding mode to the active site. The non-primed binding mode presented here paves the way for the development of selective broad-spectrum bacterial collagenase inhibitors with potential therapeutic application in humans.