MicrobiologyBytes

Human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) are the most prevalent deadly chronic viral diseases. HIV is treated by small molecule inhibitors. HBV is treated by immunomodulation and small molecule inhibitors. HCV is currently treated primarily by immunomodulation but many small molecules are in clinical development. Although HIV is a retrovirus, HBV is a double-stranded DNA virus, and HCV is a single-stranded RNA virus, antiviral drug resistance complicates the development of drugs and the successful treatment of each of these viruses. Although their replication cycles, therapeutic targets, and evolutionary mechanisms are different, the fundamental approaches to identifying and characterizing HIV, HBV, and HCV drug resistance are similar. This review describes the evolution of HIV, HBV, and HCV within individuals and populations and the genetic mechanisms associated with drug resistance to each of the antiviral drug classes used for their treatment.

Comparison of the Mechanisms of Drug Resistance among HIV, Hepatitis B, and Hepatitis C. Viruses. 2010; 2(12):2696-2739. doi:10.3390/v2122696

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From the outside and within, we are constantly bombarded with a myriad of diverse microbial species. However, our bodies are equipped with an evolutionarily conserved innate immune defense system that allows us to thwart potential pathogens. Antimicrobial peptides (AMPs) are a unique and assorted group of molecules produced by living organisms of all types, considered to be part of the host innate immunity. These peptides demonstrate potent antimicrobial activity and are rapidly mobilized to neutralize a broad range of microbes, including viruses, bacteria, protozoa, and fungi. More significantly, the ability of these natural molecules to kill multidrug-resistant microorganisms has gained them considerable attention and clinical interest. With the growing microbial resistance to conventional antimicrobial agents, the need for unconventional therapeutic options has become urgent. This article provides an overview of AMPs, their biological functions, mechanism of action, and applicability as alternative therapeutic agents.

Presently, AMPs represent one of the most promising future strategies for combating infections and microbial drug resistance. This is evident by the increasing number of studies to which these peptides are subjected. As our need for new antimicrobials becomes more pressing, the question remains: can we develop novel drugs based on the design principles of primitive molecules?

Antimicrobial Peptides: Primeval Molecules or Future Drugs? (2010) PLoS Pathog 6(10): e1001067. doi:10.1371/journal.ppat.1001067

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From the outside and within, we are constantly bombarded with a myriad of diverse microbial species. However, our bodies are equipped with an evolutionarily conserved innate immune defense system that allows us to thwart potential pathogens. Antimicrobial peptides (AMPs) are a unique and assorted group of molecules produced by living organisms of all types, considered to be part of the host innate immunity. These peptides demonstrate potent antimicrobial activity and are rapidly mobilized to neutralize a broad range of microbes, including viruses, bacteria, protozoa, and fungi. More significantly, the ability of these natural molecules to kill multidrug-resistant microorganisms has gained them considerable attention and clinical interest. With the growing microbial resistance to conventional antimicrobial agents, the need for unconventional therapeutic options has become urgent. This article provides an overview of AMPs, their biological functions, mechanism of action, and applicability as alternative therapeutic agents.

Presently, AMPs represent one of the most promising future strategies for combating infections and microbial drug resistance. This is evident by the increasing number of studies to which these peptides are subjected. As our need for new antimicrobials becomes more pressing, the question remains: can we develop novel drugs based on the design principles of primitive molecules?

Antimicrobial Peptides: Primeval Molecules or Future Drugs? (2010) PLoS Pathog 6(10): e1001067. doi:10.1371/journal.ppat.1001067

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• Antimicrobial Peptides
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Marine natural products are a continued focus for drug discovery and have provided many important therapeutic agents. Lead compounds with biomedical potential have been isolated from marine invertebrates, bacteria, and fungi. Each year numerous compounds with an array of biological activities are reported, but to-date only 13 molecules have entered into the clinical pipeline. Four molecules have been approved for clinical use, one of which is approved only in the EU. The approved molecules include two nucleosides based on sponge-derived nucleosides, a cone snail peptide, and a metabolite isolated from a tunicate. Marine microbes have received growing attention as the sources for bioactive metabolites and have great potential to increase the number of marine natural products in clinical trials. The sustainable and economic supply of the active pharmaceutical ingredient is often easier to achieve for compounds produced through microbial fermentation approaches versus the cultivation of slower growing macroorganisms.

Marine natural products provide an excellent opportunity to study diverse and unique compounds not readily accessible from any other source leading to expansion of the pharmaceutical pipeline. Marine microbes can produce unique compounds covering new chemical space, and the utility of marine natural products is expanding beyond its original role in identification of new prototype drug leads into fields of study involving sustainable supplies of unique molecules using biosynthesis in conjunction with synthesis. Perhaps the greatest impact marine natural products has played is in revealing that unexplored and previously inaccessible chemical space can contribute to growth in the pharmaceutical pipeline. Improved methodologies in fermentation technologies, biosynthesis, and synthesis provide opportunities to both create and supply drug leads that would not be available by any single method independently. As a result pharmaceutical biotechnology in the future is certain to provide increasingly sophisticated molecular architecture assembled using biosynthesis and synthesis in concert.

The expanding role of marine microbes in pharmaceutical development. Curr Opin Biotechnol. Oct 16 2010
Marine microbes have received growing attention as sources of bioactive metabolites and offer a unique opportunity to both increase the number of marine natural products in clinical trials as well as expedite their development. This review focuses specifically on those molecules currently in the clinical pipeline that are established or highly likely to be produced by bacteria based on expanding circumstantial evidence. We also include an example of how compounds from harmful algal blooms may yield both tools for measuring environmental change as well as leads for pharmaceutical development. An example of the karlotoxin class of compounds isolated from the dinoflagellate Karlodinium veneficum reveals a significant environmental impact in the form of massive fish kills, but also provides opportunities to construct new molecules for the control of cancer and serum cholesterol assisted by tools associated with rational drug design.

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Enteroviruses are associated with a number of diverse syndromes such as myocarditis, febrile illness, and are the main causative agents of aseptic meningitis. No effective therapeutics exist to combat non-poliovirus enterovirus infections. A better understanding of the mechanisms by which these viruses infect host cells could lead to the design of effective therapeutic interventions. This study found that intracellular calcium stores in polarized endothelial monolayers are depleted upon exposure to coxsackievirus B (CVB) and that this release is mediated by viral attachment to its receptor decay-accelerating factor. It also discovered that the calcium release requires the activation of signaling molecules involved in calcium signaling such as Src tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3 on the ER membrane. A calcium-activated cystein protease, calpain-2, was activated and necessary for proper viral trafficking inside the cell. Interestingly, this signaling cascade was critical for CVB internalization into the endothelium, but was not involved in CVB entry into the epithelium. This is an important advance in our understanding of how enteroviruses hijack host endothelial cell signaling mechanisms in order to facilitate their entry and eventual spread.

Release of Intracellular Calcium Stores Facilitates Coxsackievirus Entry into Polarized Endothelial Cells. (2010) PLoS Pathog 6(10): e1001135. doi:10.1371/journal.ppat.1001135
Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers.

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The traditional route for identifying early hits in antibiotic research is to target multiplying bacteria. All current antibiotics have been generated this way. Activity of a potential antibiotic in such assays is predictive of an antimicrobial effect in humans (bearing in mind many compounds are not suitable due to undesirable characteristics such as toxicity). The disadvantage of this route is that the numbers of novel classes of non-toxic compounds which kill multiplying bacteria may have been almost exhausted and those that remain, may require substantial effort and expense to bring to market. Furthermore anti-multiplying agents are almost always either inactive or only partially active against non-multiplying or slowly multiplying or persister bacteria, which leads to the need for multiple doses of antibiotics in order to achieve cure of a bacterial infectious disease. This prolongs the duration of therapy and increases the emergence of resistance. Since bacterial resistance reduces the effectiveness of antibiotics, new ones are required at regular intervals, as the old ones lose their potency for most infections. However, the number of new antibiotics which reach the market each year is falling. Whilst at least 15 classes of antibiotics were introduced into the market between 1940 and 1962, only three new classes of antibiotics have been marketed since then. Together with their subsequent analogues, each class loses effectiveness, at least for some species of bacteria such as Gram-negatives, within 50 years after entry into the market. So, if we continue to use existing technologies for the next 50 years, it is unlikely that we will produce enough new classes to prevent the antibiotic era fading away. A fundamentally new route for antibiotic drug discovery is required if the antibiotic era is to continue. Bacterial molecules have been targeted, in order to create new drugs, but this has not produced any new classes of antibiotics which have reached the market. Another potential way to develop new antibacterials is to use bacteriophages. Although this method has been utilized for decades, no marketed bacteriophages are available in Western countries for licensed medicinal purposes.

In a clinical infection, multiplying and non-multiplying bacteria co-exist. Antibiotics kill multiplying bacteria, but they are very inefficient at killing non-multipliers which leads to slow or partial death of the total target population of microbes in an infected tissue. This prolongs the duration of therapy, increases the emergence of resistance and so contributes to the short life span of antibiotics after they reach the market. Targeting non-multiplying bacteria from the onset of an antibiotic development program is a new concept. This paper describes the proof of principle for this concept, which has resulted in the development of the first antibiotic using this approach. The antibiotic, called HT61, is a small quinolone-derived compound with a molecular mass of about 400 Daltons, and is active against non-multiplying bacteria, including methicillin sensitive and resistant, as well as Panton-Valentine leukocidin-carrying Staphylococcus aureus. It also kills mupirocin resistant MRSA. The mechanism of action of the drug is depolarisation of the cell membrane and destruction of the cell wall. The speed of kill is within two hours. In comparison to the conventional antibiotics, HT61 kills non-multiplying cells more effectively, 6 logs versus less than one log for major marketed antibiotics. HT61 kills methicillin sensitive and resistant S. aureus in the murine skin bacterial colonization and infection models. No resistant phenotype was produced during 50 serial cultures over a one year period. The antibiotic caused no adverse affects after application to the skin of minipigs. Targeting non-multiplying bacteria using this method should be able to yield many new classes of antibiotic. These antibiotics may be able to reduce the rate of emergence of resistance, shorten the duration of therapy, and reduce relapse rates.

A New Approach for the Discovery of Antibiotics by Targeting Non-Multiplying Bacteria: A Novel Topical Antibiotic for Staphylococcal Infections. 2010 PLoS ONE 5(7): e11818. doi:10.1371/journal.pone.0011818

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The C-C chemokine receptor type 5 (CCR5) is a key player in HIV infection due to its involvement in the infection process. Investigations into the role of the CCR5 coreceptor first focused on its binding to the virus and the molecular mechanisms leading to the entry and spread of HIV. The identification of naturally occurring CCR5 mutations has allowed scientists to address the CCR5 molecule as a promising target to prevent or limit HIV infection in vivo. Naturally occurring CCR5-specific antibodies have been found in exposed but uninfected people, and in a subset of HIV seropositive people who show long-term control of the infection. This suggests that natural autoimmunity to the CCR5 coreceptor exists and may play a role in HIV control. Such natural immunity has prompted strategies aimed at achieving anti-HIV humoral responses through CCR5 targeting.

From Natural Resistance to a New Anti-HIV Strategy. Viruses 2010, 2(2), 574-600 doi:10.3390/v2020574

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Traditional treatment of bacterial infections relies heavily on the use of antibacterial compounds that either kill bacteria (bactericidal) or inhibit their growth (bacteriostatic). Typically, the targets for the main conventional antibiotics are essential cellular processes such as bacterial cell wall biosynthesis, bacterial protein synthesis, and bacterial DNA replication and repair. However, resistance to these drugs arises and spreads very rapidly, even to such an extent that bacteria have been identified that are simultaneously resistant to all available antibiotics. The increasing occurrence of resistant bacteria gradually renders antibiotics ineffective in treating infections and has enormous human and economic consequences worldwide. As a result, the identification of novel drug targets and the development of novel therapeutics constitute an important area of current scientific research. An alternative to killing or inhibiting growth of pathogenic bacteria is the specific attenuation of bacterial virulence, which can be attained by targeting key regulatory systems that mediate the expression of virulence factors. One of the target regulatory systems is quorum sensing (QS), or bacterial cell-to-cell communication. QS is a mechanism of gene regulation in which bacteria coordinate the expression of certain genes in response to the presence or absence of small signal molecules. As the importance of QS in virulence development of pathogenic bacteria became clear, about a decade ago, QS disruption was suggested as a new anti-infective strategy.

Although at this moment it is difficult to accurately estimate the risk of resistance development, this paper argues that scientists need to pay attention to the possibility that it will evolve. Once we have better knowledge of the risk of resistance development to QS disruption, it might be possible to direct further research on QS inhibition preferentially towards strategies that include a lower risk of resistance development.

Can Bacteria Evolve Resistance to Quorum Sensing Disruption? 2010 PLoS Pathog 6(7): e1000989. doi:10.1371/journal.ppat.1000989

The major approach to the medical management of HIV infection is the treatment of patients with antiviral drugs. The enzymatic processes of the HIV-1 replication cycle present unique approaches for targeted disruption by pharmacological agents. Due to the high rates of virus production and the mutation rate of the virus, treatment of HIV-1 infection generally includes administration of three agents in combination, referred to as highly active antiretroviral therapy (HAART). Sustained treatment of patients with three active drugs results in suppression of viral replication in peripheral blood to below detection limits of sensitive clinical assays (<50 RNA copies/ml). Continued virologic suppression has led to dramatic increases in the life expectancy of HIV-infected individuals and in time to diagnosis with AIDS, and decreases in HIV-associated morbidity and opportunistic infection. To date, 24 individual drugs have been approved by the United States Food and Drug Administration for the treatment of HIV infection. These drugs are distributed into six major classes:

1. Nucleoside-analog reverse transcriptase inhibitors (NRTI)
2. Non-nucleoside reverse transcriptase inhibitors (NNRTI)
3. Protease inhibitors (PI)
4. Fusion inhibitors
5. Entry Inhibitors – Coreceptor Antagonists
6. Integrase inhibitors

Entry inhibitors represent a new class of antiretroviral agents for the treatment of infection with HIV-1. While resistance to other HIV drug classes has been well described, resistance to this new class is still ill-defined despite considerable clinical use. Several potential mechanisms have been proposed: tropism switching (utilization of CXCR4 instead of CCR5 for entry), increased affinity for the coreceptor, increased rate of virus entry into host cells, and utilization of inhibitor-bound receptor for entry. This review addresses the development of attachment, fusion, and coreceptor entry inhibitors and explores recent studies describing potential mechanisms of resistance.

HIV-1 Entry, Inhibitors, and Resistance. Viruses 2010, 2(5), 1069-1105; doi:10.3390/v2051069

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