Antibiotics: Inhibitors of Bacterial Cell Wall Biosynthesis PDF

Antibiotics Inhibitors of Bacterial Cell Wall Biosynthesis 11/7/2024 Prof. Afaf AL Nadaf 1  Bacterial cell wall Bacterial cells have a complex and largely rigid cell wall which is not present in human being The...

Antibiotics Inhibitors of Bacterial Cell Wall Biosynthesis 11/7/2024 Prof. Afaf AL Nadaf 1  Bacterial cell wall Bacterial cells have a complex and largely rigid cell wall which is not present in human being The main functions of the bacterial cell wall are: 1) to provide a semipermeable barrier interfacing with the environment through which only desirable substances may pass 2) to provide a sufficiently strong barrier so that the bacterial cell is protected from changes in the osmotic pressure of its environment 3) to prevent digestion by host enzymes The enzymes involved in the late steps of cell wall biosynthesis are more vulnerable to inhibition, because they are at or near the cell surface 11/7/2024 Prof. Afaf AL Nadaf 2  Bacterial cell wall G+ve bacteria 11/7/2024 Prof. Afaf AL Nadaf 3  Mechanism of Action The penicillins and the other β-lactam antibiotics have a structure that closely resembles that of acylated D-alanyl-D-alanine The enzyme mistakenly accepts the penicillin as though it were its normal substrate. The intermediate acylenzyme complex is rather different structurally from the normal intermediate in that the hydrolysis does not break penicillin into two pieces, as it does with its normal substrate 11/7/2024 Prof. Afaf AL Nadaf 4  Mechanism of Action 11/7/2024 Prof. Afaf AL Nadaf 5  Mechanism of Action 11/7/2024 Prof. Afaf AL Nadaf 6  Cell wall cross-linking and mechanism of action of β-lactams 11/7/2024 Prof. Afaf AL Nadaf 7  Synthesis of penicillins ✓ Penicillins were discovered by Alexander Fleming, which were obtained from Penicillium chrysogenum ✓ The earliest penicillins were produced by fungi from media constituents. The bicyclic heterocyclic nucleus of 6-aminopenicillanic acid (6-APA) was constructed by an involved process catalyzed by enzymes. ✓ The side chain was added essentially intact from media constituents. 11/7/2024 Prof. Afaf AL Nadaf 8  β-Lactam antibiotics 11/7/2024 Prof. Afaf AL Nadaf 9  Synthesis of penicillins 11/7/2024 Prof. Afaf AL Nadaf 10  OCH3 OCH3 11/7/2024 Prof. Afaf AL Nadaf 11 11/7/2024 Prof. Afaf AL Nadaf 12  Penicillins ❑ The sodium and potassium salts of penicillins are crystalline and water- soluble. ❑ When dry, they are stable for long periods but hydrolyze rapidly when in solution. Their best stability is noted at pH values between 5.5-8.0. ❑ The procaine and benzathine salts of benzylpenicillin are water insoluble. Because they dissolve slowly, they are used as injection when long-term blood levels are required. 11/7/2024 Prof. Afaf AL Nadaf 13  Nomenclature ❑ The nomenclature of the penicillins: (For example, the chemical name for benzylpenicillin sodium is monosodium (2S,5R, 6R -)3,3 -dimethyl-7-oxo- 6-(2-phenylacetamido)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate. ❑ The use of the names benzyl penicillin and phenoxymethyl penicillin makes practical sense. ❑ There are three asymmetric centers in the benzyl penicillin molecule. 11/7/2024 Prof. Afaf AL Nadaf 14  Chemical Instabilities ❑ β-lactam amide bond: This bond cleaves moderately slowly in water unless heated, but it breaks down much more rapidly in alkaline solutions to produce penicilloic acid (irreversible under physiologic conditions), which readily decarboxylates to produce penilloic acid The bacterial enzyme, β-lactamase, catalyzes this reaction and is a principal cause of bacterial resistance in the clinic 11/7/2024 Prof. Afaf AL Nadaf 15 11/7/2024 Prof. Afaf AL Nadaf 16  Chemical Instabilities ❖ Alcohols and amines bring about the same cleavage reaction, but the products are the corresponding esters and amides. These products are inactive. A reaction with a specific primary amino group of aminoglycoside antibiotics is of clinical relevance, because it inactivates penicillins and cephalosporins ❖ allergies. ❖ Solutions of penicillins for parenteral use should be refrigerated, used promptly, and not stored ❖ In acidic solutions: The main end products of the acidic degradation are penicillamine, penilloic acid, and penilloaldehyde 11/7/2024 Prof. Afaf AL Nadaf 17 11/7/2024 Prof. Afaf AL Nadaf 18  Structure-Activity Relationship ▪ The chemical substituents attached to the penicillin nucleus can greatly influence the stability of the penicillins as well as the spectrum of activity ▪ It is important to recognize whether the structural changes affect drug stability on the shelf or in the GI tract (in vivo), improve stability toward bacterial metabolism, or enlarge the spectrum of activity. 11/7/2024 Prof. Afaf AL Nadaf 19  SAR ✓ The strained β-lactam ring is essential. ✓ The free carboxylic acid is essential. This is usually ionized and penicillins are administered as sodium or potassium salts. The carboxylate ion binds to the charged ammonium ion of a lysine residue in the binding site. ✓ The bicyclic system is important. (This confers further strain on the β- lactam ring-the greater the strain, the greater the activity, but the greater the instability of the molecule to other factors.) ✓ The acylamino side chain is essential. Sulfur is usual but not essential ✓ The stereochemistry of the bicyclic ring with respect to the acylamino side chain is important ✓ Very little variation is tolerated by the penicillin nucleus and any variations are restricted to the acylamino side chain 11/7/2024 Prof. Afaf AL Nadaf 20  SAR ✓ The substitution of a side-chain R group on the primary amine with an electron-withdrawing group decreases the electron density on the side- chain carbonyl and protects these penicillins from acid degradation (can be given orally for systemic purposes) ✓ in vitro degradation reactions of penicillins can be retarded by keeping the pH of solutions between 6.0-6.8 and by refrigerating them ✓ Metal ions, such as mercury, zinc, and copper, catalyze the degradation of penicillins, so they should be kept from contact with penicillin solutions (The lids of containers are routinely made of inert plastics) 11/7/2024 Prof. Afaf AL Nadaf 21  SAR 11/7/2024 Prof. Afaf AL Nadaf 22  SAR The more lipophilic the side chain of a penicillin, the more serum protein bound is the antibiotic. Contrary to popular assumption, the degree of serum protein binding of the penicillins has comparatively little influence on their half-lives The penicillins are actively excreted into the urine via an active transport system for negatively charged ions, and the rate of release from their bound form is sufficiently rapid that the controlling rate is the kidney secretion rate. The serum half-life of penicillin G is 0.4-0.9 hrs and that of phenoxymethyl penicillin approximately 0.5 hours Probenicid competes effectively for excretion and prolongs the half-life 11/7/2024 Prof. Afaf AL Nadaf 23  SAR Stability of the penicillins toward β-lactamase is influenced by the bulk in the acyl group attached to the primary amine. Movement of one of the methoxy groups to the para position, or replacing one of them by a hydrogen, resulted in an analogue sensitive to β- lactamases. Putting in a methylene between the aromatic ring and 6-APA likewise produced a β-lactamase-sensitive agent Prime examples of this effect are seen in the drugs methicillin, nafcillin, oxacillin, cloxicillin, and dicloxicillin 11/7/2024 Prof. Afaf AL Nadaf 24  SAR 11/7/2024 Prof. Afaf AL Nadaf 25  OCH3 OCH3 11/7/2024 Prof. Afaf AL Nadaf 26 11/7/2024 Prof. Afaf AL Nadaf 27  Resistance ❑ Unfortunately, resistance to β-lactam antibiotics is increasingly common and is rather alarming. It can be intrinsic and involve decreased cellular uptake of drug, or it can involve lower binding affinity to the PBPs. ❑ Much more common is the elaboration of a β-lactamase. β-Lactamases are enzymes (serine proteases) elaborated by MO that catalyze hydrolysis of the β-lactam bond and inactivate β-lactam antibiotics to penicilloic acids before they can reach the PCPs ❑ With Gram-positive bacteria, such as staphylococci, the β-lactamases usually are shed continuously into the medium and meet the drug outside the cell wall (turn over many times) ❑ With Gram-negative bacteria, the β-lactamases are secreted into the periplasmic space between the inner and outer membrane, so although still distal to the PBPs, they do not readily escape into the medium and need not be resynthesized as often 11/7/2024 Prof. Afaf AL Nadaf 28  Resistance 11/7/2024 Prof. Afaf AL Nadaf 29  Allergenicity ❑ This is expressed as a mild drug rash or itching and is of delayed onset. Occasionally, the reaction is immediate and profound. It may include cardiovascular collapse and shock and can even result in death ❑ A previous history of allergy to penicillins is a contraindicating factor to their use ❑ When an allergic reaction develops, the drug must be discontinued, and, because cross-sensitivity is common, other β-lactam drugs should be avoided. ❑ Erythromycin and clindamycin are useful alternate choices for therapy in many cases of penicillin allergy. 11/7/2024 Prof. Afaf AL Nadaf 30  Allergenicity ❑ In some cases, the patient may have become sensitized because of previous passive exposure through contaminated foodstuffs or cross- contaminated medications. Penicillins are manufactured in facilities separate from those used to prepare other drugs to prevent cross- contamination and possible sensitization ❑ Because the origin of the allergy is a haptenic reaction with host proteins and the responsible bond in the drug is the β-lactam moiety, this side effect is caused by the pharmacophore of the drug and is unlikely to be overcome by molecular manipulation. 11/7/2024 Prof. Afaf AL Nadaf 31  Individual Penicillins 11/7/2024 Prof. Afaf AL Nadaf 32  Benzylpenicillin Group ❑ Active versus non-β-lactamase producing Gram- positive bacilli (e.g. Meningitis, Gonorrhoea, and early strains of staphylococci) and several Gram- negative cocci (e.g. Neisseria) ❑ Currently the agent of choice for treatment of infections such those caused by Streptococcus pyogenes and susceptible strains of Streptococcus pneumoniae and enterococci ❑ Active versus anaerobic MO and only against rapidly dividing bacteria ❑ Bactericidal rather than bacteriostatic. ❑ Non-toxic. The penicillins are amongst the safest drugs known to medicine. But some people are allergic to them 11/7/2024 Prof. Afaf AL Nadaf 33  Benzylpenicillin Group ❑ Sensitive to all known β-Iactamases ❑ Penicillin G can only be administered by injection. ❑ Causes allergic reactions in some individuals, varying from a rash to immediate anaphylactic shock. ❑ Very water-insoluble penicillin salts form with procaine and with N, N′- benzathine which are used as deep IM injections in order to produce lower but prolonged levels of penicillin as the drug slowly diffuses from the injection site. 11/7/2024 Prof. Afaf AL Nadaf 34  Phenoxymethyl penicillin (Penicillin V) ❑ It also can be prepared by semisynthesis, and it is considerably more acid stable than benzylpenicillin, as indicated by oral absorption ❑ This is caused by the electronegative oxygen atom in the C-6 amide side chain inhibiting participation in β-lactam bond hydrolysis. ❑ Penicillin V has approximately the same sensitivity to β-lactamases and allergenicity as penicillin G. 11/7/2024 Prof. Afaf AL Nadaf 35  Penicillinase-Resistant, Parenteral Penicillins Methicillin and Nafcillin ❖ Methicillin was the first of the penicillinase-resistant agents to reach the clinic. It is unstable to gastric acid, having a half-life of 5 minutes at pH 2, so it must be administered via injection. ❖ Increased bulk resulting from the addition of the dimethoxybenzoyl group to 6-APA leads to methicillin, a β-lactamase-resistant drug ❖ Methicillin has a significantly narrower antimicrobial spectrum and less potency, so it was restricted to clinical use primarily for parenteral use in infections caused by β-lactamase-producing S. aureus and a few other infections 11/7/2024 Prof. Afaf AL Nadaf 36 ❑ Many cultures have developed resistance by reducing the uptake and alterating the PBPs ❑ Nafcillin has a fused benzene ring on one flank and an ethoxy moiety on the other of the side-chain amide linkage. Although slightly more acid stable than methicillin, it is virtually identical to it clinically 11/7/2024 Prof. Afaf AL Nadaf 37  Penicillinase-Resistant, Oral Penicillins Oxacillin, cloxacillin, and dicloxacillin ❑ Use an isoxazolyl ring as a bio-isosteric replacement for the benzene ring and a methyl on one flank and a substituted benzene ring on the other in place of the methoxyls of methicillin ❑ Like methicillin, these generally are less potent than benzylpenicillin against G+ve MO (generally staphylococci and streptococci) that do not produce a β-lactamase but retain their potency against those that do. ❑ They are somewhat more acid stable. Thus, they may be taken orally, and they are more potent as well 11/7/2024 Prof. Afaf AL Nadaf 38  OCH3 OCH3 11/7/2024 Prof. Afaf AL Nadaf 39 11/7/2024 Prof. Afaf AL Nadaf 40 ❑ Because they are highly serum protein bound, they are not good choices for treatment of septicemia. Microorganisms resistant against methicillin generally also are resistant to the isoxazolyl group of penicillins. ❑ The isoxazoyl group of penicillins is primarily used against S. aureus in osteomyelitis, septicemia, endocarditis, and CNS infections 11/7/2024 Prof. Afaf AL Nadaf 41  Penicillinase-Sensitive, Broad-Spectrum, Oral Penicillins ❑ Ampicillin and amoxicillin are very similar in structure, the only difference being an extra phenol group on amoxicillin. Amoxicillin has similar properties to ampicillin, but is better absorbed through the gut wall. ❑ The properties of ampicillin and amoxidillin are as follows: o Similar spectrum of activity to penicillin G but more active against Gram-negative cocci and enterobacteria. o Acid-resistant due to the NH2 group, and therefore orally active. o Non-toxic. o Sensitive to β-lactamases (no shield). o Inactive against Pseudomonas aeruginosa (a particularly resistant species). 11/7/2024 Prof. Afaf AL Nadaf 42 11/7/2024 Prof. Afaf AL Nadaf 43  Penicillinase-Sensitive, Broad-Spectrum, Oral Penicillins o Amoxicillin has been used in the treatment of bronchitis, pneumonia, typhoid, gonorrhoea, and urinary tract infections. Its spectrum of activity is increased when administered with clavulanic acid o They can cause diarrhoea due to poor absorption through the gut wall. All penicillins used at high doses for prolonged periods will abolish the normal bacterial flora in the intestines. This allows the colonization of resistant Gram-negative bacilli or fungi leading to intestinal problems. o The problem of poor absorption through the gut wall is due to the dipolar nature of the molecule -it has both a free amino group and a free carboxylic add function. This problem can be alleviated by using a prodrug where one of the polar groups is masked with a protecting group which can be removed metabolically once the prodrug has been absorbed 11/7/2024 Prof. Afaf AL Nadaf 44  Penicillinase-Sensitive, Broad-Spectrum, Oral Penicillins o In addition to the usual mode of penicillin allergenicity, concentrated preparations of ampicillin can self-condense to form high-molecular- weight aggregates through reaction of its primary amino group with the β- lactam bond of another molecule. These aggregates are thought to be antigenic and to be responsible for ampicillin allergenicity-a form of hypersensitivity that differs in some details from the usual penicillin allergenicity, which ampicillin also possesses o Ampicillin and amoxicillin are the penicillins most commonly associated with drug-induced rash. Avoiding use of old preparations is a somewhat effective means of dealing with this potential problem o Ampicillin is essentially equivalent to benzylpenicillin for pneumococcal, streptococcal, and meningococcal infections, and many strains of Gram- negative Salmonella ,Shigella ,Proteus mirabilis ,and Escherichia coli ,as well as many strains of Haemophilus influenzae and Neisseria gonorrhoeae ,respond well to oral treatment with ampicillin 11/7/2024 Prof. Afaf AL Nadaf 45  Penicillinase-Sensitive, Broad-Spectrum, Oral Penicillins o Bacampicillin is a weak base and is very well absorbed in the duodenum (80-98%). Enzymatic ester hydrolysis in the gut wall liberates carbon dioxide and ethanol, followed by spontaneous loss of acetaldehyde and production of ampicillin. The acetaldehyde is metabolized oxidatively by alcohol dehydrogenase to produce acetic acid, which joins the normal metabolic pool o The addition of clavulanic acid to amoxicillin (Augmentin) gives a combination in which the clavulanic acid serves to protect amoxicillin to a considerable extent against β-lactamases. This is now an extremely popular antimicrobial combination for outpatient use 11/7/2024 Prof. Afaf AL Nadaf 46 11/7/2024 Prof. Afaf AL Nadaf 47  Clavulanic acid o Clavulanic acid is a mold product with only weak intrinsic antibacterial activity, but it is an excellent irreversible inhibitor of most β-lactamases o It is believed to acylate the active site serine by mimicking the normal substrate o Hydrolysis occurs with some β-lactamases, but in many cases, subsequent reactions occur that inhibit the enzyme irreversibly. This leads to its classification as a mechanism-based inhibitor (or so-called suicide substrate) o The precise chemistry is not well understood but when clavulanic acid is added to ampicillin and amoxicillin preparations, the potency against β-lactamase-producing strains is markedly enhanced 11/7/2024 Prof. Afaf AL Nadaf 48 11/7/2024 Prof. Afaf AL Nadaf 49  Sulbactam o Another β-lactamase-disabling agent is sulbactam. o Sulbactam is prepared by partial chemical synthesis from penicillins. The oxidation of the sulfur atom to a sulfone greatly enhances the potency of sulbactam o The combination of sulbactam and ampicillin is now clinically popular o Not all β-lactamases are sensitive to the presence of clavulanic acid or to sulbactam (different mechanism of resistance) 11/7/2024 Prof. Afaf AL Nadaf 50  Penicillinase-Sensitive, Broad-Spectrum, Parenteral Penicillins o Mezlocillin and piperacillin are ampicillin derivatives in which the D-side chain amino group has been converted by chemical processes to a variety of substituted urea analogues (acylureidopenicillins) o They preserve the useful anti-Gram-positive activity of ampicillin but have higher anti-Gram-negative potency. Even some strains of P. aeruginosa are sensitive to these agents. It is speculated that the added side-chain moiety mimics a longer segment of the peptidoglycan chain than ampicillin does. It is recalled that this cell wall fragment usually is a tetrapeptide, so there certainly is room for an extension in this direction. This would give more possible attachment points to the penicillin binding proteins, and perhaps these features are responsible for their enhanced antibacterial properties o Resistance caused by β-lactamases is a prominent feature of their use, so disk testing and incorporation of additional agents (e.g., an aminoglycoside) for the treatment of severe infections is advisable. 11/7/2024 Prof. Afaf AL Nadaf 51  Penicillinase-Sensitive, Broad-Spectrum, Parenteral Penicillins o Tazobactam (β-lactamase inhibitor) is usually used with piperacillin o Carbenicillin is a benzylpenicillin analogue in which one of the methylene hydrogens of the side chain has been substituted with a carboxylic acid moiety o The introduction of the side-chain carboxyl produces enhanced anti-Gram- negative activity. o Carbenicillin is intrinsically one of the broadest spectrum penicillins. Carbenicillin is an order of magnitude less potent than the acylureidopenicillins. The drug is susceptible to β-lactamases and is acid unstable, so it must be given by injection. o Because it is a malonic acid hemiamide with a carbonyl (amide) moiety β to the carboxyl group, carbenicillin can decarboxylate readily to produce benzylpenicillin 11/7/2024 Prof. Afaf AL Nadaf 52 11/7/2024 Prof. Afaf AL Nadaf 53  Decarboxylation of carbenicillin to benzylpenicillin 11/7/2024 Prof. Afaf AL Nadaf 54  Penicillinase-Sensitive, Broad-Spectrum, Parenteral Penicillins o The large doses of carbenicillin sodium that had to be employed (multigrams per day) resulted in ingestion of a significant amount of sodium ion, which could be a consideration with heart patients. o Many of these problems were avoided by switching to the oral prodrug ester indanyl carbenicillin. This pro-drug is primarily used for oral treatment of urinary tract infections o Ticarcillin is a sulfur-based bio-isostere of carbenicillin that cannot decarboxylate as the carboxyl group of carbenicillin does o This agent is somewhat more potent against pseudomonas compared with indanyl carbenicillin o When potassium clavulanate is added to ticarcillin, the combination has enhanced antipseudomonas activity because of its enhanced stability to lactamases. 11/7/2024 Prof. Afaf AL Nadaf 55