In addition to its attention on new targets, the book focusses on the medicinal chemistry and chemistry of the targets. Within this framework, chapters from leading researchers in academia and industry address findings in important areas such as biofilm production, narrow spectrum antibiotics and novel antibacterials from previously uncultured soil bacteria.
This book will be a useful resource for postgraduate students and researchers in medicinal chemistry wishing to understand the latest approaches to antibiotic drug discovery.
In addition to its attention on new targets, the book focusses on the medicinal chemistry and chemistry of the targets. Within this framework, chapters from leading researchers in academia and industry address findings in important areas such as biofilm production, narrow spectrum antibiotics and novel antibacterials from previously uncultured soil bacteria.
This book will be a useful resource for postgraduate students and researchers in medicinal chemistry wishing to understand the latest approaches to antibiotic drug discovery.
Antibiotic Drug Discovery: New Targets and Molecular Entities
286
Antibiotic Drug Discovery: New Targets and Molecular Entities
286Hardcover
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Overview
In addition to its attention on new targets, the book focusses on the medicinal chemistry and chemistry of the targets. Within this framework, chapters from leading researchers in academia and industry address findings in important areas such as biofilm production, narrow spectrum antibiotics and novel antibacterials from previously uncultured soil bacteria.
This book will be a useful resource for postgraduate students and researchers in medicinal chemistry wishing to understand the latest approaches to antibiotic drug discovery.
Product Details
| ISBN-13: | 9781782624240 |
|---|---|
| Publisher: | RSC |
| Publication date: | 05/30/2017 |
| Series: | Drug Discovery Series , #58 |
| Pages: | 286 |
| Product dimensions: | 6.15(w) x 9.20(h) x (d) |
About the Author
Troy Lister, PhD, is Head of Chemistry at Spero Therapeutics in Cambridge, MA. Dr. Lister previously held roles as Team Leader of Infection Chemistry at AstraZeneca and Investigator III in Global Discovery Chemistry at the Novartis Institute for BioMedical Research. He has lead numerous discovery programs searching for novel agents targeting bacterial and viral pathogens including multidrug resistant Gram negative and Gram positive bacteria, hepatitis C virus, human rhinovirus, respiratory syncytial virus, ebola virus and human enterovirus. Dr. Lister has led programs from the earliest stages of target selection and high throughput screening, through lead identification and optimization, to candidate selection and IND enablement. He is an author of 15 peer review publications, has extensive experience in natural product chemistry and is a member of the American Chemical Society. Dr. Lister received his B.Tech, B.S. and PhD degrees from Flinders Universityin Adelaide, Australia, and completed a post-doctoral fellowship at The Scripps Research Institute in La Jolla, California with Professoer K.C. Nicolaou.
Read an Excerpt
Antibiotic Drug Discovery
New Targets and Molecular Entities
By Steven M. Firestine
The Royal Society of Chemistry
Copyright © 2017 The Royal Society of ChemistryAll rights reserved.
ISBN: 978-1-78262-424-0
CHAPTER 1
Treatment of Clostridium difficile Infections
CHRISTOPHER YIP, JACQUELINE PHAN AND ERNESTO ABEL-SANTOS
1.1 Background
Clostridium difficile infection (CDI) is a nosocomial disease mainly correlated with antibiotic-associated diarrhea. These infections are caused by Clostridium difficile, an anaerobic, rod shaped, gram-positive bacterium that is normally found in the gastrointestinal tract (Figure 1.1).
Approximately 5% of healthy adults, and 50% of newborns are asymptomatic carriers of C. difficile. C. difficile was originally thought to be a commensal bacterium, but due to the recent boom of antibiotic therapies and advancements, it was quickly recognized that C. difficile is the leading cause of hospital-acquired diarrhea worldwide. In the United States alone, there are roughly 500 000 cases of CDI annually, with associated costs estimated to be approximately $4.8 billion.
C. difficile has a unique lifecycle such that it can form metabolically dormant, non-reproductive spores when stressed (Figure 1.2).
These stressors include, but not limited to, nutrient limitation and desiccation. The resulting spores are highly resistant to harsh environmental factors such as stomach acid, extreme temperatures, and pharmaceutically relevant antibiotics. Spores can persist over prolonged periods of time, while constantly monitoring the environment for favorable conditions. Upon reintroduction to nutrient rich environments, the spores are able to revert back into actively growing cells through a process known as germination. When C. difficile has completed its lifecycle, transitioning from a spore to an actively growing cell, the newly germinated cells can now colonize the local environment.
CDI begins with the ingestion of the highly resistant C. difficile spores. As these spores travel through the gastrointestinal tract, various endogenous bile salts stimulate the spores to germinate into actively growing, vegetative cells. While the spores are metabolically dormant and act solely as a vehicle for infection, the vegetative cells are metabolically active, can produce toxins, and elicit disease.
The diversity of the endogenous bile salts depends heavily on the intestinal gut flora. Under normal circumstances, the bacteria found naturally in the gastrointestinal tract provide a barrier against C. difficile colonization by occupying nutrient-rich niches and by metabolizing specific bile salts required for C. difficile germination. With the current pharmaceutical advancements, several new broad-spectrum antibiotics have been developed. Exposure to antibiotics, such as clindamycin, 2nd and 3rd generation cephalosporins, and fluoroquinolones, can disrupt the natural gut flora. Disruption of the gut flora effectively removes the protective barrier, as the change in the bile salt diversity becomes more favorable for C. difficile (Figure 1.3).
As C. difficile spores germinate and outgrow, the resulting vegetative cells begin to produce and release two major toxins, TcdA and TcdB. The C-terminal region of both TcdA and TcdB contain a binding domain, which is able to interact with different carbohydrate and protein structures found on the surface of host cell membranes. TcdB binds to chondroitin sulfate proteoglycan 4 (CSPG4) and the poliovirus receptor-like 3 (PVRL3) found on the surface of intestinal epithelial cells. In contrast, TcdA can bind to glycoprotein 96 and sucrase isomaltase, both of which are found on the surface of human colonocytes. Once these toxins interact with the host cell receptors, the toxins are internalized by endocytosis. Acidification of the endosome causes the toxins to undergo conformational changes resulting in translocation into the host cell cytoplasm. Upon entry into the cytoplasm, the toxins undergo autocatalytic cleavage. Cleavage of the toxins allow for the release and activation of their glycosyltransferase domain (GTD) into the host cell.
The GTD can transfer glucose from UDP-glucose to several crucial Rho proteins. Glucosylation of Rho proteins result in their inactivation. Since Rho proteins play an essential role in regulating the cell cytoskeleton, inactivation of these proteins can have several cytopathic effects including the disruption of cell-to-cell contacts and tight junctions, as well as increased epithelial permeability. Glucosylation of RhoA also activates the inflammasome and upregulates a pro-apoptotic protein, RhoB.
Expression of tcdA and tcdB is heavily regulated and dependent on resource availability. When carbon sources and other nutrients are readily available, toxin expression is inhibited. Conversely, toxin production is upregulated during stationary phase when resources are low. This type of regulation suggests that C. difficile virulence is a killing strategy used to improve resource availability by scavenging the host cell for resources. A combination of several factors contributes to C. difficile's virulence. TcdA and TcdB are undoubtedly major contributors. Due to genetic variability between C. difficile strains, the extensive genotypic variances in the pathogenicity locus (PaLoc), which houses tcdA and tcdB, currently give rise to at least 31 different toxinotypes. These different toxinotypes result from mutations in tcdA and tcdB, as well as the regulatory factors that ultimately lead to the overexpression or repression of the toxin genes. Several other factors, such as rates of sporulation and toxin release, motility and host cell adherence, can contribute to C. difficile virulence.
Interestingly, so called "hypervirulent" strains of C. difficile have begun to reveal themselves in the healthcare setting worldwide. Hypervirulent strains are highly variable — some strains may have higher rates of sporulation and toxin production. TcdC, an anti-sigma factor that acts as a negative regulator of toxin production, is upregulated during exponential growth. Several hypervirulent strains contain mutations in tcdC that result in the constant, unregulated production of C. difficile toxins. Toxins produced by hypervirulent strains can also undergo necessary conformational changes at higher pH ranges, allowing the toxins to enter the host cell cytoplasm earlier and at a faster rate, relative to toxins produced by non-hypervirulent strains. Clostridium difficile transferase (CDT), a ribosyltransferase, is a binary toxin produced only by several strains of C. difficile. Similar to TcdA and TcdB, CDT binds to a host-cell by interacting with a surface molecule, specifically lipolysis-stimulated lipoprotein receptor (LSR). CDT eventually localizes into the host cell cytoplasm where it begins to ribosylate G actin. At low concentrations of the toxin, CDT induces microtubules to form protrusions from the host-cell membrane, facilitating C. difficile adherence to the surface of the intestinal epithelial cells. At high concentrations of CDT, actin polymerization is inhibited and actin depolymerization is induced ultimately causing the collapse of the host cell cytoskeleton.
1.2 CDI Symptoms and Progression
Symptom severity in CDI patients can range from mild diarrhea, to life-threatening pseudomembranous colitis, a condition which causes exudative plaques on the intestinal mucosa. Mild CDI is defined solely by the presence of diarrhea. Other symptoms that can indicate moderate disease include abdominal pain, loss of appetite, fever, nausea, vomiting, gastrointestinal bleeding, bloody stools, and weight loss.
Severe disease can include some or all of the symptoms associated with mild-to-moderate disease plus additional indicative symptoms. Severe CDI is indicated by a serum albumin < 3 g dl-1 with an elevated white blood cell (WBC) count of ≥ 15 000 cells/mm3 and/or abdominal tenderness.
In more severe cases of CDI, patients can develop several complications. If symptoms progress, they can lead to hypotension, fever above 38.5 °C, ileus (a condition in which peristaltic activity to move stool through the gastrointestinal tract is diminished) or abdominal distention, altered mental state, WBC count =35 000 cells/mm3, serum lactate levels >2.2 mmol L-1, and ultimately organ failure.
Another complication called pseudomembranous colitis is unique to CDI. Pseudomembranous colitis occurs when toxins produced by C. difficile cells damage the walls of the colon causing inflammation and thickening of the colonic mucosa producing yellowish exudates called pseudomembranes to form along the colon. This can lead to other complications such as perforated colon and toxic megacolon. Toxic megacolon can render the colon incapable of expelling gas and stool contents, potentially causing the colon to rupture.
1.3 Relapse
CDI relapse is characterized by the return of symptoms within eight weeks of primary diagnosis after initial symptoms have previously been resolved. CDI recurrence after initial treatment can reach up to 25% in treated patients. Chances of subsequent recurrences nearly doubles to 45% after the first recurrence. One explanation for CDI relapse is that resident C. difficile spores may have survived in the gut after completion or discontinuation of antibiotic treatment. C. difficile spores may also be picked up via contamination of the local environment. Therefore, relapse and reinfection may be difficult to distinguish. However, reinfections can be identified by the diagnosis with a different C. difficile strain. Other possible reasons for relapse include poor immune response leading to inadequate production of antibodies against to C. difficile toxins and frequent disruption of normal gut flora. Moreover, epidemiologic factors such as advanced age, use of other antibiotics, and prolonged hospital stay may also contribute to increased risk for recurrent CDI. Emergence of resistant strains and hypervirulent strains over the past decade has made treatment of recurrent CDI increasingly difficult.
1.4 Diagnosis
CDI may mimic flu-like symptoms or flare-ups of other gastrointestinal diseases. Therefore, early and accurate diagnosis of CDI is important to the successful management of the disease. In 2010, the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA) recommended a two-step algorithm for CDI diagnosis. First, an initial immunoassay screen of stool samples for the presence of C. difficile is performed. If C. difficile is found to be absent in the initial screen, C. difficile should be ruled out as the cause of diarrhea. Following a positive result from the initial screen, detection of toxins from the stool is tested. Positive indications for C. difficile toxins along with moderate-to-severe symptoms can warrant the need for additional and more invasive testing.
Initial screening of stools for the presence of C. difficile can be performed by a common and relatively inexpensive enzyme immunoassay (EIA) test called the common antigen test or glutamate dehydrogenase (GDH) test. The GDH test looks for the GDH enzyme that is produced in relatively large amounts by C. difficile and can be readily detected the stools of CDI patients. Although this EIA can give results within 15–45 min, it tends to have lower sensitivities than other tests since it only test for the presence of the C. difficile organisms rather than the production of toxins.
Following a positive GDH test, the second step of the two-step algorithm includes a cell cytotoxicity neutralization assay (CCNA) which tests for the presence of toxin B in stool filtrate. CCNA has long been considered to be the traditional "gold standard" for the detection of C. difficile toxins. In a CCNA, filtrate of the collected stool sample is added to mammalian cultures (e.g. human fibroblast). If C. difficile TcdB is present in the filtrate, the mammalian cells will round up and necrotize. To verify that the cytopathic effect is caused by C. difficile toxin, the cell cultures are supplemented with an antitoxin (monoclonal antibodies). If the cytopathic effect is reversed, a test is positive for TcdB. While CCNA is highly sensitive and specific, it has a slow turnaround of 24 to 48 h and requires technical expertise.
Alternatively, a three-step algorithm for CDI diagnosis may be used. The three-step algorithm includes the steps of the two-step algorithm with the addition of an intermediate EIA test that detects the presence of free TcdA and TcdB (TOX-A/BII) in stool. If results from the TOX-A/BII EIA test is positive, the stool is said to be positive for C. difficile toxins. If the test is negative, CCNA will be performed in the third step.
Alternately, the third step of the three-step algorithm may employ a molecular diagnostic test instead of CCNA. Nucleic acid amplification tests (NAAT s) allow for the detection of C. difficile toxin gene fragments via real-time quantitative polymerase chain reaction (RT-PCR). Through molecular methods, Toxin B (tcdB) and binary toxin (cdtA and cdtB) can be detected. NAAT s have better sensitivity than CCNA to test for non-free toxins. Although this method tests for the presence of the toxin, it does not indicate the expression of the toxins genes. NAAT s can be costly and must be interpreted with caution as they may detect toxigenic strains in asymptomatic carriers who may have diarrhea caused by other pathologies.
The three-step algorithm provides an effective and reliable method to diagnosing CDI and may eliminate the need to perform a CCNA test if the TOX-A/BII EIA test is positive. However, due to a lack in sensitivity, it has been recently widely accepted that the TOX-A/BII EIA is not well-suited to be a stand-alone test to diagnose CDI. Therefore, the two-step algorithm is usually preferred over the three-step algorithm as it more practical, cost effective, and requires less workload in comparison to the three-step algorithm. Colonoscopy and Computed Tomography (CT scan) may also be used to diagnose conditions caused by CDI such as pseudomembranous colitis.
These imaging methods are used less often than laboratory tests as they can be more costly, unpleasant to the patient, and less sensitive.
1.5 Prevention Measures — General Hospital Practice and Other Prevention Methods
Although CDI can occasionally occur in healthy individuals, CDI is most prevalent among elderly and immunocompromised patients. Patients are more likely to get CDI in healthcare-acquired settings (e.g. hospitals and long-term care facilities) than in community-acquired settings. Because C. difficile spores can survive for long periods of time on hospital surfaces and patient beds, proper precautions must be taken in these settings to prevent CDI from spreading.
The Association for Professionals in Infection Control and Epidemiology (APIC) suggests that hospitals implement infection control programs. Three recommendations include a criteria index for patients who have risk factors for CDI (e.g. malignancy, advanced age, and recent hospitalization or stay at a long-term care facility), advocacy for physicians to use proper C. difficile diagnostic testing with rapid turn-around times and high sensitivity for toxin detection, and appropriate notification to staff members of positive C. difficile test results to ensure that proper precautions and treatments be taken. When a C. difficile infection control protocol was implemented at the University of Pittsburgh Medical Center-Presbyterian, a decrease from 7.2 cases per 1000 discharges to 4.8 cases per 1000 discharges was obtained in a 5 year-period.
Proper hand hygiene is critical in the prevention of CDI. Although alcohol antiseptics can be used to kill most vegetative bacteria and viruses, they do not affect C. difficile spores due to their intrinsic resistance. Therefore, healthcare providers and visitors should be required to wash their hands with antimicrobial hand soap and water. Since a person can easily contaminate their hands with C. difficile spores by contacting an infected patient, healthcare personnel and visitors must also use gloves and gowns upon entry into a CDI patient's room. An intervention study incorporated an infection prevention education program with vinyl gloving wearing surveillance for six months. The study showed a significant decline in CDI rates from 7.7 cases per 1000 patients to 1.5 cases per 1000 patients six months after intervention. Other patient contact precautions include the use of single-use disposable equipment and the limit of patient contact until resolution of diarrhea.
(Continues...)
Excerpted from Antibiotic Drug Discovery by Steven M. Firestine. Copyright © 2017 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Table of Contents
Treatment of Clostridium difficile Infections;Targeting Purine Biosynthesis for Antibacterial Drug Design;
Inhibitors of Biofilm Production;
Narrow Spectrum Antibacterial Agents;
The LPS Transport Pathway: A New Target for the Development of Gram-Negative Antibiotics;
The Discovery of Teixobactin;
Emerging Targets in Anti-Tubercular Drug Design;
Antibacterial Leads Targeting Isoprenoid Biosynthesis