Subinhibitory Concentrations of Antibiotics Alter the Response of Klebsiella pneumoniae to Components of Innate Host Defense.

Carbapenem-resistant Klebsiella pneumoniae isolates classified as multilocus sequence type 258 (ST258) are a problem in health care settings in many countries globally. ST258 isolates are resistant to multiple classes of antibiotics and can cause life-threatening infections, such as pneumonia and sepsis, in susceptible individuals. Treatment strategies for such infections are limited. Understanding the response of K. pneumoniae to host factors in the presence of antibiotics could reveal mechanisms employed by the pathogen to evade killing in the susceptible host, as well as inform treatment of infections. Here, we investigated the ability of antibiotics at subinhibitory concentrations to alter K. pneumoniae capsular polysaccharide (CPS) production and survival in normal human serum (NHS). Unexpectedly, pretreatment with some of the antibiotics tested enhanced ST258 survival in NHS. For example, a subinhibitory concentration of mupirocin increased survival for 7 of 10 clinical isolates evaluated and there was increased cell-associated CPS for 3 of these isolates compared with untreated controls. Additionally, mupirocin pretreatment caused concomitant reduction in the deposition of the serum complement protein C5b-9 on the surface of these three isolates. Transcriptome analyses with a selected ST258 isolate (34446) indicated that genes implicated in the stringent response and/or serum resistance were upregulated following mupirocin treatment and/or culture in NHS. In conclusion, mupirocin and/or human serum causes changes in the K. pneumoniae transcriptome that likely contribute to the observed decrease in serum susceptibility via a multifactorial process. Whether these responses can be extended more broadly and thus impact clinical outcome in the human host merits further investigation. IMPORTANCE The extent to which commensal bacteria are altered by exposure to subinhibitory concentrations of antibiotics (outside resistance) remains incompletely determined. To gain a better understanding of this phenomenon, we tested the ability of selected antibiotics (at subinhibitory concentrations) to alter survival of ST258 clinical isolates in normal human serum. We found that exposure of ST258 to antibiotics at low concentrations differentially altered gene expression, capsule production, serum complement deposition, and bacterial survival. The findings were isolate and antibiotic dependent but provide insight into a potential confounding issue associated with ST258 infections.

Innate Host Defense against Klebsiella pneumoniae and the Outlook for Development of Immunotherapies.

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative commensal bacterium and opportunistic pathogen. In healthy individuals, the innate immune system is adept at protecting against K. pneumoniae infection. Notably, the serum complement system and phagocytic leukocytes (e.g., neutrophils) are highly effective at eliminating K. pneumoniae and thereby preventing severe disease. On the other hand, the microbe is a major cause of healthcare-associated infections, especially in individuals with underlying susceptibility factors, such as pre-existing severe illness or immune suppression. The burden of K. pneumoniae infections in hospitals is compounded by antibiotic resistance. Treatment of these infections is often difficult largely because the microbes are usually resistant to multiple antibiotics (multidrug resistant [MDR]). There are a limited number of treatment options for these infections and new therapies, and preventative measures are needed. Here, we review host defense against K. pneumoniae and discuss recent therapeutic measures and vaccine approaches directed to treat and prevent severe disease caused by MDR K. pneumoniae.

Klebsiella pneumoniae capsule polysaccharide as a target for therapeutics and vaccines.

Carbapenem-resistant (CR) Klebsiella pneumoniae has emerged as an urgent public health threat in many industrialized countries worldwide, including the United States. Infections caused by CR K. pneumoniae are difficult to treat because these organisms are typically resistant to multiple antibiotics, and the patients have significant comorbidities. Notably, there is high (∼50%) mortality among individuals with bacteremia caused by CR K. pneumoniae. Given the dearth of new antibiotics, and the recent convergence of multidrug resistance and hypervirulence, there is a critical need for alternative strategies for the treatment of CR K. pneumoniae infections. The capsule polysaccharide (CPS) of K. pneumoniae has long been viewed as an important virulence factor that promotes resistance to phagocytosis and serum bactericidal activity. Thus, the CPS has been targeted previously for the development of therapeutics and vaccines, although there is no licensed CPS-based vaccine or therapy for the treatment of CR K. pneumoniae infections. Here, we discuss immunoprophylactic and immunotherapeutic approaches that have been tested previously for the treatment of Klebsiella infections. We also suggest potential strategies to promote development of CPS-based vaccines and therapies for prevention and treatment of CR K. pneumoniae infections.

Inhibitors of Intracellular Gram-Positive Bacterial Growth Synthesized via Povarov-Doebner Reactions.

Staphylococcus aureus can survive both inside and outside of phagocytic and nonphagocytic host cells. Once in the intracellular milieu, most antibiotics have reduced ability to kill S. aureus, thus resulting in relapse of infection. Consequently, there is a need for antibacterial agents that can accumulate to lethal concentrations within host cells to clear intracellular infections. We have identified tetrahydrobenzo[a or c]phenanthridine and tetrahydrobenzo[a or c]acridine compounds, synthesized via a one-flask Povarov-Doebner operation from readily available amines, aldehydes, and cyclic ketones, as potent agents against drug-resistant S. aureus. Importantly, the tetrahydrobenzo[a or c]phenanthridine and tetrahydrobenzo[a or c]acridine compounds can accumulate in macrophage cells and reduce the burden of intracellular MRSA better than the drug of choice, vancomycin. We observed that MRSA could not develop resistance (by passage 30) against tetrahydrobenzo[a or c]acridine compound 15. Moreover, tetrahydrobenzo[c]acridine compound 15 and tetrahydrobenzo[c]phenanthridine compound 16 were nontoxic to red blood cells and were nonmutagenic. Preliminary data indicated that compound 16 reduced bacterial load (MRSA USA300) in mice (thigh infection model) to the same degree as vancomycin. These observations suggest that compounds 15 and 16 and analogues thereof could become therapeutic agents for the treatment of chronic MRSA infections.

Antibacterial Small Molecules That Potently Inhibit Staphylococcus aureus Lipoteichoic Acid Biosynthesis.

The rise of antibiotic resistance, especially in Staphylococcus aureus, and the increasing death rate due to multiresistant bacteria have been well documented. The need for new chemical entities and/or the identification of novel targets for antibacterial drug development is high. Lipoteichoic acid (LTA), a membrane-attached anionic polymer, is important for the growth and virulence of many Gram-positive bacteria, and interest has been high in the discovery of LTA biosynthesis inhibitors. Thus far, only a handful of LTA biosynthesis inhibitors have been described with moderate (MIC=5.34 µg mL-1 ) to low (MIC=1024 µg mL-1 ) activities against S. aureus. Herein we describe the identification of novel compounds that potently inhibit LTA biosynthesis in S. aureus, displaying impressive antibacterial activities (MIC as low as 0.25 µg mL-1 ) against methicillin-resistant S. aureus (MRSA). Under similar in vitro assay conditions, these compounds are 4-fold more potent than vancomycin and 8-fold more potent than linezolid against MRSA.

Proteomic analysis of bacterial response to a 4-hydroxybenzylidene indolinone compound, which re-sensitizes bacteria to traditional antibiotics.

Halogenated 4-hydroxybenzylidene indolinones have been shown to re-sensitize methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE) to methicillin and vancomycin respectively. The mechanism of antibiotic re-sensitization was however not previously studied. Here, we probe the scope of antibiotic re-sensitization and present the global proteomics analysis of S. aureus treated with GW5074, a 4-hydroxybenzylidene indolinone compound. With a minimum inhibitory concentration (MIC) of 8 µg/mL against S. aureus, GW5074 synergized with beta-lactam antibiotics like ampicillin, carbenicillin and cloxacillin, the DNA synthesis inhibitor, ciprofloxacin, the protein synthesis inhibitor, gentamicin and the folate acid synthesis inhibitor, trimethoprim. Global proteomics analysis revealed that GW5074 treatment resulted in significant downregulation of enzymes involved in the purine biosynthesis. S. aureus proteins involved in amino acid metabolism and peptide transport were also observed to be downregulated. Interestingly, anti-virulence targets such as AgrC (a quorum sensing-related histidine kinase), AgrA (a quorum sensing-related response regulator) as well as downstream targets, such as hemolysins, lipases and proteases in S. aureus were also downregulated by GW5074. We observed that the peptidoglycan hydrolase, SceD was significantly upregulated. The activity of GW5074 on S. aureus suggests that the compound primes bacteria for the antibacterial action of ineffective antibiotics. SIGNIFICANCE: Antibiotic resistance continues to present significant challenges to the treatment of bacterial infections. Given that antibiotic resistance is a natural phenomenon and that it has become increasingly difficult to discover novel antibiotics, efforts to improve the activity of existing agents are worth pursuing. A few small molecules that re-sensitize resistant bacteria to traditional antibiotics have been described but the molecular details that underpin how these compounds work to re-sensitize bacteria remain largely unknown. In this report, global label-free quantitative proteomics was used to identify changes in the proteome that occurs when GW5074, a compound that re-sensitize MRSA to methicillin, is administered to S. aureus. The identification of pathways that are impacted by GW5074 could help identify novel targets for antibiotic re-sensitization.

Amino alkynylisoquinoline and alkynylnaphthyridine compounds potently inhibit acute myeloid leukemia proliferation in mice.

BACKGROUND: Acute myeloid leukemia (AML) remains one of the most lethal, rarely cured cancers, despite decades of active development of AML therapeutics. Currently, the 5-year survival of AML patients is about 30% and for elderly patients, the rate drops to <10%. About 30% of AML patients harbor an activating mutation in the tyrosine kinase domain (TKD) of Fms-Like Tyrosine kinase 3 (FLT3) or a FLT3 internal tandem duplication (FLT3-ITD). Inhibitors of FLT3, such as Rydapt that was recently approved by the FDA, have shown good initial response but patients often relapse due to secondary mutations in the FLT3 TKD, like D835Y and F691 L mutations. METHODS: Alkynyl aminoisoquinoline and naphthyridine compounds were synthesized via Sonogashira coupling. The compounds were evaluated for their in vitro and in vivo effects on leukemia growth. FINDINGS: The compounds inhibited FLT3 kinase activity at low nanomolar concentrations. The lead compound, HSN431, also inhibited Src kinase activity. The compounds potently inhibited the viability of MV4-11 and MOLM-14 AML cells with IC50 values <1 nM. Furthermore, the viability of drug-resistant AML cells harboring the D835Y and F691 L mutations were potently inhibited. In vivo efficacy studies in mice demonstrated that the compounds could drastically reduce AML proliferation in mice. INTERPRETATION: Compounds that inhibit FLT3 and downstream targets like Src (for example HSN431) are good leads for development as anti-AML agents. FUND: Purdue University, Purdue Institute for Drug Discovery (PIDD), Purdue University Center for Cancer Research, Elks Foundation and NIH P30 CA023168.

N-(1,3,4-oxadiazol-2-yl)benzamide analogs, bacteriostatic agents against methicillin- and vancomycin-resistant bacteria.

Various reports of multidrug-resistant bacteria that are immune to all available FDA-approved drugs demand the development of novel chemical scaffolds as antibiotics. From screening a chemical library, we identified compounds with antibacterial activity. The most potent compounds, F6-5 and F6, inhibited growth of various drug-resistant Gram-positive bacterial pathogens at concentrations ranging from 1 µg/mL to 2 µg/mL. Both compounds were active against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate and vancomycin-resistant S. aureus (VISA and VRSA respectively) and vancomycin-resistant Enterococcus faecalis (VRE). Resistance generation experiments revealed that MRSA could develop resistance to the antibiotic ciprofloxacin but not to F6. Excitingly, F6 was found to be non-toxic against mammalian cells. In a mouse skin wound infection model, F6 was equipotent to the antibiotic fusidic acid in reducing MRSA burden.

Alkynylnicotinamide-Based Compounds as ABL1 Inhibitors with Potent Activities against Drug-Resistant CML Harboring ABL1(T315I) Mutant Kinase.

The introduction of imatinib into the clinical scene revolutionized the treatment of chronic myelogenous leukemia (CML). The overall eight-year survival rate for CML has increased from about 6 % in the 1970s to over 90 % in the imatinib era. However, about 20 % of CML patients harbor primary or acquired resistance to tyrosine kinase inhibitors. ABL1 point mutations in the BCR-ABL1 fusion protein, such as ABL1(T315I), typically emerge after prolonged kinase inhibitor treatment. Ponatinib (AP24534) is currently the only approved CML drug that is active against the ABL1(T315I) mutation. However, ponatinib has severe cardiovascular toxicities; hence, there have been efforts to find safer CML drugs that work against ABL1 secondary mutations. We reveal that isoquinoline- or naphthyridine-based compounds, such as HSN431, HSN576, HSN459, and HSN608 potently inhibit the enzymatic activities of ABL1, ABL1(T315I), and ABL1(E255K). These compounds inhibit the proliferation of ABL1-driven CML cell lines, K652 and KCL22 as well as the drug-resistant cell line, KCL22-IR, which harbors the secondary mutated ABL1(T315I) kinase.

3H-pyrazolo[4,3-f]quinoline haspin kinase inhibitors and anticancer properties.

Histone modification, a post-translational modification of histones and involving various covalent tags, such as methyl, phosphate and acetate groups, affects gene expression and hence modulates various cellular events, including growth and proliferation. Consequently histone-modifying proteins have become targets for the development of anticancer agents. Thus far, compounds that inhibit the methylation or acetylation of histones have advanced in the clinic, but inhibitors of histone phosphorylation have lagged behind. Haspin is a kinase that phosphorylates histone H3 and is a promising anticancer target. Thus far only a handful of haspin inhibitors have been reported. Using a one-flask Doebner/Povarov reaction, we synthesized a library of compounds that potently inhibit haspin with IC50 values as low as 14 nM. Some of these compounds also inhibited the proliferation of cancer cell lines HCT116, HeLa and A375. The ease of synthesis of the new haspin inhibitors, coupled with their anticancer activities make these compounds interesting leads to develop into therapeutics.

Tetrahydro-3H-pyrazolo[4,3-a]phenanthridine-based CDK inhibitor.

Cyclin-dependent kinases have emerged as important targets for cancer therapy. HSD992, containing a novel scaffold based on the tetrahydro-3H-pyrazolo[4,3-a]phenanthridine core, inhibits CDK2/3 but not other CDKs and also potently inhibits several cancer cell lines.

Suramin potently inhibits cGAMP synthase, cGAS, in THP1 cells to modulate IFN-ß levels.

AIM: Persistent activation of STING pathway is the basis for several autoimmune diseases. STING is activated by cGAMP, which is produced by cGAS in the presence of DNA. Results/methodology: HPLC-based medium throughput screening for inhibitors of cGAS identified suramin as a potent inhibitor. Unlike other reported cGAS inhibitors, which bind to the ATP/GTP binding site, suramin displaced the bound DNA from cGAS. Addition of suramin to THP1 cells reduced the levels of IFN-ß mRNA and protein. Suramin did not inhibit lipopolysaccharide- or Pam3CSK4-induced IL-6 mRNA expression. CONCLUSION: Suramin inhibits STING pathway via the inhibition of cGAS enzymatic activity. Suramin or analogs thereof that displace DNA from cGAS could be used as anti-inflammatory drugs.

Dual FLT3/TOPK inhibitor with activity against FLT3-ITD secondary mutations potently inhibits acute myeloid leukemia cell lines.

AIM: Approximately 30% of acute myeloid leukemia (AML) patients carry FLT3 tyrosine kinase domain (TKD) mutations or internal tandem duplication (FLT3-ITD). Currently there is a paucity of compounds that are active against drug-resistant FLT3-ITD, which contains secondary mutations in the TKD, mainly at residues D835/F691. RESULTS: HSD1169, a novel compound, is active against FLT3-ITD (D835 or F691). HSD1169 is also active against T-LAK cell-originated protein kinase (TOPK), a collaborating kinase that is highly expressed in AML cell lines. HSD1169 was active against MV4-11 and Molm-14 (FLT3-ITD cell lines) but not NOMO-1 or HL60 (FLT3-WT cell lines). HSD1169 was also active against sorafenib-resistant Molm13-res cell line (containing FLT3-ITD/D835Y). CONCLUSION: HSD1169 or an analog could become a therapeutic agent for AML containing drug-resistant FLT3-ITD.

Fluorescent 2-Aminopurine c-di-GMP and GpG Analogs as PDE Probes.

c-di-GMP is widely recognized as an important ubiquitous signaling molecule in bacteria. c-di-GMP phosphodiesterases (PDEs) regulate the intracellular concentration of c-di-GMP and some could be potential drug targets. Here, we describe a class of dinucleotide probes suitable for monitoring the enzymatic activities of c-di-GMP PDEs in real time. Such probes contain fluorescent nucleobases and can be readily cleaved by PDEs, resulting in a change in fluorescence. Fluorescent cyclic and linear dinucleotide probes could be used in diverse applications, such as confirming the activity of an expressed PDE or oligoribonuclease (Orns) or identifying inhibitors of PDEs or Orns using high-throughput screening formats.

Targeting c-di-GMP Signaling, Biofilm Formation, and Bacterial Motility with Small Molecules.

Bacteria possess several signaling molecules that regulate distinct phenotypes. Cyclic di-GMP (c-di-GMP) has emerged as a ubiquitous second messenger that regulates bacterial virulence, cell cycle, motility, and biofilm formation. The link between c-di-GMP signaling and biofilm formation affords novel strategies for treatment of biofilm-associated infections, which is a major public health problem. The complex c-di-GMP signaling pathway creates a hurdle in the development of small molecule modulators. Nonetheless, some progress has been made in this regard and inhibitors of c-di-GMP metabolizing enzymes that affect biofilm formation and motility have been documented. Herein we discuss the components of c-di-GMP signaling, their correlation with biofilm formation as well as motility and reported small molecule inhibitors of c-di-GMP signaling.

Aminoisoquinoline benzamides, FLT3 and Src-family kinase inhibitors, potently inhibit proliferation of acute myeloid leukemia cell lines.

AIM: Mutated or overexpressed FLT3 drives about 30% of reported acute myeloid leukemia (AML). Currently, FLT3 inhibitors have shown durable clinical responses but a complete remission of AML with FLT3 inhibitors remains elusive due to mutation-driven resistance mechanisms. The development of FLT3 inhibitors that also target other downstream oncogenic kinases may combat the resistance mechanism. RESULTS: 4-substituted aminoisoquinoline benzamides potently inhibit Src-family kinases and FLT3, including secondary mutations, such as FLT3D835. Modifications of aminoisoquinoline benzamide to aminoquinoline or aminoquinazoline abrogated FLT3 and Src-family kinase binding. CONCLUSION: The lead aminoisoquinolines potently inhibited FLT3-driven AML cell lines, MV4-11 and MOLM-14. These aminoisoquinoline benzamides represent new kinase scaffolds with high potential to be translated into anticancer agents.

Cyclic dinucleotide (c-di-GMP, c-di-AMP, and cGAMP) signalings have come of age to be inhibited by small molecules.

Bacteria utilize nucleotide-based second messengers to regulate a myriad of physiological processes. Cyclic dinucleotides have emerged as central regulators of bacterial physiology, controlling processes ranging from cell wall homeostasis to virulence production, and so far over thousands of manuscripts have provided biological insights into c-di-NMP signaling. The development of small molecule inhibitors of c-di-NMP signaling has significantly lagged behind. Recent developments in assays that allow for high-throughput screening of inhibitors suggest that the time is right for a concerted effort to identify inhibitors of these fascinating second messengers. Herein, we review c-di-NMP signaling and small molecules that have been developed to inhibit cyclic dinucleotide-related enzymes.

Inhibition of cyclic diadenylate cyclase, DisA, by polyphenols.

Cyclic di-AMP has emerged as an important signaling molecule that controls a myriad of functions, including cell wall homeostasis in different bacteria. Polyphenols display various biological activities and tea polyphenols in particular have been shown to possess among other properties antioxidant and antibacterial activities. Certain tea polyphenols, such as catechin and epigallocatechin gallate, have been used to augment the action of traditional antibiotics that target the cell wall. Considering the expanding role played by cyclic dinucleotides in bacteria, we investigated whether the action of polyphenols on bacteria could be due in part to modulation of c-di-AMP signaling. Out of 14 tested polyphenols, tannic acid (TA), theaflavin-3'-gallate (TF2B) and theaflavin-3,3'-digallate (TF3) exhibited inhibitory effects on B. subtilis c-di-AMP synthase, DisA. TF2B and TF3 specifically inhibited DisA but not YybT (a PDE) whilst TA was more promiscuous and inhibited both DisA and YybT.

Potent inhibition of cyclic diadenylate monophosphate cyclase by the antiparasitic drug, suramin.

C-di-AMP synthases are essential in several bacteria, including human pathogens; hence these enzymes are potential antibiotic targets. However, there is a dearth of small molecule inhibitors of c-di-AMP metabolism enzymes. Screening of 2000 known drugs against DisA has led to the identification of suramin, an antiparasitic drug as potent inhibitor of c-di-AMP synthase.

Inhibition of P. aeruginosa c-di-GMP phosphodiesterase RocR and swarming motility by a benzoisothiazolinone derivative.

Various important cellular processes in bacteria are controlled by c-di-GMP, such as motility, biofilm formation and virulence factors production. C-di-GMP is synthesized from two molecules of GTP by diguanylate cyclases (DGCs) and its actions are terminated by EAL or HD-GYP domain phosphodiesterases (PDEs), which hydrolyze c-di-GMP to linear pGpG or GMP. Thus far the majority of efforts have been dedicated to the development of inhibitors of DGCs but not PDEs. This is probably because the old view was that inhibiting any c-di-GMP PDE would lead to biofilm formation, an undesirable phenotype. Recent data however suggest that some PDEs only change the localized (not global) concentration of c-di-GMP to increase bacterial virulence and do not affect biofilm formation. A challenge therefore is to be able to develop selective PDE inhibitors that inhibit virulence-associated PDEs but not inhibit PDEs that regulate bacterial biofilm formation. Using high throughput docking experiments to screen a library of 250 000 commercially available compounds against E. coli YahA (also called PdeL), a benzoisothiazolinone derivative was found to bind to the c-di-GMP binding site of YahA with favorable energetics. Paradoxically the in silico identified inhibitor (a benzoisothiazolinone derivative) did not inhibit the hydrolysis of c-di-GMP by YahA, the model PDE that was used in the docking, but instead inhibited RocR, which is a PDE from the opportunistic pathogen P. aeruginosa (PA). RocR promotes bacterial virulence but not biofilm dispersal, making it an ideal PDE to target for anti-virulence purposes. This newly identified RocR ligand displayed some selectivity and did not inhibit other P. aeruginosa PDEs, such as DipA, PvrR and PA4108. DipA, PvrR and PA4108 are key enzymes that reduce global c-di-GMP concentration and promote biofilm dispersal; therefore the identification of an inhibitor of a PA PDE, such as RocR, that does not inhibit major PDEs that modulate global c-di-GMP is an important step towards the development of selective c-di-GMP PDEs that could have interesting biomedical applications. The identified RocR ligand could also inhibit P. aeruginosa (PAO1) swarming but not swimming or biofilm formation. Rhamnolipid production was decreased, explaining the inhibition of swarming.