Medchem Final

Bagel

Molecular Pathology

the study of the molecular basis of disease

‣ Clinical data, genome-wide association study (GWAS)

‣ Cellular models (knockout, transcriptomics / proteomics)

‣ Animal models (knockout, transcriptomics / proteomics)

‣ Pharmacological hypothesis testing with tool compound

Viral RNA encodes for …

One long polyprotein. It has PLpro and MPro chop it up and make it functional.

PCSK9

It is a protease that binds to LDL-Receptors and takes em to lysosome for degredation. (We want less PCSK9 because more receptors means less LDL is the bloodstream)

Assay

are brief, quantitative tests of a biological hypothesis

‣ Does my enzyme transform its substrate with compound X?

‣ Does my receptor signal with compound X?

‣ Does my target bind its partner in the presence of compound X?

‣ Does my compound enter the cell?

Biophysical Assay and Biochemical Assay

Biophysical:

‣ Binding (calorimetry), structure (NMR, X-ray)

Biochemical:

‣ Enzymatic activity, receptor activity, channel conductance

Cell based Assay and Animal Assay

Cell based:

‣ Cytotoxicity, reporter gene, phenotypic

Animal:
‣ Behavior (learning, memory, pain), organ-level, toxicity

Considering factors for the type of assay to use for screening

difficulty

physiological relevance

cost

throughput

Fluorescence concept

‣ High-energy photon is absorbed

‣ Low-energy photon is emitted

‣ Engineer a fluorescence-based sensor of activity

‣ Examples = fluorescent dyes, fluorescent proteins

‣ Potential interferences = fluorescent compounds, quenchers

Chemiluminescence concept

‣ Enzyme (luciferase = luc) transforms substrate (luciferin)

‣ Enzymatic transformation results in light emission

‣ ATP + O2 required, almost no background

‣ Potential interferences = luciferase inhibitors

Normal distribution standard deviation rule

‣ 68-95-99.7 rule

ex) 68% of the data is within 1σ of the µ

Z’ Factor

Z’ = 1 is ideal assay

Z’ = 05 is acceptable assay for screening

Want sharp peaks far apart

Hit Validation steps

‣ Step 1 = synthesize / procure the compound

‣ Step 2 = validate in the primary assay (=confirmation)

‣ Step 3 = dose-response (IC50) in primary assay (=conforming behavior)

‣ Step 4 = secondary assay (orthogonal to primary, biochemical assay or cellular assay)

‣ Step 5 = evaluate common toxicity / nuisance behavior

‣ Step 6 = structure determination (identify functional grps for binding)

PAINS

Parmaceutic Phase

‣ Administration → distribution

‣ Oral = mouth → stomach → gut

Pharmacokinetic Phase

‣ Distribution → target

‣ Oral admin = gut → liver → target

‣ Metabolism in liver

‣ Transiting cell membranes

‣ Excretion

Pharmacodynamic Phase

‣ Drug acting on target

ADME

A = ABSORPTION

‣ Uptake of drug into circulation (blood)

‣ Usually by mouth or lung

D = DISTRIBUTION

‣ Movement of drug around body

‣ Points of accumulation

M = METABOLISM

‣ Transformation of drug in body

‣ Enzyme action (mostly in liver + stomach)

‣ Usually inactivates, increases polarity for…

E = EXCRETION

‣ Removal of drug from body

‣ Urine (kidneys)

‣ Feces (liver → gut)

Oral Administration Path

1. Mouth

‣ Most drugs are swallowed

‣ Some chewed (absorption)

‣ Enzymes (amylase, lipase)

2. Stomach

‣ H+ (pH 1.5–3 = v acidic!)

‣ Enzymes (peptidases)

3. Gut

4. Liver

‣ More enzymes (MANY!)

‣ Absorption through gut wall

‣ MORE ENZYMES!

‣ phase I metabolism (cytochrome P450)

‣ phase II metabolism (transferases)

‣ Leads to distribution

Bioavailability

the fraction of drug making it to circulation

Intravenous injection by definition is 100% available

logP

Partition coefficient, P

‣ Observe fraction of drug in n-octanol compared to water

(n-Octanol = good approximation of fatty tissues / membranes)

P= [drug] in octanol/ [drug] in water

Higher LogP = more greasy

Lipinski’s Rule of 5

‣ MW < 500 (not very big)

‣ LogP < 5 (not too greasy, but also not too polar)

‣ HBA < 10 (not too much H bonding)

‣ HBD < 5 (not too much H bonding)

Veber Rules

ROTATABLE BONDS (Nrot) ≤ 10

PERMEATION COEFFICIENT = Pe > 10-6 cm/s

• or POLAR SURFACE AREA ≤ 140 Å

Liver metabolism

‣ phase I metabolism (cytochrome P450)

‣ phase II metabolism (transferases)

Polar molecules in the blood will be…

excreted by kidneys by dialysis between the renal vein and nephron. Nephron takes polar stuff for piss and Renal vein takes nonpolar stuff for blood.

Phase 1 metabolism

Oxidation, Reduction, Hydrolysis

• Cytochrome p450 (Terminal Aliphatic, oxidize the most exposed)

  • Benzylic C-H favored

  • Also demethylation of heteroatoms

• Alcohol Dehydrogenase (turns into aldehyde)

• Aldehyde dehydrogenase (turns into acid)

• Flavin Monooxygenases (heteroatom oxidizers)

• Hydrolase activity (proteases and esterases)

Flavin Monooxygenases

• S oxidations

  • Add =O onto S for R-S-R or (R)₂-S=O

  • Add -O^- onto S atom when thiocarbonyl

  • Add both =O and -O^- onto S when -SH

• N oxidations

  • Add -O^- onto tertiary amine

  • Add -OH onto secondary amine

  • Add -O^- onto primary amine bonded to N (N-NH2)

Phase 2 metabolism

Conjugation reactions

• Glucaronidation (catalysed by UGT or UDP-glucoronosyltransferase).

• Glutathione (GSH) conjugation (catalysed by GST or Glutathione-S-transferase)

  • Other enzymes (GGT and CCBL) will cleave GSH to just Cystine

  • Then N-Acyl Transferase will make the acylate the amine

Glucaronidation substrates

Glutathione Conjugation substrates

• Michael acceptors

• Alkyl Halides

• Epoxides

Drug Elimination

the rate at which drug is removed from the body

What order chemical reaction is the body and what is half life of it

1st order chem rxn

t1/2= ln2/k, (k is first order rate constant)

Theraputic level

above the EC50 of the drug

Toxic level

defines onset of adverse effects

Theraputic window

the range between theraputic level and toxic level

Steady-state drug concentration

Usually achieved after about 7 half lives

Alcohol analogues

ether, ester or just H

Can you rotate the C-N bond in amide?

No, electrons on N delocalize

Acid analogues

Methyl ester, primary alcohol, primary amide, methyl ketone

Heteroaryls are dangerous because

they can undergo SNAR reactions (nucleophilic aromatic substitution)

Isostere

same shape or bulk

Isosteres for methyl

amine, alcohol, thiol, fluorine, chlorine

Isostere for isopropyl

Bromine

Isostere for butyl

Iodine

Isostere for proton

deuterium, fluorine

Bivalent Carbonyl Connector Isosteres

ketone, amide, ester, thioester

Bivalent one atom connector isosteres

methylene, amine, ether, thioether

Ring isosteres

Benzene and thiophene are very interchangable

Others are pyridine, pyrrole, furan and cyclopentadiene

Exploring Hydrophobic pocket

cyclopropyl is especially interesting bc it investigates with minimal increase in logP and no Nrot

Simplifying Molecules for production

• Eliminate unnecessary chiral centers (ease synthesis / scale-up)

• Enantiomers are difficult to separate and must be tested for activity

  • Can do this by introducing symmetry

Why no floppy

Binding free energy depends on entropy, so more entropy means its not gonna wanna just snap into place

• can fix with cyclization

Morpholine

the goat

• aliphatic amine with pKa= 7.4

• No exposed C-H for metabolism

• Polar oxygen but not nucleophilic

Desolvation

There is an energetic penalty for having to remove H2Os from enzyme or ligand for binding

Bioisosteres

bioactive functional group replacements

Bioisostere of COOH

tetrazole, same pKa but much better cLogP

Amide bioisostere

pyrrole ring, thiazole, triazole, oxadiazole

Oligonucleotide drugs

For gene silencing

• go in a ds-oligo and form RNA-Induced Silencing Complex (RISC) which bind to complementary mRNA and cleave it. Leaving it for degredation

Will need to modify phosphate backbone and sugars for solubility and selectivity

Gram positive and Gram negative bacteria

+ Bacteria that have a thick cell wall

- Bacteria have thin cell wall

Bacterial Cell wall Biosynthesis

NAG and NAM sugars bound to L-Ala + D-Glu (where the chain continues from Glu side chain) + L-Lys + D-Ala and crosslinks from there to the Lys behind.

Origionally it is a D-Ala + D-Ala that transpeptidaese acts on to crosslink em

Beta-Lactam Antibacterials

4 membered ring with amide in it is a beta lactam ring

• these inhibit cell wall biosynthesis by forming a covalent link to transpeptidase

Beta lactamases

inactivate beta lactam by cleaving the amide bond in the beta lactam ring

Aminoglycosides

Protein synthesis inhibitors for bacteria

• They have alot of -OH and positively charged functional groups

• They bind to the 30S/mRNA complex near the decoding site

• Block translocation

Tetracycline

Protein synthesis inhibitors for bacteria

• They have a functionally rich and a functionally poor side

• They bind to the decoding site and inhibit tRNA binding

• Binds exclusively to backbone and nucleobases of rRNA

Fluoroquinolones

They stabilize topoisomerase which pause DNA in the ds break form

Quinolone group

Its a benzene ring attatched to a pyridine with a carbonyl

Viral Life Cycle (Influenza)

1. Adsorption

2. Fusion & Entry

3. vRNA Replication

4. Virion Assembly

5. Budding & Release

Viral Life Cycle (HIV)

1. Adsorption & Fusion

2. Reverse Transcription

3. Integration

4. Transcription

5. Polyprotein Synthesis & Processing

6. Virion Assembly, Budding, & Release

Nucleoside Reverse Transcriptase Inhibitors

They will mimic a nucleoside but will have chain terminating groups like an azide or sulfur instead of a 3’ OH

Non-Nucleoside Reverse Transcriptase Inhibitors

Allosteric binders to reverse transcriptase

• they are non-competitive binders as a result of allosteric binding

Viral protease inhibitors

They make it so that the polyprotein remains inactive

Lenacapavir

Accelerates the capsid assembly and will yield malformed capsids that will clump everything together making even drug resistant capsids stuck using the druggable capsids

Cell Cycle

Checkpoints in cell cycle

Foot on the Accelerator

1. Epidermal Growth Factor will interact with its receptor (which is an RTK)

2. EGFR will autophosphorylate

3. Grb2 and SOS will interact with the phosphorylated EGFR and RAS which is bound to Grb2 and SOS will release a GDP

4. GTP will bind to Ras and activate Raf

5. Cascade down to activate Cyclin D and CDK4/6

6. This activates RB by phosphorylating it and releases the E2F which tells cell cycle to go

Foot on brakes

DNA Damage

• will activate p53, then activate p21, which will inhibit Cyclin D

Transforming Growth Factor

1. Will bind to TGF beta receptor

2. SMAPS activate p15, p27, and p21

3. These inhibit CDK4/6

Foot on cell’s neck (cell death aka apoptosis)

Immunologic T cells

1. Inject Granzyme

2. increase permiability of mitochondria

3. Cytochrome C will come out of mitochondria, activating Casp 9

4. Casp9 activates Casp 3 and Casp 7.

5. Casp 3 and Casp 7 will lead to Apoptosis

DNA Damage

1. p53 activates

2. BAX is activated by p53 and will increase permiability of mitochondria

3. …

Tumor Necrosis Factor

1. Will interact with its recepor (a trimer)

2. Activate scaffolding proteins that will activate Casp9

p53

aka Guardian of the Genome

EGFR inhibitors

• will stop Ras from being activated and the cascade that leads to cell replication

• cancer can counter by just upregulating EGFR

• This is hard because Kinases are all very similar, so this can be doing bad things to yo body

Paclitaxel

Binds to tubulin and stabilizes it (freezing it)

• WIll induce mitotic arrest and apoptosis

Quasispecies

Host cells are infected with multiple viral genomes

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs)

‣ Design antibodies to bind overexpressed receptors on cancer

‣ Antibody binding flags cell for immunological disposition

‣ Conjugate cytotoxin to mAb = ADC; silver bullet to cancer cells

What are the liabilities and optimizations of peptide-derived drugs

Liabilities:

• human proteases potentially cleave the drug

• lots of HBA, HBD and high MW

Optimizations:

• steric shields, isosteres

• removing chirality