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22 Jan 2019

QUESTION 17

How do we classify people with respect to the ability to metabolize drugs? Many of the genes that encode the cytochrome P450 proteins involved in metabolizing drugs are polymorphic. What effects does this have on metabolism of drugs and efficacy/adverse reactions of drug treatment? For cytochrome P450 enzymes, how might gene copy number variation affect metabolism of drugs?

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Nelly StrackeLv2

24 Jan 2019

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Question : Using the Article below," Write a review of the current status of development of antibiotics." There is not Table only from the article below that you should take informations from to write a review of the status of the development of Antibiotics!!

Article: "Bacterial fatty acid metabolism in modern antibiotic discovery."

Abstract:

Bacterial fatty acid synthesis is essential for many pathogens and different from the mammalian counterpart.

These featuresmake bacterial fatty acid synthesis a desirable target for antibiotic discovery. The structural divergence

of the conserved enzymes and the presence of different isozymes catalyzing the same reactions in the

pathwaymake bacterial fatty acid synthesis a narrowspectrumtarget rather than the traditional broad spectrum

target. Furthermore, bacterial fatty acid synthesis inhibitors are single-targeting, rather than multi-targeting like

traditional monotherapeutic, broad-spectrum antibiotics. The single-targeting nature of bacterial fatty acid synthesis

inhibitors makes overcoming fast-developing, target-based resistance a necessary consideration for antibiotic

development. Target-based resistance can be overcome through multi-targeting inhibitors, a cocktail of

single-targeting inhibitors, or by making the single targeting inhibitor sufficiently high affinity through a pathogen

selective approach such that target-based mutants are still susceptible to therapeutic concentrations of drug.

Many of the pathogens requiring new antibiotic treatment options encode for essential bacterial fatty acid

synthesis enzymes. This reviewwill evaluate themost promising targets in bacterial fatty acid metabolismfor antibiotic

therapeutics development and review the potential and challenges in advancing each of these targets to

the clinic and circumventing target-based resistance. This article is part of a Special Issue entitled: Bacterial Lipids

edited by Russell E. Bishop.

Introduction:

Phospholipid synthesis is one of themajor biosynthetic pathways in

all living organisms, and fatty acids form the acyl chains found on all

phospholipids. Fatty acid synthesis is essential inmany bacterial pathogens,

and significant differences exists between the structures of

mammalian type I fatty acid synthesis and bacterial type II fatty acid

synthesis (FASII). The essential nature of bacterial fatty acid synthesis

coupledwith the ability to target bacterial fatty acid synthesis selectively

over themammalian counterpart has made FASII a focus for developing

novel antibiotics. In this review, we will discuss the potential of the

various molecular targets in fatty acid synthesis to be targeted for antibiotic

discovery.Wewill startwith a brief reviewof antibiotic discovery

to understand how history has shaped thinking in the field. Next, we

will discuss the antibiotic discovery targeting bacterial fatty acidmetabolismand

the potential of enzymes in bacterial fatty acidmetabolismas

antibiotic targets. Finally, we will discuss current thinking in antibiotic

discovery and how to develop novel therapeutics against the targets

in bacterial lipid metabolism.

2. A brief history of antibiotic discovery

2.1. Activity based synthetic compound screening â the first step

The history of antibiotic discovery started fromthe crossroads ofmicrobiology

and organic chemistry in the early 1900s. Having recently

shown microorganisms as the cause of infectious diseases, early researchers

screened for compounds that kill the infectious agentwith reduced

toxicity to the human patient (as measured in animal models).

The German Nobel laureate Paul Ehrlich was the first person to formalize

the idea of a hypothetical agent (âmagic bulletâ) that kills the specific

microbe responsible for the diseases without harming the body [1].

Through synthesizing and screening analogues of the organoarsenic

compound Atoxyl which had antisyphilitic activity but also severe

side effects, Ehrlich's team discovered arsphenamine (Salvarsan) and

neoarsphenamine (Neosalvarsan), the first modern chemotherapeutic

agents. The first antibiotic to reach commercial successwas sulfonamide

drugs discovered by scientists in Bayer AG [2]. A red aniline dye was

found to effectively inhibit bacterial infections in mice after screening

hundreds of dyes. The compound was named Prontosil and was

highly effective against Gram-positive streptococci. Further research

found that Prontosil was a prodrug of the active sulfanilamide.

Sulfanilamide was the first broad-spectrum antibiotic, and was

used on the battlefields of World War II for treating wound

infections.

2.2. Activity based natural product screening â the Golden Age

The Golden Age of antibiotic discovery occurred from the activity

screening of natural products. Alexander Fleming was the first to

show that the pure Penicillium mold was able to produce an agent

with antimicrobial activity in 1928. Fleming was able to isolate the active

compound and demonstrated its antimicrobial activity. Penicillin

had greater clinical efficacy than sulfanilamide and reduced toxic side

effects, but Fleming was unable to garner interest for research into the

industrial production of penicillin due to the success of the recently

commercialized sulfanilamide antibiotics. The mass scale production

of penicillin in the United States was a triumph of the allied war

effort during World War II. Penicillin was so successful that it

defined the characteristics of the ideal antibiotic â broad-spectrum,

monotherapeutic, and low toxicity.

The success of penicillin stimulated natural product screening

efforts and spawned the âGolden Ageâ of antibiotic discovery from the

1940s to the 1970s [3]. Advances in structural biology and medicinal

chemistry allowed researchers to chemically modify the natural

products to produce semi-synthetic antibiotics with improved clinical

properties. The majority of the broad-spectrum antibiotic classes we

use even today including the Î²-lactams, tetracycline, and macrolides

were discovered through this process. Unfortunately, resistance quickly

caught up with these newly discovered antibiotics, as the discovery of

new antibiotic classes through natural product screening decreased

over time [4,5]. Additional screens largely rediscovered previously

found chemical entities and failed to find promising new chemical

entities. The decreasing return caused many pharmaceutical companies

to leave antibiotic research.

2.3. Target-based discovery â the draught

Major advances in genetics and molecular biology by the 1990s

allowed the identification of the molecular targets of the antibiotics

[6,7]. The first sequenced bacterial genomes were also released at

this time, with the various proteins encoded by the genomes rapidly

characterized [8]. Several hundred proteins essential for bacterial

growth were identified, and thought to be potential antibiotic targets

[9]. The success of fluoroquinolone, a synthetic antibiotic rationally

designed against DNA topoisomerase suggested that other

essentialmolecular targets could be exploited as novel antibiotic targets

as well [10]. Antibiotic discovery entered a new phase. Rather

than screen for active compounds and determine their molecular

target, hits against essential molecular targets were identified and

chemically modified to become successful antibiotics [11]. However,

the end goal for target-based antibiotic discovery remained the same

- to discover another broad-spectrum, monotherapeutic, and low

toxicity penicillin-like antibiotic. To rationally design such an antibiotic,

researchers startedwith an essentialmolecular target found in a

broad spectrum of bacteria [9]. Furthermore, this target would be

nonexistent or significantly different in humans to decrease the

probability of toxicity. High-throughput screening technology was

used to find the lead compounds against the molecular target, and the

lead compound was modified via medicinal chemistry to penetrate

the bacterial cell membrane, possess the ideal broad-spectrum, and

have drug like pharmacokinetic properties. This compound would

then be tested in mousemodels, and would be brought to clinical trials

in humans if successful.

Several large pharmaceutical companies conducted systemic targetbased

discovery campaigns in the late 1990s. GlaxoSmithKline carried

out the best documented target-based antibiotic discovery campaign

[9]. The goal of the campaign was to discover novel antibacterial inhibitors

with either Gram-positive or broad-spectrum activity. Over 350

conserved gene targetswere identified through comparing the genome

sequences of Haemophilus influenzae, Streptococcus pneumoniae, and

Staphylococcus aureus. These conserved genes were genetically verified

for essentiality, and 127 genes were essential in at least one of the

three bacterial species. Several genes encoding for fatty acid synthesis

were validated as conserved and essential in these screens. A total of

67 high throughput screening campaigns against the SmithKline

Beecham compound collection were conducted against the subset of

essential and conserved targets with known function and amenable assays.

Only 5 leads resulted from the HTS screens, and two of the leads

targeted fatty acid synthesis enzymes FabI and FabH. Nearly a decade

later, none of the leads have passed phase III trials, although some are

still under clinical development. The target-based antibiotic discovery

efforts by the other pharmaceutical companies also did not yield clinical

products despite significant investment [12]. The systematic failure

of target-based antibiotic discovery suggests that some underlying

assumptions about the discovery process must be incomplete or

incorrect.

3. Antibiotic targets in fatty acid metabolism

Fatty acids make up the acyl chains of glycerophospholipids,

the major and essential component of bacterial membrane [13,14].

Bacteria synthesize fatty acids using the type II fatty acid synthesis system,

where fatty acids are synthesized from acetyl-CoA precursors

through rounds of 2 carbon elongations using discrete, monofunctional

enzymes [15] (Fig. 1). In contrast, fatty acids are synthesized by the

multifunctional type I fatty acid synthase in mammals, allowing for

selective targeting of FASII [16]. FASII is a validated target, with two of

the five leads discovered in the GlaxoSmithKline HTS campaign

targeting FASII (FabH and FabI) [9]. Targeting fatty acid synthesis for

novel antibiotic therapies via target-based discovery is also an active

area of research, with the FabI inhibitor AFN-1252 having passed

phase II clinical trials and currently undergoing further clinical development

[17â22]. This section summarizes the antibiotic targets in bacterial

fatty acid metabolism.

3.1. Bacterial fatty acid metabolism is a narrow spectrum antibiotic target

The initiation and elongation steps of FASII are shown in Fig. 1

[13,15]. Some of the validated inhibitors of FASII are shown in

Fig. 2. Two features of bacterial fatty acid synthesis reduce the potential

spectrum of antibiotics targeting fatty acid synthesis [23]. First,

fatty acid synthesis is not essential in certain bacteria, such as the

key pathogen Streptococcus pneumoniae, if they are provided an extracellular

source of fatty acids [24,25]. These bacteria can potentially

bypass the inhibition of endogenous fatty acid synthesis by using

host fatty acids to make their phospholipids,making them refractory

to FASII inhibition. Second, there are different enzyme isoforms

performing the same enzymatic reactions in several key steps of

FASII in different bacteria. For example, there are four structurally

distinct enoyl-ACP reductase enzyme forms [26â29] (Fig. 1). This

feature further reduces the spectrum of potential inhibitors of

bacterial fatty acid synthesis. Inhibitors targeting bacterial fatty

acid metabolism will have at its inception narrower spectrum than

the broad-spectrum, monotherapeutic antibiotics commonly deployed

in clinics today. This factor must be considered when designing

inhibitors of bacterial fatty acid synthesis.

Enzymes catalyzing rate-limiting reactions in key regulatory steps

make for the best chemotherapeutic targets. In contrast, fast turnover

enzymes catalyzing equilibrium reactions make for bad chemotherapeutic

targets because a large amount of inhibition is required to

cause relatively little phenotypic effect. There are three fast turnover enzymes

catalyzing equilibrium reactions â FabD, FabZ, and FabA â which

make for poor drug targets. In contrast, the condensation enzymes

FabF and FabH, enoyl-ACP reductases, and the acetyl-CoA carboxylase

enzyme complex are rate-determining reactions at key regulatory

steps, making them desirable drug targets [30]. A number of natural

products and synthetic inhibitors are known for these enzymes, validating

them as antibacterial targets [31,32].

3.2. Acetyl-CoA carboxylase

The acetyl-CoA carboxylase complex catalyzes the irreversible carboxylation

of acetyl-CoA to make malonyl-CoA using the energy from

hydrolyzing ATP to ADP [33]. This reaction is the first committed, regulated,

and rate-limiting step in fatty acid synthesis, making it a good

chemotherapeutic target. The acetyl-CoA carboxylase complex is composed

of 4 protein subunits catalyzing two half reactions in bacteria. Biotin

carboxylase (AccC) catalyzes the carboxylation of the biotin

prosthetic group attached to biotin-carboxyl carrier protein (AccB) in

the first half reaction. The carboxyltransferase complex (AccA and

AccD) transfers the carboxyl group from the biotin to acetyl-CoA to

form malonyl-CoA. The bacterial acetyl-CoA carboxylase complex is

highly conserved [31,34] and structurally different from the mammalian

acetyl-CoA carboxylase complex which is organized as a single polypeptide

rather than separate protein subunits [35],making it a potential

broad-spectrum antibiotic target.

The effective spectrum of acetyl-CoA carboxylase inhibition is

still being investigated [25]. Several natural products such as

moiramide B and andrimid block the growth of bacteria through

on-target inhibition of fatty acid synthesis in standard laboratory

culture media without fatty acids [36]. Plants also encode for a

bacteria-like acetyl-CoA carboxylase complex, and the plant enzyme

is targeted by commercial herbicides [37]. Extensive effort at designing

ACC inhibitors for antibacterial therapy has been attempted [38,

39], including designing dual-ligand inhibitors that simultaneously

inhibit two subunits of the acetyl-CoA carboxylase complex [40].

Exogenous fatty acids can overcome Andrimid inhibition in both

Staphylococcus aureus and Streptococcus pneumoniae in planktonic

growth, and a Staphylococcus aureus strain encoding an inactive

acetyl-CoA carboxylase can grow in laboratory culturemedia supplemented

with exogenous fatty acids [25]. However, this same strain of

Staphylococcus aureus cannot proliferate in a mouse sepsis model

illustrating the importance of in vivo testing [41]. Acetyl-CoA carboxylase

inhibition is expected to be effective against the model

Gram-negative bacterium Escherichia coli because the essential lipopolysaccharide

synthesis requires Î²-hydroxyacyl-ACP made from

endogenous fatty acid synthesis [42,43]. Acetyl-CoA carboxylase is

also essential for Pseudomonas aeruginosa [44]. Whether acetyl-CoA

carboxylase is essential for Gram-negative bacteria with nonessential

lipopolysaccharides, such as Neisseria [45,46], remains to be

validated.

3.3. Condensation enzymes

The condensation enzymes, FabH, FabF, and FabB, catalyze a

Claisen condensation using malonyl-ACP as the nucleophile to elongate

the acyl chain by two carbons at a time [30,47]. FabH initiates

fatty acid synthesis by condensing a malonyl-ACP with acetyl-CoA

to acetoacetyl-ACP [15]. Branched-chain acyl-CoA precursors such

as 2-methylbutyryl-CoA are used in place of acetyl-CoA for the synthesis

of branched chain fatty acids [48]. FabF initiates each round

of 2 carbon elongation through the condensation of malonyl-ACP

with acyl-ACP [15]. FabB has similar function as FabF, but is essential

for the elongation of unsaturated fatty acids in bacteria expressing a

FabA [49,50]. FabF and FabB have a Cys-His-His catalytic triad, and

FabH has a Cys-His-Asn triad [51]. Condensing enzymes proceed

via a Ping-Pong mechanism where the active site cysteine attacks

the thioester of the acetyl-CoA to make the acyl-cysteine thioester

intermediate [52â54]. After the release of the CoA, decarboxylation

of the carboxyl group of malonyl-ACP makes an enolate intermediate.

Nucleophilic attack from the enolate group on the acyl-cysteine

thioester elongates the acyl group by two carbons.

The condensing enzymes have been an active area of antibacterial

research. FabH was genetically essential in the GlaxoSmithKline antibacterial

screening efforts and FabH inhibitors have demonstrated

in vivo activity [9]. A variety of natural product and synthetic inhibitors

also target the condensing enzymes [13,32]. Due to the similar enzymatic

reactions catalyzed by the condensing enzymes, inhibitors against

the condensing enzymes have the potential to be dual targeting. Several

natural product inhibitors have been demonstrated to target both

classes of condensing enzymes [55,55â57], including cerulenin which

is a covalent inhibitor of FabB and FabF [58,59].

3.4. Reductases

There are two reduction reactions in each elongation cycle of fatty

acid synthesis [15]. Enoyl-ACP reductase catalyzes the reduction of

trans-2-enoyl-ACP into acyl-ACP. Because the dehydration step preceding

enoyl-ACP reductase in the elongation cycle is an equilibrium

reaction, enoyl-ACP reductase is one of the rate-determining steps in

fatty acid synthesis [60,61]. There are four characterized isoforms of

enoyl-ACP reductase. The FabI enoyl-ACP reductase isoform was first

discovered in E. coli and a target of the biocide triclosan [26,62,63].

The FabL isoform is distantly related to FabI and found in addition to

FabI in Bacillus species [27]. The FabK isoform is a flavoprotein with no

structural relationship to FabI [28]. FabK is the primary enoyl-ACP reductase

in Streptococcus species and a significant number of other

firmicutes [64]. The FabV isoform is the least characterized isoform

and is found predominately in Î³-Proteobacteria clades such as Vibrio,

Yersinia, and Pseudomonas species [29,65]. The FabV isoform may have

originated from Pseudomonas fluorescens encoding for the synthesis of

natural product inhibitors of FabI [66].

FabI is a validated antibiotic target [63,67], and FabI inhibitors are

currently being evaluated in clinical trials [17â22]. Although FabI is

not the most widely distributed enoyl-ACP reductase isoform, FabI is

the essential enoyl-ACP reductase in a number of key pathogens

including Staphylococcus, Neisseria, Enterococcus, Acinetobacter, and Enterobacter

species [25,68,69]. The biocide triclosan targets the FabI in

Staphylococcus aureus and E. coli [63,67] in addition to acting as a nonspecific

biocide [28,70]. The anti-tuberculosis drug isoniazid also targets

InhA in mycolic acid synthesis, which is the homologue of FabI in

Mycobacterium tuberculosis [71]. Although concerns have been raised

that exogenous fatty acids could bypass FabI inhibition [24], experiments

in Staphylococcus and Neisseria have conclusively shown that

exogenous fatty acids cannot bypass FabI inhibition in these organisms

[25,68]. Because FabI is not broadly distributed in bacteria,most FabI antibiotic

discovery has focused on Staphylococcus.

The Î²-ketoacyl-ACP reductase FabG catalyzes the reduction of Î²-

ketoacyl-ACP into Î²-hydroxyacyl-ACP [15]. In contrast to enoyl-ACP

reductase, there is only one highly conserved Î²-ketoacyl-ACP reductase

isoform that has been characterized, suggesting that inhibitors

targeting this enzyme would have broad-spectrum activity. However,

FabG does not appear to have a rate-controlling role in fatty acid

synthesis, making it less desirable as a target. While several natural

product FabG inhibitors have been reported [72,73], there has been

very little follow through research to validate the selectivity of

these inhibitors or develop the compounds further. FabG remains

to be validated as a suitable target for drug discovery.

3.5. Acyltransferases

Acyl-ACP of the appropriate length becomes substrate for the

acyltransferases and the synthesis of phosphatidic acid, the precursor

to all phospholipids in bacteria [48]. The structures of the enzymes involved

in bacterial phospholipid synthesis are poorly defined because

they are typically membrane bound proteins. Phosphatidic acid is

made by two successive acylations of glycerol-3-phosphate using acyl-

ACP. The PlsC enzyme involved in the second acylation step is conserved

in both bacteria andmammals and make a poor antibiotic target. Three

enzymes, PlsB, PlsE, and PlsY, are involved in the first acylation step. The

PlsX/PlsY acyltransferase system is the most widely distributed in

bacteria [74]. All characterized bacteria use the PlsX/PlsY acyltransferase

system except for Î³-Proteobacteria which uses PlsB and Chlamydia

which uses PlsE [75]. PlsB and PlsE share the same HX4D catalytic

motif and substrate as PlsC and may not be expected to allow for

selective antibacterial targeting [76]. Incontrast,PlsYbelongs to-another

protein family and uses acyl-phosphate made by PlsX for the acylation

reaction [77]. Analogues of acyl-phosphate show that the on-target inhibition

of PlsY inhibits bacterial growth [78,79]. However, current

acyl-phosphate analogues are too hydrophobic to be âdrug likeâ, and

the lack of PlsY structure hampers improved compound design [78,

79]. Because PlsX catalyzes the reversible conversion between acyl-

ACP and acyl-phosphate [80,81], acyl-phosphate analogues could be

dual targeting inhibitors of PlsX and PlsY.

4. Antibiotic discovery in bacterial fatty acid metabolism

4.1. Resistance in antibiotic discovery

In addition to targeting an essential molecular target, resistance

must also be considered in antibiotic discovery. Themechanismof resistance

against an inhibitor determines the time frame that the inhibitor

is therapeutically relevant. If high level resistance to the inhibitor is

able to develop during the course of treatment, then the inhibitor stands

a significant chance of failure during therapeutic treatment and will

likely fail clinical trials. Resistance development to the inhibitor must

be considered during target selection, inhibitor design, and clinical development.

There are fourmechanisms for bacterial resistance to antibiotics

[82]. First, bacteria canmodify the cellwall/membrane to decrease

the permeability of the antibiotic to render the intracellular concentration

of the antibiotic ineffective. Second, bacteria can increase the efflux

of the antibiotic to again decrease the intracellular concentration of the

antibiotic. Third, bacteria can modify the antibiotic to render it ineffective

such as hydrolyzing the Î²-lactamring or methylation of aminoglycosides.

Finally, bacteria can modify the molecular target of the

antibiotic to decrease the affinity of the antibiotic to the molecular

target.

These four resistance mechanisms arise and spread by lateral gene

transfer and spontaneousmutation. Themode that the resistancemechanismoccurs

affects the rate of resistance development [82]. Of the four

resistance mechanisms, modification of cell wall, increase in efflux, and

molecular target modification occur through spontaneous mutations.

Spontaneous mutation arises from errors in DNA replication leading to

changes in gene function or the expression level of the gene function.

Missense mutations occur at approximately one in 109 bacterial cells,

approximating the error rate in DNA replication [83]. Environmental

stresses can further accelerate themutation rate. This feature of bacterial

physiology means that a strain encoding for a mutant gene product

resistant to the antibiotic might already be present at low populations

in nature, and could quickly develop if it is not already present. While

resistance through modification of cell wall or increased efflux lead to

low level of antibiotic resistance, single amino acid changes via a missense

mutation in the drug binding pocket of the molecular target can

increase resistance against the antibiotic by several hundred fold. This

makes the modification of the molecular target resistance mechanism

a key consideration during antibiotic discovery because thismechanism

gives rise to fast-developing, high level resistance. One of the key reasons

for the failure of target based antibiotic discovery (which targets

a single molecular target) is due to high level resistance quickly developing

via modification of themolecular target [84]. Therefore, overcoming

resistance development, particularly missense mutations in the

gene target, must be considered in the early phases of antibiotic

discovery.

In contrast, chemicalmodification of antibiotics causes high level resistance

by spreading through lateral gene transfer. Spontaneousmutations

to an existing gene that give it the ability to modify a xenobiotic

are possible in theory, but evolving such new gene functions requires

long periods of selection in practice [85]. Therefore, resistance through

chemical modification of antibiotics develops slowly at first, but spreads

with frightening speed once a certain threshold has been reached. The

development of this mode of resistance is largely unpredictable, and

the appropriate focus is to slowthe spread of this resistance mechanism

through proper use of the antibiotic [82].

4.2. Multi-targeting monotherapeutic antibiotics â FabH/FabF/FabB

inhibitors

The well-reasoned and provocative âmulti-targetâ hypothesis posits

that all broad-spectrum, monotherapeutic antibiotics in clinical use

today target multiple gene targets, and stipulates that the multitargeting

feature of these antibiotics limit the fast development of

high level resistance through modifying the molecular target [84]. Antibiotics

targeting two or more gene targets require simultaneous resistance

causing missense mutations in all the gene targets to cause high

level resistance. The probability of developing a resistantmissense mutation

in a gene target is approximately one in 109 bacteria [83,86]

meaning that a resistant bacteria can be found in an approximately

1ml OD600=1 culture. However, the probability of developing two resistantmissensemutations

simultaneously is approximately one in 1018

bacteria,meaning a 1,000,000 l OD600=1 culture would be required to

find the resistancemutant. Themultiplicative probability of developing

resistant missense mutations means that targeting two or more essential

targets simultaneously is an effectiveway of slowing the emergence

of resistance throughmodifying themolecular target. A summary of the

design principles of a broad-spectrum antibiotic is summarized in

Table 1. Resistance against these antibiotics occurs via a combination

of decreasing cell permeability against the antibiotic, efflux of the

antibiotic, modification of the antibiotic, and stepwise accumulation of

multiple target-based resistance mutations [84,87].

A monotherapeutic antibiotic under this model must inhibit molecular

target(s) encoded for by 2 or more genes in the bacterial genome.

This means that the active site and function of these molecular targets

must be sufficiently similar to be inhibited by a single pharmacophore.

Thesemolecular target(s)must also be highly conserved in awide spectrum

of bacteria if broad-spectrum activity is desired. Despite having

nearly a dozen different classes of antibiotics, all broad-spectrum

monotherapeutic antibiotics target 3 molecular target classes - penicillin

binding proteins, ribosomes, and topoisomerases [84]. Each of

these targets represents a family of essential and related protein functions

encoded for by multiple genes. These three targets are also highly

conserved in a wide range of bacteria. With the genomes of many

bacterial pathogens sequenced and the protein functions of model

organisms like E. coli largely characterized, an educated guess can be

ventured onwhether there aremoremolecular target(s) that are highly

conserved, widely distributed, and encoded for 2 or more times in the

bacterial genome. There appear to be few enzyme classes that fulfill all

three criteria.

There are three dual targeting inhibitors of bacterial fatty acid

metabolism. The natural product inhibitor platencin is a dual inhibitor

of the condensing enzymes FabH and FabF in Staphylococcus aureus

[55,88]. Platencin has relatively balanced activity against the two enzymes

(IC50 of 4.6 Î¼M vs FabF and 9.2 Î¼M vs FabH). Unfortunatelyplatencin has poor pharmacokinetic properties and a continuousinfusion

is required to effectively treat Staphylococcus aureus in the

mousemodel [88]. Little progress has beenmade inmodifying platencin

to have better pharmacokinetic properties due to the complex chemical

nature of platencin that makes generating chemical analogue libraries

difficult [89].

The natural product inhibitor thiolactomycin is a dual inhibitor

of FabF and FabB in bacteria encoding both enzymes such as E. coli

[56,57,90â92]. Staphylococcus aureus only encodes for FabF, so

thiolactomycin is only a single-target inhibitor of FabF in Staphylococcus

aureus. Thiolactomycin is a structural mimic of malonyl-ACP

and binds to the acyl-enzyme (Pong form) of the condensing enzyme

[53,93]. Thiolactomycin has an antibacterial spectrum against both

Gram-positive and Gram-negative bacteria, low cytotoxicity, effectiveness

in a mouse model, and favorable pharmacokinetic properties

[94,95]. However, despite considerable effort to structurally

improve thiolactomycin [96â98], successful therapeutic products

have not emerged from this lead. Optimizing thiolactomycin remains

an active area of continuing research [99].

The natural product inhibitor cerulenin is a covalent dual inhibitor of

FabF and FabB [58,59]. Like thiolactomycin, cerulenin is only singletargeting

in bacteria with only FabF. The catalytic cysteine in the condensing

enzymes attacks the epoxide group in cerulenin to forma covalent

intermediate that inhibits the enzyme reaction [100]. The covalent

natural of cerulenin inhibitionmeans that selectivity is for the active site

catalytic cysteine, and the inhibitor could tolerate some differences in

the rest of the active site architecture (similar to Î²-lactam antibiotics).

Unfortunately, cerulenin reacts with eukaryotic fatty acid synthase

and sterol synthesis enzymes using similar enzymatic mechanisms as

the condensing enzymes [101â103], so it lacks bacterial selectivity.

The covalentmode of inhibition and the lack of bacterial selectivity suggest

that designing a bacterial selective cerulenin derivative will be

challenging and little progress has been made to improve its antibacterial

properties.

4.3. Pathogen-selective antibiotic discovery â FabI

Three companies continue to be involved in developing FabI inhibitors

for antibiotic therapy.Mutabilis [104], CrystalGenomics [105â107],

and Affinium [17,20,21] are all focused on developing FabI inhibitors

with Staphylococcus selective activity rather than broad-spectrum

activity. These inhibitors are the first reported single-target, pathogenselective

inhibitors to enter into clinical evaluation. CG400549 from

CrystalGenomics has successfully passed phase 1 trials and recently

completely phase 2a trials (the results are currently under review).

AFN-1252 from Affinium has successfully completed phase 2 trials and

is undergoing further clinical development by Debiopharm [19]. The

extensive research surrounding AFN-1252 provides an informative

case study in the potential benefits and challenges of designing singletarget

pathogen-selective inhibitors, Fig. 3.

Pathogen-selective inhibitors could potentially overcome targetbased

resistance by being so potent that therapeutic doses of antibiotics

are still curative against the missense resistant mutant. Rather

than compromising affinity to target a broad spectrum of bacteria,

pathogen-selective inhibitors can be designed to have extremely high

affinity and activity against the particular pathogen. AFN-1252 has a

4 nM apparent Ki against the Staphylococcus FabI and 4 ng/ml minimal

inhibitory concentration against Staphylococcus aureus in planktonic

growth [18,20,108]. The only mutations causing high level resistance

to AFN-1252 are missense mutations to two residues (M99T and

Y147H) in FabI. The M99T mutation causes a 64 fold increase in AFN-

1252 resistance (MIC of 250 ng/ml) withminimal change in growth fitness.

The Y147Hmutation causes a 128 fold increase (MIC of 500 ng/ml)

in AFN-1252 resistance along with severe growth defects [25,108].

Mutants with higher levels of resistance were not found by selecting

the single missense mutants, while a mutant enzyme with both the

M99T and Y147H mutations is catalytically inactive [108]. This result

suggests that there is a ceiling in short term resistance development

(at least in Staphylococcus aureus), and that a sufficiently potent

inhibitor can overcome target-based resistance. The key question for

AFN-1252 is whether the drug can be dosed at the levels required to

eradicate the M99T and Y147H mutants.

Pathogen-selective inhibitors also have the potential to minimize

the collateral damage to the microbiome. Recent advancesin

microbiome research show that broad-spectrum antibiotics damage

the beneficial microbiomeof the human body, leading to a variety of adverse

short and long termconsequences [109,110]. In particular, broadspectrumantibiotics

are known to cause antibiotic associated secondary

infections. These diseases arise from broad-spectrum antibiotic treatment

wiping out the beneficial host microbiome and allowing drug

resistant pathogens that can't normally compete with the microflora

to proliferate [111,112]. The effect of AFN-1252 on the mouse gut

microbiome was examined as a case study [64]. AFN-1252 caused no

significant change in gut bacterial abundance and minimal disturbance

in bacterial composition in contrast to broad-spectrum antibiotics that

reduced the gut bacterial abundance several thousand fold along with

severe disturbance in bacterial composition. Similar results are expected

in humans, but this conclusion requires verification. One of the

major demands for newantibiotics comes frompatientswithweakened

immune systems [112,113]. A pathogen-selective approach would

benefit these patients by minimizing the disturbance to the beneficial

microbiome.

A summary of the design principles of a pathogen-selective antibiotic

are summarized in Table 1. This pathogen-selective paradigm faces

two major hurdles. First, how resistance will develop against an inhibitor

must be taken into account during the course of development. The

inhibitor must be designed to be high-affinity while minimizinginteractionswith the side chains of non-conserved residues that can undergo

mutations that decrease the affinity of the inhibitor [114]. Resistance

against the inhibitor must be evaluated early in the discovery

phase, and minimizing the impact of resistance must be an additional

criterion that guides the structure optimization process. The inhibitor

must also be tested against the resistant mutant in animal models to

verify that the inhibitor is still effective against the missense resistant

mutants. Second, the specific nature of a pathogen-selective inhibitor

requires accurate pathogen diagnostic technology. Because the antibioticmust

bematchedwith the correct pathogen, rapid and accurate identification

of the pathogen responsible for the disease must precede

antibiotic prescription. One major challenge in pathogen detection is

determining which bacterial species is responsible for diseases because

many diseases causing pathogens are also permanent residents of the

human microflora [115]. Fortunately, rapid pathogen detection is an

area of major research and advances [116].

4.4. Combinations of single-target antibiotics

The success of the Î²-lactam antibiotics has made combinations of

antibiotics a less favorable choice due to the difficulty in dosingmultiple

compounds. However, combinational antibiotic therapy has long been

the standard for treating Mycobacterium tuberculosis. Because Mycobacterium

tuberculosis encodes for and produces extended spectrum Î²-

lactamases, Î²-lactamdrugs are largely ineffective [117]. Mycobacterium

tuberculosis also encodes only a single rRNA gene so ribosomeinhibitors

must be used in combination therapy with other antibiotics to prevent

fast-developing, target-based resistance [118]. Fluoroquinolones are effective

against tuberculosis, but has been largely held in reserve for

treatment of drug resistant tuberculosis. Instead, the front line treatment

against tuberculosis is a combination of isoniazid, rifampin, ethambutol,

and pyrazinamide. Each of these drugs target a single gene

target and experiences high rates of resistance development when

used individually [119], but the drug cocktail is effective at preventing

resistant mutants. The large scale use of this effective drug cocktail

against M. tuberculosis suggests that the combination of single antibiotics

can be an effective therapeutic strategy to fight target-based

resistance. Another example of this strategy is the sulfamethoxazoletrimethoprim

combination [120]. Sulfamethoxazole is a sulfanilamide

analogue that inhibits dihydropteroate synthetase in tetrahydrofolic

acid synthesis. Trimethoprim inhibits dihydrofolate reductase, another

enzyme in tetrahydrofolic acid synthesis. Target-based resistance arises

quickly for sulfamethoxazole or trimethoprim treatment alone, which

limits the clinical efficiency of these drugs. However, combining these

two drugs for treatment decreases the occurrence of spontaneous

target-based mutations.

Given the difficulties of finding new monotherapeutic, broadspectrum

antibiotics, combination therapy is gaining more interest.

Combining Î²-lactam and Î²-lactamase inhibitors is a significant area of

recent antibiotic development [121]. Given the rather limited target options

in developing broad-spectrum, monotherapeutic antibiotics,

combination therapy of single-target inhibitors with orthogonal gene

targets should be considered as an alternative. The rate-determining

steps in bacterial fatty acid synthesis are validated targets for these

single-target inhibitors. Combining two inhibitors targeting two different

gene targets provides the benefit ofminimizing resistance via single

missense mutations. Furthermore, if resistance is the key reason why

single-target, broad-spectrum antibiotics failed to reach the clinic,

then combining these failed single-target antibiotics should provide an

effective therapy. This hypothesis could be tested directly using the

low toxicity, FASII inhibitors that have already been discovered [13,

32]. The challenge tomulti-drug therapy is avoiding adverse drug interactions

and matching the dosage of the drug components so both drugs

are at active concentrations over the course of treatment to avoid stepwise

resistance. Stepwise resistance against one component of the cocktail

at a time when the other components are low due to improper

antibiotic use is a major concern for the development of resistance in

Mycobacterium tuberculosis [122].

A variety of single-target FASII inhibitors have been described in

literature [13,32]. These include both natural product inhibitors and

synthetic inhibitors. The two best characterized molecular targets are

FabI and FabH, which have been subject to numerous discovery campaigns

by a variety of investigators. One major recurring result from

these campaigns is the difficulty in getting both spectrum and potency

[9,21,99,104â107,123]. For example, a recent FabH discovery campaign

reported a series of 22 thiolactomycin analogues and tested these analogues

againstMycobacteriumtuberculosis, Francisella tularensis, Yersinia

pestis, and Staphylococcus aureus [99]. While several analogues were

able to achieve low Î¼g/ml minimal inhibitory concentration against

one of the bacterial species, only 1 analogue was able to achieve low

Î¼g/ml minimal inhibitory concentration against two bacterial species

and none against 3 or more species. These results show that FASII enzymes

are sufficiently structurally divergent that even broad-spectrum

single-targeting is exceptionally difficult. Outside of cell wall synthesis,

ribosome, and DNA synthesis, there simply do not appear to be

more highly conserved molecular targets in bacteria. A cocktail of narrow

spectrum antibiotics should be considered in future antibiotic

applications.

5. Summary

The Centers for Disease Control released the âAntibiotic

resistance in the United Statesâ report in 2013 [124]. The report

identified Clostridium difficile, Neisseria gonorrhoeae, Enterobacter,

Acinetobacter, Campylobacter, Psuedomonas aeruginosa, Salmonella,

Shigella, Staphylococcus, Enterococcus, and Streptococcus as major

pathogen threats in need of new antibiotic treatments. Bacterial

fatty acid metabolism has been validated as essential in most of

these pathogens. Of these pathogens, only Streptococcus has the ability

to bypass FASII inhibition through using exogenous fatty acids

[23â25]. FASII has been demonstrated to be essential in Staphylococcus,

Enterocococcus, Neisseria, and E. coli [23,25,68,69], so FabI, FabH, FabF,

and acetyl-CoA carboxylase inhibitors are predicted to be effective

against these pathogens. Bioinformatic predications suggest that the organization

of fatty acid, phospholipid, and lipopolysaccharide synthesis

in Enterobacter, Acinetobacter, Campylobacter, Salmonella and Shigella

are similar to E. coli, so inhibition of the enzymes in FASII would be expected

to work in these pathogens as well. Additionally, dual FabF and

FabB targeting would be possible for the Gram-negative pathogens in

the above list because these Gram-negative bacteria synthesize unsaturated

fatty acids using a FabB [59,125]. Psuedomonas aeruginosa encodes

for multiple enoyl-ACP reductase and FabH isoforms [66,126], but is

predicted via bioinformatics or validated experimentally to encode

only a single, essential copy of acetyl-CoA carboxylase [44], FabF, and

FabB. Finally, Clostridium difficile is an opportunistic pathogen that

proliferates in the gut when the normal microflora is eliminated by

broad-spectrum antibiotic treatment [112,127,128]. The narrow

spectrum of a FabI inhibitor designed using a pathogen-selective

paradigm preserves the gut microflora [64] and prevents Clostridium

difficile colitis from occurring in the first place. The various application

of FASII inhibition holds great potential for the future development of

antibiotics against the most urgent pathogens.

brownhamster745

Summarize the following reading (Nutrient (zinc and vitamin E)-gene interactions related to inflammatory and antioxidant response in aging and inflammation) and it should be at least one paragraph (100-200 words) and include the following information:

* Main point of the reading

* Describe how the health deviations you read about alter normal physiology.

* Provide your key take away from the reading. This could be something that made you pause, changed your thinking, supported your thinking, or made you question something.

INTRODUCTION Aging is an inevitable biological process that is accompanied by gradual and spontaneous biochemical and physiological changes including increased susceptibility to diseases, adverse environmental conditions, and loss of mobility and agility. Alterations in neuroendocrine-immune interactions as well as in antioxidant capacity also play a fundamental role in aging. The inability of an organism to remodel these changes may lead to the appearance of some degenerative age-related diseases. As a result, the remodeling theory of aging has been proposed (1). Various nutritional factors are directly linked to these phenomena, as, for instance, in restoring the neuroendocrine-immune network, metabolic harmony, and the capacity to respond to oxidative stress (2). Approximately 40 micro nutrients (vitamins, essential minerals, and other compounds required in small amounts for normal metabolism) have been reported as essential components in the diet (3). The dietary intake of essential macro nutrients and micronutrients is usually inadequate in the elderly (4). Several causes contribute to this gap. First of all, the poor socio-economic conditions in which a large part of the older population live may lead to a consumption of inexpensive foods deficient in micronutrients, such as carbohydrates (5). The gap is worsened by the loss of appetite, lack of teeth, intestinal malabsorption, and decreased requirements for energy that lead finally to frailty, disability, and mortality (6). Some authors have reported that the deficiency of macro nutrients and micronutrients in aging is strictly related to global impairments of immune functions, metabolic harmony, and antioxidant defense by external noxae with subsequent appearance of age-related diseases (7). Indeed, many micronutrients contribute directly or indirectly to the biological activity of some antioxidant enzymes (e.g., SOD, GPx, and catalase) (8), to the efficiency of the immune system (9), and to the maintenance of metabolic function especially in preventing mitochondrial decay (4). In this last context, feeding studies in older rats have shown that mitochondrial metabolites and antioxidants protect neuronal cells from neurotoxin- and oxidant-induced toxicity and oxidative damage, delay the normal senescence of human diploid fibroblasts, and inhibit the oxidant-induced acceleration of senescence (10). With advancing age, increased oxidative damage to proteins and lipid membranes, particularly in mitochondria, causes a deformation of the structure of enzymes, with a consequent decrease in enzyme activity as well as substrate binding affinity for their substrates. An increased level of the substrate by micro nutrients restores the speed of the reaction as well as mitochondrial function, thus delaying mitochondrial decay and aging (10, 11). In contrast, recent longitudinal studies of dietary daily intake in human nonagenarians and centenarians (successful aging) have shown that an adequate consumption of micronutrients and macronutrients, as well as a satisfactory content of some trace elements within the cells, leads to good performance in several immune functions, metabolic compensation, and preservation of antioxidant activity (12, 13). In this context, polyunsaturated fatty acids, highly sensitive to reactive oxygen species, decrease in liver mitochondria from human centenarians, a feature acquired during evolution as a protective mechanism to favor longevity (14). Therefore, nutritional factors may play a pivotal role for healthy aging and longevity. However, the effects of the nutritional factors are strongly influenced by genetic factors, in particular by the genes involved in inflammatory/immune responses. Proinflammatory cytokines such as IL-1, IL-6, and TNF-Î±; the antiinflammatory cytokine IL-10; the HSP70 chaperones; and the regulators of trace element homeostasis, metallothioneins (MTs), seem particularly relevant, taking into account that the same genes are involved in the susceptibility to major geriatric disease/disorders such as diabetes, osteoporosis, osteoarthritis, dementia, cardiovascular diseases, and infections (15, 16). Indeed this reemphasizes that up to 25% of the variation in human life span is heritable (17); the rest is due to environmental and lifestyle factors, which impact the aging process, contributing as such to a large interindividual variability. Therefore, one current challenge is to understand how the interaction between genetic factors and nutrients may influence aging and longevity, in view of the high impact on gene expression, protein production, and epigenetic mechanisms implicated in regulating the life span (18). Some dietary patterns with likely different impacts on longterm disease occurrence and survival have been identified. In this context, strong evidence for a beneficial effect of higher conformity with the Mediterranean dietary pattern on causes of death, including those of cardiovascular diseases and cancer, has been reported (19). Anyway, it is also commonly accepted that the complex interactions of multiple polymorphisms play a key role in how individuals may respond to dietary interventions (20). For each nutrient, there is a window of intake between the recommended dietary allowance (RDA), which is defined as the dietary intake sufficient to meet the requirements of 97% of healthy individuals in a particular stage of life and sex group, and the tolerable upper limit (UL), which is the highest nutrient intake that can be achieved without incurring a risk of adverse health effects for most individuals in the general population (21). Although worldwide research on genetic variations that require a different RDA or UL is still in progress, several genes and alleles have been suggested to affect nutrient utilization, including genes involved in the metabolism of folate and vitamin B12 (22), vitamin E (23), lipids (24), iron (25), and zinc (26). In this last case, it is important to note that the RDA (11 mg/day for men, and 8 mg/day for women) and UL (40 mg/day for adults) for zinc are very close (Food and Nutrition Board and Institute of Medicine, 2001). Without excluding the beneficial effects of all of these nutrients for healthy aging and longevity (27), but taking into account the pivotal role played by zinc and vitamin E in fighting oxidative stress in aging and in some age-related diseases, such as atherosclerosis (23), we herein report the studies carried out on the interrelationships between zinc and some genes related to the inflammatory/immune response (such as MT and IL-6), as well as the interactions between vitamin E and the gene expressions related to inflammation. Clinical trials of zinc and vitamin E are also reported for their possible beneficial effects on longevity or in preventing the initiation and progression of cardiovascular diseases.

ZINC-MT GENE INTERACTION MTs MTs are essential to intracellular zinc homeostasis by sequestration and release of the metal when it occurs and thereby controlling available free zinc ions (28). The cysteine sulfur ligands in the cluster structure of MT can be reduced (zinc sequestration) or oxidized (zinc release), thus with concomitant changes in the relative amount of bound and free zinc (29). MTs are genetically polymorphous protein families with subfamilies, subgroups, and various isoforms. Humans possess genes for 4 subfamilies (encoded by at least 10 functional MT genes), all located on chromosome 16: the brainspecific MT3, the squamous epitheliumâspecific MT4, and the ubiquitous MT1 and MT2 (30). One of the first functions of MT1 and MT2 is to regulate zinc homeostasis and to limit oxidative damage within the cells (28). Following an injurious stimulus, such as a transient inflammation, the subsequent oxidative stress induces the release of zinc from MT via nitric oxide (NO), to promote the activity and expression of antioxidant enzymes, including MT itself, thus reducing the oxidative damage and the consequences of the injurious stimulus (31). However, the increased expression of proinflammatory cytokines occurring in aging, leads to increased expression of MTs, which in turn sequester considerable amounts of zinc, making it less available for an efficient immune response (32). If, on the one hand, the reduced zinc ion availability in old age might indicate an excessive sequestration of zinc ions by MTs, on the other, consideration of recent findings on oxidative modification of MTs leading to their loss of function suggests also a mechanism whereby these proteins can also lose their ability to buffer the intracellular free zinc concentration (33). In this case, MTs would be unable either to bind or to consequently release zinc in response to stressors. However, it is still unclear if dysfunctional MTs can be considered a typical alteration associated with specific disease/disorders, such as hyperhomocysteinemia and type 2 diabetes, or if they are a common feature of aging. Anyway, as a consequence of the altered zinc metabolism, a large number of genes that act as zinc sensors and transcriptional activators and/or repressors are also altered in aging (34). This may have a deep impact on the regulation of zincdependent transcription factors, because they not only regulate âzinc sensitiveâ genes but also control their own transcription through positive autoregulatory mechanisms (35). Taking into account that healthy centenarians display a low MT expression and satisfactory zinc ion availability despite increases in proinflammatory cytokines (IL-6, TNF-Î±) (36), it may be suggested that this feature reflects the existence of compensatory phenomena able to counteract the effects of inflammation in these exceptional individuals. Moreover, these data suggest that a preservation of zinc homeostasis is an important feature of healthy centenarians. Therefore, the role played by the zinc-gene interaction is pivotal to successful aging and, at the same time, to escape from some age-related diseases. Since, the persistence of inflammatory stimuli over time represents the biological background favoring susceptibility to age-related diseases/ disabilities, the absence of specific ârobustâ gene variants and/or the presence of specific âfrailâ gene variants might predict, on the one hand, longevity, and on the other, the predisposition to the appearance of the more common age-related diseases, such as infections and cardiovascular diseases. In this context, it has to be considered that the genes selected, because they confer a reproductive advantage early in life, may have dangerous effects in the postreproductive period. In fact, negative selection against these harmful effects fails due to the decline of natural selection with age. This fact means that one gene that is âfavorableâ in young/adult age may be âdisadvantageousâ in aging (the antagonistic pleiotropy theory of ageing (37)). Following this perspective, the beneficial effect of inflammation, via an optimal MFT-1-MT-zinc-gene interaction, in young and adult age may become detrimental in old age (32). The recent discovery of novel polymorphisms of MT2A and MT1A supports this assumption. Indeed, older subjects carrying the AA genotype for the MT2A polymorphism display low zinc ion bioavailability, chronic inflammation by high IL-6 and altered lipid assessments, with subsequent elevated risk for atherosclerosis and type 2 diabetes (38). By contrast, a polymorphism corresponding to an A/T (asparagine/ threonine) transition at the +647 nt position in the MT1A-coding region is the most often involved in female longevity (39). Such allelic variants may be very useful tools to screen older subjects at risk for zinc deficiency on a genetic basis, taking into account that the actual methodological procedures to test âzinc statusâ are often misleading and that laboratory investigations to assay zinc ion bioavailability are scarcely reproducible and poorly applicable to clinical practice (40). In this context, a novel reproducible system for testing intracellular zinc ion bioavailability has been developed using a zinc fluorescent probe (Zynpir-1) associated with MT values, with both tests representing valid methods to detect the intracellular zinc status (41).

ZINCâINTERLEUKIN-6 GENE INTERACTION Interleukin-6 (IL-6) is a pleiotropic cytokine capable of regulating proliferation, differentiation, and activity of a variety of cell types, and it plays a pivotal role in immune response. In particular, the most important function of IL-6 is most likely as a mediator of acute phase inflammatory responses. These include the balance of the proinflammatory/antiinflammatory pathways, lymphocyte activation, and hepatocellular stimulation of acute phase protein synthesis (42). Studies of the effects of aging on inflammatory response show IL-6 to be an important âcytokine for gerontologistsâ. Recent evidence has shown that abnormally increased concentrations of IL-6 are reliable markers for functional disability and predictors of disability and mortality among the elderly (43). Moreover, IL-6 dysregulation is involved in age-related inflammatory diseases, such as cancer, lymphoma, cardiovascular diseases, osteoporosis, Alzheimerâs disease, diabetes and atherosclerosis (44). The human IL-6 gene is located on chromosome band 7p21 and consists of 5 exons and 4 introns (45). Genetic studies have identified 4 polymorphisms in the Lattanzio et al 121 promoter region of the IL-6 gene (-597G/A, -572G/C, - 373A/T, -174G/C) that have a significant effect on IL-6 expression in an in vitro system (46). A variable number of tandem repeat (VNTR) polymorphisms were found in the 3â flanking region of the IL-6 gene (C allele) (47). In humans, a polymorphism in the IL-6 promoter (A/C polymorphism at position -174) altered IL-6 gene transcription rates in vitro (48) and IL-6 levels in vivo (49). In this last paper, people carrying CC or GC genotypes are defined as C+, while those carrying GG genotypes are indicated as C-. It has been suggested that IL-6 -174G/C locus variability is capable of modulating, on the one hand, individual susceptibility to common causes of morbidity and mortality among elderly, while, on the other hand, it may play a crucial role in longevity (50). Therefore, the genetic variations of this locus of IL-6 gene are fundamental in the elderly population to better understand the intrinsic causes of the longevity. The association of these genetic variations to the possible, different immune responses is an attractive focus in elucidating the molecular mechanisms involved in immunosenescence. The genetic variations of the IL-6 -174G/C locus have been extensively studied by different groups with, however, contradictory data. BonafÃ¨ et al (51) studied IL-6 promoter genetic variability at the -174C/G locus and its effect on IL-6 levels in 700 Italians aged 60 to 110 years, including 323 centenarians. Individuals who are genetically predisposed to produce high levels of IL-6 during aging â i.e., C- men (GG genotype) â at the IL-6 -174C/G locus are disadvantaged for longevity. On the other hand, the capability of C+ individuals (CC and CG genotypes) to produce low levels of IL-6 throughout their life span appears to be beneficial for longevity, at least in men. Women have, conversely, high IL-6 serum levels later in life with respect to men, independently of -174C/G locus polymorphism (51). The inhibitory tone of estrogens on IL-6 gene expression could explain the sex-based difference (52), assuming that its long-term effects last until the extreme limits of the human life span. The major production of IL-6 in C- subjects for their whole life, including centenarians, has also been confirmed by other in vivo longitudinal studies (53). A more recent study in older and nonagenarian subjects has confirmed that IL-6 production is higher in C- carriers and that these subjects are prone to contract one of the more common age-related inflammatory pathologies such as atherosclerosis (54). Interestingly, in this last study, C- older and nonagenarian subjects also display impaired innate immune response (NK cell cytotoxicity) coupled with increased MT, zinc deficiency, and low zinc ion availability, in comparison with C+ carriers (54). A functional effect of the IL-6 -174G/C locus on MT expression and zinc-regulated genes has also been confirmed recently by targeted studies with in vitro and in vivo zinc supplementation (55, 56). These findings clearly suggest that the genetic variations of the IL-6 -174G/C locus play a key role in longevity also at a functional level, and confirm the existence of a higher number of C+ centenarians than Câ ones as previously found by BonafÃ¨ et al (51). In this context, an intriguing point is the low expression of the signal-transducing component (gp130) of the IL-6 receptor in centenarians, in comparison with the elderly, despite high circulating levels of IL-6 (57), which in turn may be inactive in centenarians (36). Therefore, inflammatory status is not so detrimental in very old age, as, in contrast, occurs in normal aging. However, the discrepancies among all of the genetic studies on the variations of the IL-6 -174G/C locus in relation to longevity in the elderly means that the subject is still unclear. Ethnic, lifestyle, and cultural differences among these populations could also play a role, as well as other undefined factors. Large-scale studies at the European level in many different ethnic populations are needed to clarify this important topic. For this reason, ZINCAGE project (www.zincage.org) has been developed, and the clarification of this point has been one of is tasks. The determination of the genetic variations of the IL-6 - 174G/C locus associated with a dietary assessment or with a comprehensive evaluation of zinc status has been reported as an useful strategy to identify older subjects who can benefit from zinc supplementation without health risks (see next paragraph).

ZINC SUPPLEMENTATION IN THE ELDERLY ON THE BASIS OF GENETIC BACKGROUND One possible cause of the discrepancies existing in the literature on the effect of zinc supplementation upon immune response and antioxidant performance in the elderly (see review in (58)) may be the choice of older subjects who effectively need zinc supplementation in strict relationship with dietary habits and inflammatory status. This fact is supported by the discovery that older subjects carrying GG genotypes (i.e., C- carriers) in the IL-6 -174G/C locus display increased IL-6 production, low intracellular zinc ion availability, and impaired innate immune responses coupled with enhanced MT (56). By contrast, older subjects carrying the GC and CC genotypes (C+ carriers) in the same IL-6 -174 locus display satisfactory intracellular zinc as well as innate immune responses. But, the more intriguing finding is that males carriers of the C+ allele are more likely to reach centenarian age than C- carriers. Therefore, older C- subjects are more likely to benefit from zinc supplementation than older C+ carriers. The distribution of the IL-6 -174 genotype is very different among the various European countries, with large differences between northern and southern European countries (58). Zinc supplementation in older C- subjects restores NK cell cytotoxicity to values present in older C+ carriers and considerably improves both zinc status, assessed by the percentage increment of granulocyte Zn (56), and stress response, assessed by the percentage increment of clusterin (CLU) expression and MT protein (58). When the genetic variations for the IL-6 polymorphism are also associated with the genetic variations of MT1A in position +647, the plasma zinc deficiency and the altered immune response are more evident (58), suggesting that the genetic variations of IL-6 and MT1A would be very useful tools for the choice of older people who effectively need zinc supplementation. These results open the hypothesis that the daily requirement for zinc might be different in elderly with a different genetic background. Such a role played by genetic background on the beneficial effects of zinc supplementation is also evident in the better control of proinflammatory cytokine and chemokine production (59), as well as in reducing the expression of genes related to inflammatory status, such as IL-1 and its receptor (55). A very intriguing point is also the beneficial effect of zinc supplementation in the cognitive performance of older individuals selected on the basis of IL-6 polymorphism (60). In any case, from a microarray analysis in vitro, zinc at a dose of 50 mM down-regulates the gene expression of proinflammatory cytokines (Fig. 1), strengthening its antiinflammatory role (56, 58).

Fig. 1 - In vitro zinc treatment (50 ÂµM) down-regulates proinflammatory cytokine expression (custom array) when refereed to the value 1 (baseline control) in human peripheral blood mononuclear cells from young adult donors. The red square represents the proinflammatory cytokines (IL-6, IL-1, and TNF pathways). Data redrawn from the Zincage Project (www.zincage.org) (61).