Lead Flashcards
Key criteria for a lead series
Key criteria for a lead series
* Binding/functional potency in primary assay (IC50 < 100nM)
* Potency in secondary assay (cell proliferation GI50 <500nM)
* Meets Lipinski rules (of 5) (MW<500, cLogP<5)
* In vitro ADME liabilities (t½ >60min)
* Synthesis in less than 10 steps
* Multiple points of modification
* Patentable
Chemical Modification
Goal: Determine Structure- activity relationships:
What functional groups are important to biological activity?
Procedure: Alter or remove groups using chemical synthesis
and test the activity of the altered molecule (analog). Infer role of those groups in binding.
Bioisosteric Replacement
Substitution of atoms or groups of atoms in the parent molecule to produce compounds with broadly similar biological properties to the parent with structural diversity
* Monovalent -F, -OH, -NH2, -SH, -t-C4H9
* Bivalent -O-, -S-, -CH2-, -NH-
* Trivalent -N=, -P=, -CH=
* Hydroxyl -OH, -CH2OH, -NHCOR, -NHCN
* Carbonyl -C=O, C=C(CN)2, =CHCN
* Carboxyl -COOH, -SO 3H, -CONHOH
* Halogen -Cl, -CF3, -CN, -N(CN) 2, -C(CN) 3
* Spacer -(CH2) 3-, -C6H4-
Bioesteric Replacement Example
Resveratrol + Phenyl ring substitution = Compound 13g
-> Bioisosteric replacement increases apoptosis and leukemia cell differentiation
Absorption
H2O Solubility: Essential of intravenous administration and dissolution in GI tract
Acid/Base Properties: Affects solubility in GI Tract.
Physiological range: pKa 1.5-8
Lipophilicity: Influences ability of small molecule drugs to pass through lipid bilayer (Both in and out).
Molecular Size: Smaller=better MW<500 daltons
Absorption: Solubility x Permeability
Permeability and Absorption
- Drug absorption
– Oral administration most convenient and cost-effective Absorption takes place mostly from the small intestine
– Rate of dissolution (tablet, capsule, suspension or solution)
– Dependent on the lipophilicity and extent of ionization of the drug
– Complex with ingested food
– Passes through the portal vein and enters the liver, where it may be metabolized
Solubility and stability
- Crucial criterion for drug testing and oral absorption
- Adherence to rule-of-5 should minimize solubility problems
- Solubility is a complex phenomenon not easily modeled
– Experimentally easy to measure and routinely carried out - Stability is measured at different pH and temperatures
pH application
Orally Available Gemcitabine
-Nucleoside analog(cytosine)
-Impairs DNA replication and induces apoptosis
-Intravenous injection
-Prodrug promotes oral-mediated absorption of gemcitabine with less toxicity
GI Tract pH (Fasted)
- Stomach (1.4-2.1)
- Duodenum (2.4-6.8)
- Jejunum (6.0-7.0)
- Ileum (6.5)
Bioavailability
Bioavailability/Absorption
* Should be studied as early as possible in the development process because a lack of desired response may be due to a lack of bioavailability
(not reaching the required drug concentration)
* Compounds can be suitably modified to maximize bioavailability
Distribution
Distribution
* A drug is distributed to tissues/organs from the blood stream
* Different drug concentrations are attained in different tissues/organs
* A drug may be preferentially distributed to its target tissue/organ or not at all
Clearance
Clearance
* Drugs may be eliminated either unchanged (as the parent drug) or as metabolites depending on the lipophilicity
* Most drugs are eliminated through the kidneys which can excrete only relatively polar substances
* Thus lipophilic drugs must be metabolized into more polar metabolites for elimination
Metabolism
- Drugs are metabolized to different extent mostly in the liver
- Metabolism mostly lead to inactivation of a drug but many drugs have active metabolites
- Therefore important to study the metabolism of a drug under development in order to know the impact it may have
- First studied in liver microsomes
- CYP enzymes inhibition – Drug-drug interactions
Met phase I
Metabolism
* Phase I: Functionalization (CYP450 enzymes): Alteration of functional groups through monooxygenase rxn = loss of activity
Paclitaxel
-Tubulin targeted therapy
-Mitotic inhibitor
-FDA approved for ovarian, breast, lung, bladder, head and neck, esophagus and other cancers
-metabolites are inactive
Met Phase II
*Phase II: Conjugation
* Addition of highly polar conjugates = rapid excretion
Irinotecan
-Active metabolite (SN-38)
-Topoisomerase 1 inhibitor
-Approved for colon, lung and other cancers
-UGTs inactivate SN-38
-addition of glucuronic acid
-UGT1A1*28 polymorphism
-Causes greater toxicity
Animal-based models
- Required for efficacy and toxicity drug evaluation
- Validate in vivo biomarkers for drug efficacy
- In vivo evaluation of PK/PD in normal and disease animal models
- In vivo efficacy evaluation
- Dynamic evaluation of drug efficacy
– Histological analysis
– Tissue sample analysis (RNA, DNA, Protein)
– In vivo imaging of disease progression
Chimeric Mice
Animal-based models – Human Chimera Mice
- Transplant human hepatocytes into mice.
- Evaluate ADME in human chimera mouse – drug absorption,
distribution and hepatic metabolism and clearance
- Evaluate disease in human liver model (ie, hepatitis viral infection)
- Evaluate new drugs efficacy (ie, antiviral drugs)
HPLC-radioactivity metabolite profiles in urine after oral dosing of [3H]lamotrigine to chimeric humanized mice (A) and control mice (B).
- Chimeric humanized mice more accurately metabolise lamotrigine!
Genomic Approach
Genomic Approach
– Analyzing the entire genetic information of an organism in the context of healthy vs. disease state
– Understanding genotype-phenotype correlation to identify disease target genes
Proteonomic approach
Post-Genomic Approach - Proteomics
– Separation and characterization of proteins in an organism
– Compare protein expression in normal vs disease state to identify novel target proteins
- Analysis of gene or protein expression
– Comparing normal cells to disease cells, or normal tissues to disease tissue - Identifies potential disease-causing
genes/proteins
– Coincidental, but not necessarily causal
RNAi screens
- RNAi screens
– Specific, individual genes are “knocked down” to find genes that regulate key disease processes
– Unlike expression studies, can identify potential targets by function.
– Limited to studies in cell lines. - RNAi screen example - Cancer
– An oncogene may be identified by decreased cell growth (blue)
– A drug resistance gene my be identified by decreased growth in the presence of a drug (yellow)
– A tumor suppressor gene may be identified by increased cell growth (green)
Target Validation
- In vitro assays
– Cell-based assays to verify that your target gene/protein is involved in disease progression at the cellular level. - In vivo assays
– Animal disease models that can be used to verify that your target gene/protein contributes to disease progression in a more complex environment - Engineer cell lines
– Loss-of-function: siRNA, shRNAi
– Gain-of-function: overexpression - Allows analysis of specific role of target
HIT
A compound that interacts with the chosen target at a given concentration (usually in the micromolar range)
LEAD
A compound with drug-like properties, initial SAR and a promising IP position
Preclinical Development Candidate
NCE with optimized pharmacological and pharmacokinetic properties and a secure IP position
Rational Drug Screening
- Structure–activity relationship (SAR)
– Requires coordination of structural biology and organic chemistry
– Drug design is based on the pharmacophore of endogenous ligand/substrate
– Designed to bind active site and block receptor activation or enzyme activity - Structure–activity relationship (SAR) used to identify inhibitors of SARS-CoV-2 Nsp3’s ADRP domain
SAR Approach: BCR-ABL inhibition
(Allosteric Inhibitor)
New approaches to BCR-ABL inhibition:
-Identification of c-ABL autoinhibitory mechanism
-Myristoylation on N-term of c-ABL causes the binding of myristate moiety into deep hydrophobic pocket of kinase domain.
-This results in a 90° bending of the α-I helix of the C-term and autoinihibition
-BCR-ABL lacks N-term myristoylation site
-Allosteric inhibitors can replaces myristate
HTS
- Often relies on cell-free or cell-based assays
– Target-specific effects are measured quantitatively by a reporter assay - Ex: fluorescence, luminescence, cell shape, cell metabolism, color formation
– Drug candidates are evaluated for ability to block activity
– Formats: - 96-well and 384-well plates (high-throughput)
- 1,536-well plates (ultra high-throughput (UHTS))
Requires Large Library
HTS Assays
- Primary assay should be based on isolated target molecule (can be whole or active fragment). Examples
are binding and enzymatic activity assays (cell-free assay) - Can use cell based reporter gene assay but the disadvantage is that the drug hit may be acting either directly on target or indirectly by interfering pathway (up or downstream of target)
- It is difficult to do structure-activity relationship (SAR) without knowing the exact molecular target and mode of drug interaction
Cell Based
– To identify inhibitor/antagonist
RDD vs HTS
Rational Design
* Focused analysis of limited set of candidates
* Is not predictive of favorable pharmacological properties
* Require extensive knowledge of target
HTS
* Can identify competitive or allosteric inhibitors from 1 screen
* Requires screening many thousands of candidates
* Eliminates compounds that are unstable or cannot access cellular target
* Labor-intensive and expensive
Compound Libraries
Compound Libraries
* Common strategies in creating a compound libraries (small molecules/natural products)
– Acquisition from external vendors
– Generation from chemical library synthesis
* Random libraries
* Focused libraries
– Generation from medicinal chemistry efforts
* Targeted synthesis
* Combinatorial synthesis
- A good library should be
– Large
– Diverse
– Examples of libraries: FDA-approved drugs, Natural product libraries
– Containing only “lead-like” or “drug-like” compounds - Non-reactive
- No known toxic moieties
- Following Lipinski’s Rule-of-5
- Aqueous soluble
Rule of 5
Lipinski Rule of 5
Lipinski and his Pfizer co-workers looked over a data set of drug candidates and noticed that there were some reasonably clear cutoffs for oral absorption and general cell permeability. They suggested that you need:
1. Fewer than 5 hydrogen bond donors
2. Fewer than 10 hydrogen-bond acceptors
3. A molecular weight of less than 500 daltons
4. A partitioning coefficient (logP) of less than 5
Lipophilicity
Lipophilicity (LogP)
-The ability of a compound to partition between lipophilic organic phase (octanol) and polar aqueous phase (water)
-Often measured as Log
