Restoring susceptibility to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus.

Infections by Staphylococcus aureus have been treated historically with ß-lactam antibiotics. However, these antibiotics have become obsolete in methicillin-resistant S. aureus by acquisition of the bla and mec operons. The presence of the ß-lactam antibiotic is detected by the sensor domains of BlaR and/or MecR, and the information is transmitted to the cytoplasm, resulting in derepression of the antibiotic-resistance genes. We hypothesized that inhibition of the sensor domain would shut down this response system, and ß-lactam susceptibility would be restored. An in silico search of 11 million compounds led to a benzimidazole-based hit and, ultimately, to the boronate 4. The X-ray structure of 4 is covalently engaged with the active-site serine of BlaR. Compound 4 potentiates by 16- to 4,096-fold the activities of oxacillin and of meropenem against methicillin-resistant S. aureus strains. The combination of 4 with oxacillin or meropenem shows efficacy in infected mice, validating the strategy.

A dual-action antibiotic that kills Clostridioides difficile vegetative cells and inhibits spore germination.

Clostridioides difficile infection (CDI) is the most lethal of the five CDC urgent public health treats, resulting in 12,800 annual deaths in the United States alone [Antibiotic Resistance Threats in the United States, 2019 (2019), www.cdc.gov/DrugResistance/Biggest-Threats.html]. The high recurrence rate and the inability of antibiotics to treat such infections mandate discovery of new therapeutics. A major challenge with CDI is the production of spores, leading to multiple recurrences of infection in 25% of patients [C. P. Kelly, J. T. LaMont, N. Engl. J. Med. 359, 1932-1940 (2008)], with potentially lethal consequence. Herein, we describe the discovery of an oxadiazole as a bactericidal anti-C. difficile agent that inhibits both cell-wall peptidoglycan biosynthesis and spore germination. We document that the oxadiazole binds to the lytic transglycosylase SleC and the pseudoprotease CspC for prevention of spore germination. SleC degrades the cortex peptidoglycan, a critical step in the initiation of spore germination. CspC senses germinants and cogerminants. Binding to SleC is with higher affinity than that to CspC. Prevention of spore germination breaks the nefarious cycles of CDI recurrence in the face of the antibiotic challenge, which is a primary cause of therapeutic failure. The oxadiazole exhibits efficacy in a mouse model of recurrent CDI and holds promise in clinical treatment of CDI.

Catalytic process of anhydro-N-acetylmuramic acid kinase from Pseudomonas aeruginosa.

The bacterial cell envelope is the structure with which the bacterium engages with, and is protected from, its environment. Within this envelop is a conserved peptidoglycan polymer which confers shape and strength to the cell envelop. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics and exploratory targets for emerging antibiotic structures. We report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N-acetylmuramic acid (anhNAM) kinase (AnmK). AnmK is an enzyme in the peptidoglycan-recycling pathway of this pathogen. It catalyzes the pairing of hydrolytic ring opening of anhNAM with concomitant ATP-dependent phosphoryl transfer. AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures (apo AnmK, AnmK:AMPPNP, AnmK:AMPPNP:anhNAM, and AnmK:ATP:anhNAM) demonstrate that both substrates enter the active site independently in an ungated conformation of the substrate subsites, with protein loops acting as gates for anhNAM binding. Catalysis occurs within a closed conformational state for the enzyme. We observe this state crystallographically using ATP-mimetic molecules. A remarkable X-ray structure for dimeric AnmK sheds light on the precatalytic and postcatalytic ternary complexes. Computational simulations in conjunction with the high-resolution X-ray structures reveal the full catalytic cycle. We further report that a P. aeruginosa strain with disrupted anmK gene is more susceptible to the ß-lactam imipenem compared to the WT strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.

Application of 2D-ITC to the Elucidation of the Enzymatic Mechanism of N-Acetylmuramic Acid/N-Acetylglucosamine Kinase (AmgK) from Pseudomonas aeruginosa.

Characterization of the turnover mechanism of bisubstrate enzymes is a tedious task. Molecular tools for studying the enzymatic mechanism are not readily available for all enzymes (e.g., radioactive substrates, substrate-competitive inhibitors, etc.). Wang and Mittermaier recently introduced two-dimensional isothermal titration calorimetry (2D-ITC) for determining the bisubstrate mechanism at high resolution while simultaneously quantifying the kinetic parameters for substrate turnover in a single reporter-free experiment. We demonstrate the utility of 2D-ITC in studying N-acetylmuramic acid/N-acetylglucosamine kinase (AmgK) from Pseudomonas aeruginosa. This enzyme is involved in cytoplasmic cell-wall-recycling events as a step in the peptidoglycan salvage pathway. Furthermore, AmgK phosphorylates N-acetylglucosamine and N-acetylmuramic acid, linking the recycling events to de novo cell-wall synthesis. We document in a 2D-ITC experiment that AmgK follows an ordered-sequential mechanism, where ATP binds first and ADP is released last. We also show that classical enzyme kinetic methods support the results of 2D-ITC and that 2D-ITC could overcome the shortcomings of these classical methodologies. We provide evidence for inhibition of AmgK by the catalytic product ADP, but not by the phosphorylated sugar product. These results provide a full kinetic characterization of the bacterial kinase AmgK. This work highlights 2D-ITC as a versatile tool for the mechanistic evaluation of bisubstrate enzymes, as an alternative for classical methods.

Synthesis of Muramyl-δ-Lactam in Spore Peptidoglycan of Clostridioides difficile.

Clostridioides difficile is a spore-forming human pathogen responsible for significant morbidity and mortality. Infections by this pathogen ensue dysbiosis of the intestinal tract, which leads to germination of the spores. The process of spore formation requires a transition for the cell-wall peptidoglycan of the vegetative C. difficile to that of spores, which entails the formation of muramyl-δ-lactam. We describe a set of reactions for three recombinant C. difficile proteins, GerS, CwlD, and PdaA1, with the use of four synthetic peptidoglycan analogs. CwlD and PdaA1 excise the peptidoglycan stem peptide and the acetyl moiety of N-acetyl muramate, respectively. The reaction of CwlD is accelerated in the presence of GerS. With the use of a suitable substrate, we document that PdaA1 catalyzes a novel zinc-dependent transamidation/transpeptidation reaction, an unusual reaction that requires excision of the stem peptide as a pre-requisite.

ß-Lactams against the Fortress of the Gram-Positive Staphylococcus aureus Bacterium.

The biological diversity of the unicellular bacteria-whether assessed by shape, food, metabolism, or ecological niche-surely rivals (if not exceeds) that of the multicellular eukaryotes. The relationship between bacteria whose ecological niche is the eukaryote, and the eukaryote, is often symbiosis or stasis. Some bacteria, however, seek advantage in this relationship. One of the most successful-to the disadvantage of the eukaryote-is the small (less than 1 µm diameter) and nearly spherical Staphylococcus aureus bacterium. For decades, successful clinical control of its infection has been accomplished using ß-lactam antibiotics such as the penicillins and the cephalosporins. Over these same decades S. aureus has perfected resistance mechanisms against these antibiotics, which are then countered by new generations of ß-lactam structure. This review addresses the current breadth of biochemical and microbiological efforts to preserve the future of the ß-lactam antibiotics through a better understanding of how S. aureus protects the enzyme targets of the ß-lactams, the penicillin-binding proteins. The penicillin-binding proteins are essential enzyme catalysts for the biosynthesis of the cell wall, and understanding how this cell wall is integrated into the protective cell envelope of the bacterium may identify new antibacterials and new adjuvants that preserve the efficacy of the ß-lactams.

ß-Lactams from the Ocean.

The title of this essay is as much a question as it is a statement. The discovery of the ß-lactam antibiotics-including penicillins, cephalosporins, and carbapenems-as largely (if not exclusively) secondary metabolites of terrestrial fungi and bacteria, transformed modern medicine. The antibiotic ß-lactams inactivate essential enzymes of bacterial cell-wall biosynthesis. Moreover, the ability of the ß-lactams to function as enzyme inhibitors is of such great medical value, that inhibitors of the enzymes which degrade hydrolytically the ß-lactams, the ß-lactamases, have equal value. Given this privileged status for the ß-lactam ring, it is therefore a disappointment that the exemplification of this ring in marine secondary metabolites is sparse. It may be that biologically active marine ß-lactams are there, and simply have yet to be encountered. In this report, we posit a second explanation: that the value of the ß-lactam to secure an ecological advantage in the marine environment might be compromised by its close structural similarity to the ß-lactones of quorum sensing. The steric and reactivity similarities between the ß-lactams and the ß-lactones represent an outside-of-the-box opportunity for correlating new structures and new enzyme targets for the discovery of compelling biological activities.

Unconventional Antibacterials and Adjuvants.

The need for new classes of antibacterials is genuine in light of the dearth of clinical options for the treatment of bacterial infections. The prodigious discoveries of antibiotics during the 1940s to 1970s, a period wistfully referred to as the Golden Age of Antibiotics, have not kept up in the face of emergence of resistant bacteria in the past few decades. There has been a renewed interest in old drugs, the repurposing of the existing antibiotics and pairing of synergistic antibiotics or of an antibiotic with an adjuvant. Notwithstanding, discoveries of novel classes of these life-saving drugs have become increasingly difficult, calling for new paradigms. We describe, herein, three strategies from our laboratories toward discoveries of new antibacterials and adjuvants using computational and multidisciplinary experimental methods. One approach targets penicillin-binding proteins (PBPs), biosynthetic enzymes of cell-wall peptidoglycan, for discoveries of non-ß-lactam inhibitors. Oxadiazoles and quinazolinones emerged as two structural classes out of these efforts. Several hundred analogs of these two classes of antibiotics have been synthesized and fully characterized in our laboratories. A second approach ventures into inhibition of allosteric regulation of cell-wall biosynthesis. The mechanistic details of allosteric regulation of PBP2a of Staphylococcus aureus, discovered in our laboratories, is outlined. The allosteric site in this protein is at 60 Å distance to the active site, whereby ligand binding at the former makes access to the latter by the substrate possible. We have documented that both quinazolinones and ceftaroline, a fifth-generation cephalosporin, bind to the allosteric site in manifestation of the antibacterial activity. Attempts at inhibition of the regulatory phosphorylation events identified three classes of antibacterial adjuvants and one class of antibacterials, the picolinamides. The chemical structures for these hits went through diversification by synthesis of hundreds of analogs. These analogs were characterized in various assays for identification of leads with adjuvant and antibacterial activities. Furthermore, we revisited the mechanism of bulgecins, a class of adjuvants discovered and abandoned in the 1980s. These compounds potentiate the activities of ß-lactam antibiotics by the formation of bulges at the sites of septum formation during bacterial replication, which are points of structural weakness in the envelope. These bulges experience rupture, which leads to bacterial death. Bulgecin A inhibits the lytic transglycosylase Slt of Pseudomonas aeruginosa as a likely transition-state mimetic for its turnover of the cell-wall peptidoglycan. Once damage to cell wall is inflicted by a ß-lactam antibiotic, the function of Slt is to repair the damage. When Slt is inhibited by bulgecin A, the organism cannot cope with it and would undergo rapid lysis. Bulgecin A is an effective adjuvant of ß-lactam antibiotics. These discoveries of small-molecule classes of antibacterials or of adjuvants to antibacterials hold promise in strategies for treatment of bacterial infections.

Turnover Chemistry and Structural Characterization of the Cj0843c Lytic Transglycosylase of Campylobacter jejuni.

The soluble lytic transglycosylase Cj0843c from Campylobacter jejuni breaks down cell-wall peptidoglycan (PG). Its nonhydrolytic activity sustains cell-wall remodeling and repair. We report herein our structure-function studies probing the substrate preferences and recognition by this enzyme. Our studies show that Cj0843c exhibits both exolytic and endolytic activities and forms the N-acetyl-1,6-anhydromuramyl (anhMurNAc) peptidoglycan termini, the typical transformation catalyzed by lytic transglycosylase. Cj0843c shows a trend toward a preference for substrates with anhMurNAc ends and those with peptide stems. Mutagenesis revealed that the catalytic E390 is critical for activity. In addition, mutagenesis showed that R388 and K505, located in the positively charged pocket near E390, also serve important roles. Mutation of R326, on the opposite side of this positively charged pocket, enhanced activity. Our data point to different roles for positively charged residues in this pocket for productive binding of the predominantly negatively charged PG. We also show by X-ray crystallography and by molecular dynamics simulations that the active site of Cj0843c is still capable of binding GlcNAc containing di- and trisaccharides without MurNAc moieties, without peptide stems, and without the anhMurNAc ends.

A type VI secretion system delivers a cell wall amidase to target bacterial competitors.

The human pathogen Pseudomonas aeruginosa harbors three paralogous zinc proteases annotated as AmpD, AmpDh2, and AmpDh3, which turn over the cell wall and cell wall-derived muropeptides. AmpD is cytoplasmic and plays a role in the recycling of cell wall muropeptides, with a link to antibiotic resistance. AmpDh2 is a periplasmic soluble enzyme with the former anchored to the inner leaflet of the outer membrane. We document, herein, that the type VI secretion system locus II (H2-T6SS) of P. aeruginosa delivers AmpDh3 (but not AmpD or AmpDh2) to the periplasm of a prey bacterium upon contact. AmpDh3 hydrolyzes the cell wall peptidoglycan of the prey bacterium, which leads to its killing, thereby providing a growth advantage for P. aeruginosa in bacterial competition. We also document that the periplasmic protein PA0808, heretofore of unknown function, affords self-protection from lysis by AmpDh3. Cognates of the AmpDh3-PA0808 pair are widely distributed across Gram-negative bacteria. Taken together, these findings underscore the importance of their function as an evolutionary advantage and that of the H2-T6SS as the means for the manifestation of the effect.

Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa.

ß-Lactam antibiotics inhibit cell-wall transpeptidases, preventing the peptidoglycan, the major constituent of the bacterial cell wall, from cross-linking. This causes accumulation of long non-cross-linked strands of peptidoglycan, which leads to bacterial death. Pseudomonas aeruginosa, a nefarious bacterial pathogen, attempts to repair this aberrantly formed peptidoglycan by the function of the lytic transglycosylase Slt. We document in this report that Slt turns over the peptidoglycan by both exolytic and endolytic reactions, which cause glycosidic bond scission from a terminus or in the middle of the peptidoglycan, respectively. These reactions were characterized with complex synthetic peptidoglycan fragments that ranged in size from tetrasaccharides to octasaccharides. The X-ray structure of the wild-type apo Slt revealed it to be a doughnut-shaped protein. In a series of six additional X-ray crystal structures, we provide insights with authentic substrates into how Slt is enabled for catalysis for both the endolytic and exolytic reactions. The substrate for the exolytic reaction binds Slt in a canonical arrangement and reveals how both the glycan chain and the peptide stems are recognized by the Slt. We document that the apo enzyme does not have a fully formed active site for the endolytic reaction. However, binding of the peptidoglycan at the existing subsites within the catalytic domain causes a conformational change in the protein that assembles the surface for binding of a more expansive peptidoglycan between the catalytic domain and an adjacent domain. The complexes of Slt with synthetic peptidoglycan substrates provide an unprecedented snapshot of the endolytic reaction.

Hyperbaric oxygen therapy accelerates wound healing in diabetic mice by decreasing active matrix metalloproteinase-9.

Diabetic foot ulcers are characterized by hypoxia. For many patients, hyperbaric oxygen (HBO) therapy is the last recourse for saving the limb from amputation, for which the molecular basis is not understood. We previously identified the active form of matrix metalloproteinase-9 (MMP-9) as responsible for diabetic foot ulcer's recalcitrance to healing. Transcription of mmp-9 to the inactive zymogen is upregulated during hypoxia. Activation of the zymogen is promoted by proteases and reactive oxygen species (ROS). We hypothesized that the dynamics of these two events might lead to a lowering of active MMP-9 levels in the wounded tissue. We employed the full-thickness excisional db/db mouse model to study wound healing, and treated the mice to 3.0 atm of molecular oxygen for 90 minutes, 5 days per week for 10 days in an HBO research chamber. Treatment with HBO accelerated diabetic wound healing compared to untreated mice, with more completed and extended reepithelialization. We imaged the wounds for ROS in vivo with a luminol-based probe and found that HBO treatment actually decreases ROS levels. The levels of superoxide dismutase, catalase, and glutathione peroxidase-enzymes that turn over ROS-increased after HBO treatment, hence the observation of decreased ROS. Since ROS levels are lowered, we explored the effect that this would have on activation of MMP-9. Quantitative analysis with an affinity resin that binds and pulls down the active MMPs exclusively, coupled with proteomics, revealed that HBO treatment indeed reduces the active MMP-9 levels. This work for the first time demonstrates that diminution of active MMP-9 is a contributing factor and a mechanism for enhancement of diabetic wound healing by HBO therapy.

Cell-Wall Recycling of the Gram-Negative Bacteria and the Nexus to Antibiotic Resistance.

The importance of the cell wall to the viability of the bacterium is underscored by the breadth of antibiotic structures that act by blocking key enzymes that are tasked with cell-wall creation, preservation, and regulation. The interplay between cell-wall integrity, and the summoning forth of resistance mechanisms to deactivate cell-wall-targeting antibiotics, involves exquisite orchestration among cell-wall synthesis and remodeling and the detection of and response to the antibiotics through modulation of gene regulation by specific effectors. Given the profound importance of antibiotics to the practice of medicine, the assertion that understanding this interplay is among the most fundamentally important questions in bacterial physiology is credible. The enigmatic regulation of the expression of the AmpC ß-lactamase, a clinically significant and highly regulated resistance response of certain Gram-negative bacteria to the ß-lactam antibiotics, is the exemplar of this challenge. This review gives a current perspective to this compelling, and still not fully solved, 35-year enigma.

Lytic transglycosylases: concinnity in concision of the bacterial cell wall.

The lytic transglycosylases (LTs) are bacterial enzymes that catalyze the non-hydrolytic cleavage of the peptidoglycan structures of the bacterial cell wall. They are not catalysts of glycan synthesis as might be surmised from their name. Notwithstanding the seemingly mundane reaction catalyzed by the LTs, their lytic reactions serve bacteria for a series of astonishingly diverse purposes. These purposes include cell-wall synthesis, remodeling, and degradation; for the detection of cell-wall-acting antibiotics; for the expression of the mechanism of cell-wall-acting antibiotics; for the insertion of secretion systems and flagellar assemblies into the cell wall; as a virulence mechanism during infection by certain Gram-negative bacteria; and in the sporulation and germination of Gram-positive spores. Significant advances in the mechanistic understanding of each of these processes have coincided with the successive discovery of new LTs structures. In this review, we provide a systematic perspective on what is known on the structure-function correlations for the LTs, while simultaneously identifying numerous opportunities for the future study of these enigmatic enzymes.

Susceptibility of Methicillin-Resistant Staphylococcus aureus to Five Quinazolinone Antibacterials.

The in vitro activities of five quinazolinone antibacterials, compounds Q1 to Q5, were tested against 210 strains of methicillin-resistant Staphylococcus aureus (MRSA). The MIC50/MIC90 values (in µg/ml) were as follows: Q1, 0.5/2; Q2, 1/4; Q3, 2/4; Q4, 0.06/0.25; and Q5, 0.125/0.5. Several strains with high MIC values (from 8 to >32 µg/ml) for some of these compounds exhibited amino acid changes in the penicillin-binding proteins, which are targeted by these antibacterials.

The Quinazolinone Allosteric Inhibitor of PBP 2a Synergizes with Piperacillin and Tazobactam against Methicillin-Resistant Staphylococcus aureus.

The quinazolinones are a new class of antibacterials with in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA). The quinazolinones target cell wall biosynthesis and have a unique mechanism of action by binding to the allosteric site of penicillin-binding protein 2a (PBP 2a). We investigated the potential for synergism of a lead quinazolinone with several antibiotics of different classes using checkerboard and time-kill assays. The quinazolinone synergized with ß-lactam antibiotics. The combination of the quinazolinone with commercial piperacillin-tazobactam showed bactericidal synergy at sub-MICs of all three drugs. We demonstrated the efficacy of the triple-drug combination in a mouse MRSA neutropenic thigh infection model. The proposed mechanism for the synergistic activity in MRSA involves inhibition of the ß-lactamase by tazobactam, which protects piperacillin from hydrolysis, which can then inhibit its target, PBP 2. Furthermore, the quinazolinone binds to the allosteric site of PBP 2a, triggering the allosteric response. This leads to the opening of the active site, which, in turn, binds another molecule of piperacillin. In other words, PBP 2a, which is not normally inhibited by piperacillin, becomes vulnerable to inhibition in the presence of the quinazolinone. The collective effect is the impairment of cell wall biosynthesis, with bactericidal consequence. Two crystal structures for complexes of the antibiotics with PBP 2a provide support for the proposed mechanism of action.

A Structural Dissection of the Active Site of the Lytic Transglycosylase MltE from Escherichia coli.

Lytic transglycosylases (LTs) are bacterial enzymes that catalyze the cleavage of the glycan strands of the bacterial cell wall. The mechanism of this cleavage is a remarkable intramolecular transacetalization reaction, accomplished by an ensemble of active-site residues. Because the LT reaction occurs in parallel with the cell wall bond-forming reactions catalyzed by the penicillin-binding proteins, simultaneous inhibition of both enzymes can be particularly bactericidal to Gram-negative bacteria. The MltE lytic transglycosylase is the smallest of the eight LTs encoded by the Escherichia coli genome. Prior crystallographic and computational studies identified four active-site residues-E64, S73, S75, and Y192-as playing roles in catalysis. Each of these four residues was individually altered by mutation to give four variant enzymes (E64Q, S73A, S75A, and Y192F). All four variants showed reduced catalytic activity [soluble wild type (100%) > soluble Y192F and S75A (both 40%) > S73A (4%) > E64Q (≤1%)]. The crystal structure of each variant protein was determined at the resolution of 2.12 Å for E64Q, 2.33 Å for Y192F, 1.38 Å for S73A, and 1.35 Å for S75A. These variants show alteration of the hydrogen-bond interactions of the active site. Within the framework of a prior computational study of the LT mechanism, we suggest the mechanistic role of these four active-site residues in MltE catalysis.

Activities of Oxadiazole Antibacterials against Staphylococcus aureus and Other Gram-Positive Bacteria.

The activities of four oxadiazoles were investigated with 210 methicillin-resistant Staphylococcus aureus (MRSA) strains. MIC50 and MIC90 values of 1 to 2 and 4 µg/ml, respectively, were observed. We also evaluated the activity of oxadiazole ND-421 against other staphylococci and enterococci and in the presence of oxacillin for selected MRSA strains. The MIC for ND-421 is lowered severalfold in combination with oxacillin, as they synergize. The MIC90 of ND-421 against vancomycin-resistant enterococci is ≤1 µg/ml.

Potentiation of the activity of ß-lactam antibiotics by farnesol and its derivatives.

Farnesol, a sesquiterpene alcohol, potentiates the activity of ß-lactam antibiotics against antibiotic-resistant bacteria. We document that farnesol and two synthetic derivatives (compounds 2 and 6) have poor antibacterial activities of their own, but they potentiate the activities of ampicillin and oxacillin against Staphylococcus aureus strains (including methicillin-resistant S. aureus). These compounds attenuate the rate of growth of bacteria, which has to be taken into account in assessment of the potentiation effect.

Acceleration of diabetic wound healing using a novel protease-anti-protease combination therapy.

Nonhealing chronic wounds are major complications of diabetes resulting in >70,000 annual lower-limb amputations in the United States alone. The reasons the diabetic wound is recalcitrant to healing are not fully understood, and there are limited therapeutic agents that could accelerate or facilitate its repair. We previously identified two active forms of matrix metalloproteinases (MMPs), MMP-8 and MMP-9, in the wounds of db/db mice. We argued that the former might play a role in the body's response to wound healing and that the latter is the pathological consequence of the disease with detrimental effects. Here we demonstrate that the use of compound ND-336, a novel highly selective inhibitor of gelatinases (MMP-2 and MMP-9) and MMP-14, accelerates diabetic wound healing by lowering inflammation and by enhancing angiogenesis and re-epithelialization of the wound, thereby reversing the pathological condition. The detrimental role of MMP-9 in the pathology of diabetic wounds was confirmed further by the study of diabetic MMP-9-knockout mice, which exhibited wounds more prone to healing. Furthermore, topical administration of active recombinant MMP-8 also accelerated diabetic wound healing as a consequence of complete re-epithelialization, diminished inflammation, and enhanced angiogenesis. The combined topical application of ND-336 (a small molecule) and the active recombinant MMP-8 (an enzyme) enhanced healing even more, in a strategy that holds considerable promise in healing of diabetic wounds.