DRUGS TARGETING BACTERIA

Chemistry behind immune response

Paul Ehrlich (Father of Chemotherapy)

Discovery of Penicillin

Alexander Fleming

Discovered penicillin from

Discovered penicillin from

Penicillin as Medical Treatment

Penicillin as Medical Treatment

Streptomycin discovery

First to discover antibacterial agent from, or antimicrobial agent from soil microorganisms, Streptomycin

Selman Waksman

Define Antibiotics

“... a substance produced by microorganisms, which have the capacity of inhibiting the growth and even of destroying other microorganisms.” - S. Waksman

Product of metabolism

Synthetic product produced as structural analogues of naturally occurring antibiotics

Antagonizes the growth or survival of one or more species of microorganisms

Effective in low concentrations

“... a substance produced by microorganisms, which have the capacity of inhibiting the growth and even of destroying other microorganisms.” - S. Waksman

Antibiotics

Product of metabolism

Antibiotics

Synthetic product produced as structural analogues of naturally occurring antibiotics

Antibiotics

Antagonizes the growth or survival of one or more species of microorganisms

Antibiotics

Effective in low concentrations

What sets antibiotics apart from antiseptics or disinfectants is that antibiotics are structurally specific where they target a particular molecular drug target.

So in antibiotics, even in synthetic types, it works at a very small, specific dose to kill bacteria or other microorganisms.

Antibiotics

ATTRIBUTES OF AN IDEAL ANTIBIOTIC

Selective toxicity

Chemically stable

Acceptable rate of biotransformation

COMMERCIAL PRODUCTION OF ANTIBIOTICS

Natural: fermentation

Semisynthetic

Synthetic

If we only extracted them from microbes, we'd likely face supply shortages. Still, some antibiotics are still produced that way, by

Natural: fermentation

The precursor molecule is produced by fermentation, but to convert it into a fully functional antibiotic, you need one to three steps of chemical synthesis. So the precursor is natural, but the final product involves synthesis and manipulation.

Semisynthetic

We also have fully synthetic antibiotics, but that only happens if the synthesis is easy

Synthetic

Prokaryotic Not defined Nucleus

Bacterial Cell

Not as compartmentalized as that of eukaryotic cells (Found in cytoplasm)

Simple Structures

Bacterial Cell

DNA replication and transcription happen in the

nucleus

Only translation happens at the

ribosome

mRNA undergoes what before leaving the nucleus

post-transcriptional modification (capping, poly-A tail, splicing)

Then translation occurs at the

rough ER.

post-transcriptional modification for folding and modification (adding fats or carbohydrates) (post-translational modifications) before being released by the cell OCCURS IN

Golgi apparatus

replication, transcription, and translation can happen simultaneously in what type of cell

bacterial cell

have no defense mechanisms when it comes to mutations in their DNA.

They dont have DNA proofreading, repair mechanisms kaya sila very resilient

bacterial cell

With cell membrane and cell wall

Bacterial cell

synthesizes essential vitamins

Bacterial cell

50s and 30s (70s)

This could be used as a specific drug target that allows us to not affect 60s and 40s

Bacteria

60s and 40s (80s)

humans

proposes that eukaryotic cells evolved when large prokaryotic cells engulfed smaller, energy-producing prokaryotic cells (like aerobic bacteria) rather than digesting them. These engulfed cells became endosymbionts, eventually evolving into organelles like mitochondria and chloroplasts, providing the host cell with ATP in a symbiotic relationship.

The Endosymbiotic Theory

Ribosomes, Dna similar to bacteria. Endosymbiotic Theory

Kaya yun pinanghahawakan na pwedeng ganun yung nature ng evolution ng eukaryotics from prokaryotic. And so if that is the case, those antibiotics affecting human and bacteria ribosomes, baka kung minalas, pwede niya maapektuhan mitochondrial makeup

presence of a lipopolysaccharide layer and thin peptidoglycan

are also much more resistant to other types of antibiotics.

Gram negative

act as competitive inhibitors of the enzyme dihydropteroate synthase, which bacteria use to synthesize folic acid from para-aminobenzoic acid (PABA). Because these drugs are structural analogs of PABA, they "trick" the enzyme into binding with them instead, effectively halting the production of folic acid. Since folic acid is a vital cofactor for converting dUMP to dTMP, its absence leaves the bacteria without enough thymine to replicate their DNA, leading to inhibited growth and DNA strand breaks. Because humans absorb folic acid from food rather than synthesizing it themselves, this pathway is a selective target that leaves human cells unharmed.

SULFONAMIDES

is a competitive inhibitor of the enzyme dihydrofolate reductase (DHFR), which is responsible for converting dihydrofolate (DHF) into tetrahydrofolate (THF). By blocking this specific step, the drug prevents the formation of the active form of folic acid needed for the synthesis of thymidine. B

ecause this occurs right after the step blocked by sulfonamides, the two drugs are often combined (e.g., Co-trimoxazole) to create a sequential blockade that is much more powerful than either drug alone.

a trait it shares with the chemotherapy drug Methotrexate.

Trimethoprim

Problem with Trimethoprim

While humans also possess DHFR, Trimethoprim has a significantly higher affinity for the bacterial version of the enzyme, though it can still occasionally impact human cells

Explain this

The process shown is Thymidylate Synthesis. First, Me-THF (Methyl-tetrahydrofolate) acts as a methyl donor; it transfers its methyl group to dUMP (deoxyuridine monophosphate) via the enzyme thymidylate synthase. This reaction converts dUMP into dTMP (deoxythymidine monophosphate) and leaves behind DHF (dihydrofolate), which must then be recycled back to THF to keep the cycle moving. The dTMP is subsequently phosphorylated twice more to become dTTP (not ATP), which is the specific "T" nucleotide used as a building block for DNA replication.

is only used to pertain to the topoisomerase II in bacterial cells

DNA gyrase

Topoisomerase or DNA gyrase job

remove the supercoiling that comes as a result of the uncoiling done by DNA helicase

T/F In the nomenclature of these enzymes, bacteria use Roman numerals while in eukaryotes, it's Roman letters.

F: bacteria use Roman letters while in eukaryotes, it's Roman numerals.

inhibit DNA gyrase, allowing for the DNA to break from increased tension.

inhibit bacterial DNA synthesis by targeting two key enzymes: DNA gyrase (Topoisomerase II) and Topoisomerase IV. During replication, DNA helicase unwinds the double helix, which creates intense "supercoiling" or physical tension further down the strand. DNA gyrase normally travels ahead of the replication fork to cut, rotate, and reseal the DNA to relieve this stress. These drugs bind to these enzyme-DNA complexes, locking them in place and preventing the DNA from being resealed. This results in permanent double-stranded breaks and massive tension that causes the DNA to snap, leading to rapid bacterial cell death.

Quinolones

Inhibits RNA polymerase, which is responsible for the transcription process of DNA to RNA.

inhibits bacterial protein synthesis at the very first step by targeting DNA-dependent RNA polymerase. It binds specifically to the beta subunit of this enzyme, preventing it from transcribing DNA into messenger RNA (mRNA). Without mRNA, the "instructions" for building proteins never reach the cell's machinery, effectively starving the bacteria of the enzymes and structural components they need to survive. Because Rifampicin targets the bacterial version of RNA polymerase and not the human version, it is highly effective at stopping bacterial growth while leaving our transcription process alone.

Rifampicin

Yung image sa baba

Targets the association of the large and small ribosomal subunits, inhibiting them from joining together and inhibiting the translation process.

Linezolid is an example of this drug class.

are unique protein synthesis inhibitors that act at the very beginning of the translation process. They bind to the P site of the 50S ribosomal subunit, which prevents the formation of the 70S initiation complex. By blocking the association of the large (50S) and small (30S) subunits, the drug ensures that the "machinery" never gets put together to read the mRNA. Because this mechanism is different from other antibiotics like macrolides or tetracyclines, there is often less cross-resistance with this class.

OXAZOLIDINONES

These bind to the large ribosomal subunit after it has merged with the small subunit.

inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit. Unlike Oxazolidinones, which prevent the "burger bun" from closing, these drugs act after the ribosome has already assembled. They specifically block translocation, the step where the ribosome slides down the mRNA strand to add the next amino acid to the growing peptide chain. By physically "plugging" the exit tunnel or interfering with the movement of tRNA, they cause the assembly line to grind to a halt, preventing the bacteria from producing functional proteins.

MACROLIDES AND LINCOSAMIDES

Macrolides (like Erythromycin) and Lincosamides (like Clindamycin)

Similar to macrolides and lincosamides, but they bind to the small ribosomal subunit instead.

both inhibit protein synthesis by binding to the 30S ribosomal subunit (the "small" half of the ribosome).

AMINOGLYCOSIDES AND TETRACYCLINES

Aminoglycosides (e.g., Gentamicin) and Tetracyclines (e.g., Doxycycline)

act as bacteriostatic agents by blocking the attachment of aminoacyl-tRNA to the A-site, effectively stopping the addition of new amino acids.

TETRACYCLINES

are often bactericidal because they cause the ribosome to misread the mRNA code; this leads to the production of misprioritized or "junk" proteins that insert into the bacterial cell membrane, causing it to leak and the cell to die. While they can work on many bacteria, their biggest hurdle is penetrating the complex, lipophilic outer membrane of Gram-negative organisms.

Aminoglycosides

are so lipophilic; this is why, even though these agents can work on both types of bacteria, there is a larger amount of rejection when used on these types of bacteria. All it needs is to find a way to somehow penetrate the cell membrane

Gram negative bacteria

Target protein involved in translocating NAM and NAG

Remember that NAM and NAG are synthesized in the cytoplasm and then brought out and form the bacterial cell wall.

This drug does not allow the transportation of these NAM and NAG chains from the cytoplasm and through the cell membrane, thus inhibiting the synthesis of the cell wall.

NAG is N-acetylglucosamine, while NAM is N-acetylmuramic acid

inhibits bacterial cell wall synthesis by interfering with the recycling of bactoprenol, the lipid carrier molecule. Bacterial cell wall building blocks (NAM and NAG) are synthesized inside the cytoplasm but must be transported across the inner membrane to the exterior to form the peptidoglycan layer. THis drug binds to the pyrophosphate on bactoprenol, preventing it from dephosphorylating and returning to the inner side of the membrane to pick up another precursor. By blocking this "shuttle" system, the cell runs out of materials to build its protective outer wall, leading to cell lysis.

BACITRACIN

The beta-lactams inhibit the cross-linking of these NAM and NAG chains.

While these are generally not as effective in Gram (-) bacteria, the later generations of the other drug we see that they also have some effectiveness against gram negative bacteria.

(collectively known as Beta-lactams) inhibit the final stage of bacterial cell wall synthesis. They act by binding to and inhibiting (PBPs), specifically the transpeptidase enzyme. This enzyme is responsible for "cross-linking" the peptidoglycan chains (the NAM and NAG strands). Without these cross-links, the cell wall loses its structural integrity and cannot withstand the high osmotic pressure inside the bacteria. This causes the cell to swell and eventually burst, a process known as osmotic lysis.

PENICILLINS AND CEPHALOSPORINS

These agents target the outer wall of the bacteria, specifically in the D-Ala-D-Ala terminus. This causes steric hindrance and weakens the cell wall.

There are also glycopeptides that are non-ribosomal peptides, meaning that they are not formed in the ribosomal pathway. They are short and cyclic, and because of that, they have a lipophilic head and hydrophilic tail, which allows them to penetrate the cell membrane.

inhibit bacterial cell wall synthesis through a high-affinity binding to the D-Ala-D-Ala terminus of the peptidoglycan precursor. Unlike Beta-lactams that target the enzyme, Glycopeptides target the substrate itself. By capping the D-Ala-D-Ala end, they create "steric hindrance"—physically blocking the transpeptidase enzyme from accessing the site to create cross-links. This results in a weak, unstable cell wall that leads to bacterial death. Because these molecules are very large and bulky, they generally cannot pass through the outer membrane of Gram-negative bacteria, making them primarily effective against Gram-positive organisms like MRSA.

GLYCOPEPTIDES

Vancomycin etc.

are non-ribosomal antibiotics composed of a lipid (fatty acid) linked to a short peptide chain that binds to bacterial cell membranes and increases permeability, leading to cell death.

function by directly disrupting the bacterial cell membrane rather than inhibiting a specific metabolic enzyme or the cell wall. They consist of a lipid tail that inserts itself into the cytoplasmic membrane in a calcium-dependent manner. This insertion causes rapid depolarization of the membrane, leading to a massive efflux of intracellular ions (especially potassium). This loss of membrane potential halts all synthetic processes (DNA, RNA, and protein synthesis) and triggers cell death. Because they work on the membrane itself, they are effectively bactericidal even against resting or slow-growing bacteria that might resist other antibiotics.

Lipopeptides

Daptomycin

are cyclic polypeptide antibiotics that bind to lipopolysaccharides (LPS) in the outer membrane of Gram (-) bacteria, disrupting membrane integrity and causing leakage and cell death.

(CELL WALL)

are "detergent-like" antibiotics that specifically target Gram-negative bacteria. They work by binding to lipopolysaccharides (LPS) and phospholipids in the outer bacterial membrane. The drug's cationic (positively charged) peptide portion displaces calcium and magnesium ions that normally stabilize the LPS molecules. This insertion physically disrupts the membrane's integrity, creating holes that increase permeability. The resulting leakage of intracellular contents leads to rapid cell death. Because they target the outer membrane, they are often a "last resort" for multi-drug resistant Gram-negative infections.

POLYMYXINS

Reasons for MICROBIAL RESISTANCE

Impermeability

Modification

Pumping out

Inactivation

MICROBIAL RESISTANCE

Modified cell wall protein

Impermeability

MICROBIAL RESISTANCE

Modified drug target

Modification

MICROBIAL RESISTANCE

Increasing active efflux of the drugs

Pumping out

MICROBIAL RESISTANCE

Add a phosphate group on the antibiotic, which will reduce its ability to bind to the bacterial ribosomes

Inactivation

In Gram (-) bacteria,beta-lactamases can be overexpressed and hydrolyze the beta-lactam ring of what drug

penicillin in the periplasmic space.

To counter this, some drugs inhibit beta-lactamases instead of acting as antibacterials. An example is

clavulanic acid

Explain SYNTHESIS OF CELL WALL

Peptidoglycan monomeric unit

Transglycosylation

Crosslinking by transpeptidase (catalyzed by the enzyme transpeptidase)

cell wall synthesis

The precursor disaccharide units are formed in the cytoplasm. Peptides are attached, and several steps follow before the structure is transported outside. The nascent peptidoglycan chain refers to the newly formed chain.

Peptidoglycan monomeric unit

cell wall synthesis

After translocation to the periplasmic space, the disaccharide units are linked to the existing peptidoglycan chain through WHAT STEP. This extends the structure of the cell wall.

Transglycosylation

Cell wall synthesis

For the cell wall to become strong, THE WHAT is required. This process is catalyzed by the enzyme transpeptidase, also known as penicillin-binding protein. It links the D-alanine–D-alanine residues. The presence of D-amino acids is unique to bacteria and contributes to selectivity.

Crosslinking by transpeptidase

DRUGS ACTING ON CELL WALL SYNTHESIS

Penicillins

Cephalosphorins

Β-lactams

Vancomycin

Carbapenems

Cycloserine

Bacitracin

Nonribosomal peptides - produced ng other microbes

Cycloserine, Bacitracin, Vancomycin

It’s a transpeptidase inhibitor; it particularly inhibits the peptide linkage between the peptidoglycan monomers. The pictures below show the formation of the peptidoglycan monomer until its maturation in the layer.

This drug target the cross-linking (transpeptidase) - D-Alanyl-D-Alanine

PENICILLINS

We can see the resemblance of the beta-lactam ring to the D-Ala transition state (D-Ala is in the middle of breaking and forming new bonds) and drugs that look like the transition state have higher affinity towards the enzyme because they resemble the active conformation of the enzyme, meaning less energy is needed to change into the active form. Therefore, there is higher affinity of beta-lactams compared to the D-alanyl-D-alanine peptide.

Consequence: high affinity in exchange for stability.

Beta lactams kinemerut mej MOA

This cross-linking involves three major steps:

First step

Removal of D-Ala - the one at the C-terminus will be removed.

The main substrate is WHAT, which is a terminal peptide of the donor peptides in cross-linking

D-Alanyl-D-Alanine

which indicates that this particular enzyme only accepts peptide bonds belonging to both D amino acids

DD-TPase = DD-transpeptidase

Explain penicillin’s selectivity towards bacterial transpeptidases.

Implication: In terms of selectivity, in terms of ease of designing drugs against bacteria more than in human protein targets, the transpeptidase, although we also have transpeptidases in our body, there’s no chance that penicillins will attack them because our transpeptidase only accommodates L-amino acids.

The bacteria's cell wall is built using D-amino acids (specifically the D-Ala-D-Ala terminus). The enzyme responsible for cross-linking these building blocks is DD-transpeptidase. The "DD" prefix is critical; it signifies that the enzyme's active site is stereospecifically designed to only recognize and bind to D-configuration peptide bonds. While humans do have transpeptidases, our enzymes are evolved to process L-amino acids (the standard building blocks of human proteins). Because Penicillins are designed to mimic the D-Ala-D-Ala transition state, they fit perfectly into the bacterial "DD" enzyme but are physically ignored by human "LL" enzymes. This stereochemical mismatch provides a natural safety barrier, ensuring the drug only targets the pathogen.

This cross-linking involves three major steps:

Second step

Peptide linking of D-Ala (that was left, which is the new C-terminus) to the meso-Dap.

This cross-linking involves three major steps:

Second step

The remaining D-Ala binds to the enzyme when

the D-alanine residue is removed in the first

step. This is now ready to link with the

meso-Diaminopimelic acid (meso-DAP).

Acceptor peptide; already attached to the existing peptidoglycan layer. The one to cross-link with newly made peptidoglycan units.

meso-DAP

The donor peptide to be attached; currently bound to the enzyme.

D-alanine

What happens when there is a formation of peptide bond

Once there is formation of a peptide bond (condensation reaction) between the carbonyl carbon and the amino residue of the meso-DAP, when the peptide linkage is formed, it will eventually return to the peptide form itsel

This cross-linking involves three major steps:

3rd step

(3) Crosslinked D-Ala-meso-Dap

■ It looks like it’s still in the transition state but eventually it will adapt to a form such that it will be an ordinary straight chain like other peptide bonds. It will be liberated from the enzyme binding site.

Penicillin
MECHANISM: COMPETITIVE INHIBITION

mimic the transition state, which is the active state—in the middle of bond formation and bond breaking.

Once the penicillin binds at the same site as the peptide linkage, and due to steric hindrance, the bond formed between the penicillin and the seryl residue of the penicillin-binding protein cannot be hydrolyzed. Due to this, the substrate (D-Ala-D-Ala) cannot bind, keeping it away from the active site.

Penicillin
MECHANISM: UMBRELLA EFFECT

Non-competitive, allosteric, but because of the size of the molecule, it blocks the substrate from binding to the active site. ● Instead of the seryl residue found directly at the active site, it binds to the seryl residue in the allosteric site.

INTRINSIC RESISTANCE OF GRAM (-) BACTERIA against Penicillins

Gram (-) bacteria are intrinsically resistant against penicillins, because, unlike Gram (+) bacteria, Gram (-) bacteria, to begin with, are already producing beta-lactamases, and the more they are exposed to penicillins, the production of beta-lactamases is promoted.

Another reason for the intrinsic resistance of Gram (-) bacteria is that their porin channels do not allow the entry of large, hydrophobic, and negatively charged molecules. ○ Penicillins are acidic (free carboxylate form) and are relatively large. So it can’t just enter the porin channels.

RESISTANCE TO PENICILLINS: β-LACTAMASE ENZYMES

Beta-lactamases target the hydrolysis of beta-lactam rings. Once it opens, it will no longer bind to the penicillin binding protein.

The penicillins we know today, are actually, more likely (especially benzylpenicillins and other penicillins), produced semi-synthetically and are synthesized from

6-aminopenicillanic acid.

The fermentation product of molds that produce penicillins.

Much more abundant than penicillin itself.

More stable so it is isolated easily.

6-aminopenicillanic acid

Resitance to Penicillins

Production of high levels of transpeptidase

Change in affinity of the transpeptidase to penicillin

Efflux Mechanism

Mutations and genetic transfers

Bacterial tolerance

Resitance to Penicillins

Inherent to Gram-negative bacteria

Production of high levels of transpeptidase

Resitance to Penicillins

relative proportions varies across bacterial species

variable susceptibility to different penicillins

Change in affinity of the transpeptidase to penicillin

Resitance to Penicillins

Over-expression of P-glycoprotein or ATP-binding cassette proteins. ■ Pronounced in Gram-negative bacteria.

Efflux Mechanism

Resitance to Penicillins

Plasmids: genetic carrier of resistance genes

“Memory”

Mutations and genetic transfers

Resitance to Penicillins

Bactericidal to bacteriostatic

Bacterial tolerance

ALLERGY TO PENICILLINS

It's better to know if you are allergic to penicillin.

They resemble the structure of peptides; these penicillins are amino acid derivative products. The enzyme involved in their synthesis is that they are produced by non-ribosomal peptide synthetases.

Because of their resemblance, there is a higher chance that they can be immunogenic.

Determinants of allergic reaction

Explain Allergy to Penicillins

These are just some of the examples of the formation of an allergy towards penicillins:

○ In the form of penicillin itself.

○ Penicilloic acid (hydrolyzed form)

○ Benzylpenicilloic acid

It can form adducts with endogenous proteins (they resemble peptides); the body will consider it foreign, leading to an immune response. In certain cases, there can be formation of penicilloyl polylysine and penicilloyl octa-L-lysine. These are the determined (so far) forms of penicillin that cause allergic reactions.

The immunogenicity of Penicillins stems from their nature as dipeptide mimics. Because they are synthesized by non-ribosomal peptide synthetases in fungi, they possess a structure that closely resembles human peptide bonds.

The actual allergic trigger is rarely the "free" drug. Instead, it’s the formation of hapten-protein adducts. Penicillin (or its hydrolyzed form, penicilloic acid) is a highly reactive electrophile. It reacts with the amino groups (like lysine residues) of your own endogenous proteins (self-proteins). This "labels" your protein with a penicillin molecule. The immune system no longer recognizes the protein as "self" and attacks the penicilloyl-protein complex. Specific diagnostic markers for this include penicilloyl polylysine, which are used in skin testing to confirm a Type I hypersensitivity.

Penicilln SAR: Beta Lactam Ring and Carboxylate Residue are important for

binding

If this is modified (e.g beta-lactam ring is opened), it will not bind to transpeptidase or penicillin-binding proteins.

Carboxylate residue binds to the lysine residue of the target enzyme.

whole beta lactam ring including thiazole structure

The whole beta-lactam ring which includes thiazole introduces greater strain on the ring alone. This is the reason why it is unstable.

Acyl connected amino (which is also attached to the beta-lactam ring:

this is the part that can be modified”:

Acyl connected amino (which is also attached to the beta-lactam ring ATTACHED WITH BULKY GROUP

more resistant to beta lactamases

If bulky groups are attached in this portion → increase tendency to be effective against gram-negative bacteria

Acyl connected amino (which is also attached to the beta-lactam ring ATTACHED WITH electron-withdrawing group

(EWG)

If changed to an electron-withdrawing group (EWG) → lower tendency to act as nucleophile
Acting as a nucleophile has added benefits for the molecule since it has added binding

T/F sulfur is has the most important role (based on SAR studies)

F. All penicillins have sulfur but have no important role (based on SAR studies).

If this will be changed with an atom that has almost similar electronic properties or if changed to Carbon

not affect the activity of Penicillins

Aminobenzylpenicillin (Ampicillin)

Phenoxymethylpenicillin (Penicillin V)

D-α-amino-p-benzylpenicillin (Amoxicillin)

ACID-RESISTANT PENICILLINS

Bulky groups attached to acyl residue attached to amide (also attached to beta-lactam ring).