Drug Discovery Exam 3

Library based drug discovery

best to pick the best compund based on acutal data than try to predetermine which compounds might be best

• let assays tell u which compound is best instead

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Peptide chemistry

Automated Peptide synthesis served as start of library-based discovery

Greater number of operations required to synthesize, the less efficient it is.

Average 70% yield per step means overall 0.5% yield for 15 step process.

How do you lose product

Extraction, filtration, purification

How to minimize losses in peptide chemistry?

Synthesize normally but use resin beads made of polystyrene as a protecting group. Once it’s attached to bead, can filter/separate from other compounds.

Advantages of attaching compound to solid support (poly bead)

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Advances in peptide synthesis

Coupling reagents, protecting groups, resins, …

Library

Collection of certain types of compounds. Peptides, indoles,

Pooled Synthesis

Synthesizing a bunch of compounds at once. Different combos of different steps to make all your compounds. Splitting and regrouping called split pool.

Combinatorics

Encoding strategies, pooling strategies.

Encoding - assigning DNA to certain compounds to identify later

RF Tags

Separate compounds out of pools and identify

Using a computer to track what compound will be made on each rf tag, an instrument cant tell you what flask each resin goes in to each step

Solid phase synthesis drawback

Compounds are typically cleaved with triflouroacetic acid, which can lead to some decomposition of product. inefficient removal of TFA leads to limited shelf life of the compounds prepared in this manner.

Alternative methods for performing solid phase organic synthesis

Fluorous technology - fluorinate compound, synthesize, then remove fluorine and separate out.

Parallel organic synthesis - not combinatorial.

Solution-phase synthesis

Immobilized reagents, immobilized scavengers (resin reacts with byproduct/excess to so you can filter it out), tea bags (resin).

But can’t be pooled. Greater shelf-life cause no TFA, cleavage needed.

Convergent synthesis.

Convergent Synthesis

Can decrease total step by reducing linear sequence, so higher yield.

Late-stage diversification

introduce part that you’re changing as late in the synthesis are possible

Molecular diversity

Late stage diversification and convergent syntheis or parallel organic synthesis

Advantages of analog availability

Removes synthesis as limit to progress

more likely that activity is not a fluke

rapid SAR access

Suzuki reaction

boronic acid/ester with aryl halide with base and pd catalyst leads to carbon carbon bond.

Doesn’t add to PSA, since it doesn’t necessarily add a H bond donor or acceptor. Thousands of available boronic acids/esters and aryl halides. Don’t need protecting groups like other reactions. Works on solid phase.

Buchwald-Hartwig Reaction

Amine or alcohol to compound

Alkyl/aryl halide with amine to couple amine to compound. Tolerant of many compound types, don’t need protecting groups. Works on solid-phase.

How to speed up coupling reactions

Automated chromatography, high-pressure, doing it in the assays,.

Automation for LC/MS NMR

Lead optimization trends/modifications

First - Confirm what is and what isn’t required for activity.

Determine optimal distance between interacting groups when u have more than one binding interaction. (ex. ring expansion contraction)

Test how rigidity impacts if your compound if flexible

Avoid molecule with stereochemistry

Isosteres, bioisosteres

one of two or more substances that exhibit similarity of some properties as a result of having the same number of total valence electrons in the same arrangement and that consist of different atoms and not necessarily the same number of atoms

bioisosteres - substituents or groups with similar physical or chemical properties that broadly produce similar biological properties in a chemical compound. ex: azide sub for alcohol

Common bioisosteres

Hydrogen replaced with fluorine (electronegativity makes it almost like a hydrogen).

C-F cannot be broken by CYPs, preventing metabolization and increasing half life. Usually para position of aromatic ring.

Carboxylic acid bioisosteres since they are very reactive

Amide bioisosteres, since amides are easily digested.

Heterocycles

Carboxylic acid bioisosteres

tetrazole - widely accepted bioisostere. aromatic ring. very similar pka. can’t form esters or amides.

Heterocycles as bioisosteres

Serve as bioisosteres for aromatic rings.

TPD/PROTAC

Targeting protein-protein interactions by controlling protein degradation pathways. Inhibit desired proteins.

Take protein of interest (POI) and bind to E3 ligase using PROTAC. E3 ligase ubiquitinates POI, PROTAC now free to repeat while POI goes to proteasome.

Ubiquitin/proteasome system (UPS)

E3 ligase marks protein with a bunch of ubiquitin for degradation by proteasome.

Prodrugs

When active substance isn’t actually the drug. Drug metabolite is actually the active substance. Tamoxifen, Acyclovir.

Pharmacodynamics

what drug does to body. whether drug candidate hits the desired target in VIVO. Need animal models

Xenografts

Grow human tissue in animal. Like organs, or human tumors.

Requires immunodeficient (nude) mouse. Often examines a biomarker.

Biomarker

biochemical response measured from …

Efficacy in lead discovery questions, ways to measure

does drug do what we want it to do?

Pain (rat tail flick test), rat paw edema?, mental state (forced swim test), disease progression, waterfall plot, progr…survival.

is it statistically significant?

does it cause adverse effects?

weight loss

Biologics

any susbtance. serum or vaccine, derived from animal products or other biological sources designed to treat…

DPT vaccine, tetanus shot, bovine insulin, RNAi

Antibody engineering

Engineer an antibody to target whatever you want in immune system. Can tell if if a drug is a biologic cause generic name typically ends in “ab”. “imab means derived from primate” “umab” means “humanized” antibody.

Biologics made with recombinant DNA technology

Generated protein will undergo post-translational modifications in the system, so not guaranteed to be effective in human host.

Biologics pros and cons

Very specific, low toxicity/adverse effects

But poor PK - IV only. Pretty much defy all Lipinski’s Rules

Very expensive

Advances in biology that have advanced out ability to use biologics.

Potential use of RNA knockout therapies, antibody engineering. Recombinant DNA technology

Phenotypic and Genotypic drug discovery

Phenotypic - observe that a compound has a specific response (hit) so you have to to determine target

Genotypic - start with target, develop compound that elicits desired response.

When can you start moving to clinical trials?

Have leads that…

Preclinical studies

• Toxicity (including everything that goes into the drug)

• Salt selection

• stability

• CMC

IND application and requirements

Need to submit an Investigational New Drug (IND) application and get it approved in order to start human dosing.

• Need all protocols you’ve used to analyze compound to date and your detailed plans and protocols to use during clinical trial

• all details of drug candidate

• full details about how and where compound will be prepared

• full details about pharmacology and toxicology

Name of drug, generic and trade name

FDA Human Trial Guidelines

GLP - Good Laboratory Practice, not hiding anything

GMP - Good Manufacturing Practice, document everything

GCP Good Clinical Practice

FDA certifies that laboratories meet this standards.

Protocol and Plans Questions

Dosing method (IV, PO, etc)?

How many doses?

How will you analyze data?

What data and info are you trying to determine?

Who will be performing studies and where?

Drug Candidate Details

Structure

MW

MP

pKa, logP, logD7.4

Optical Roation

Proof of Structure (x-ray, NMR, IR)

Form (solid - crystalline, powder; solution)

Stability

Assessing stability

Have to decide what compound will be

Salt selection - counterion plays a big role in PK

How to studie stability?

Form drug via x-ray powder diffraction (XRPD or XRD) - not same as crystal x-ray. Allows identification of different polymorphs.

One you identify the polymorph you want, assess in its stability in :

light, heat, mechanical, hygroscopicity

Florida Test

Polymorphism

Ability of solid materials to exist in more than one form. Can get different polymorphs from different extraction methods.

CMC

Chemical Manufacturing and Control group - deals with how and where compound will be perpared.

optimize synthesis of compound at large scale (convergent synthesis in cGMP).

Must then thoroughly examine purity of drug candidate.

Things to avoid for large scale synthesis

Reactions that need very low temps

slow, controlled additions

Chromatographic purifications

cGMP facility

How to limit synthesis expenses

Developing a convergent synthesis

finding multiple commercial supplies for advanced intermediates

conductiing only final step in a cGMP Lab

Details that must be provided for every component in drug synthesis

Source, purity, grade

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Ways to detect purity

Triple quadrupole mass spec

NMR using other nulcei

elemental analysis

chemical derivatization

FDA purity requirements

Every impurity that exists above 0.05% or every impurity that a human will be exposed to above 1.0 mg/day must be accounted for and tested for safety…

ADME details required by FDA

Metabolism extensively studies, every metabolite more that 1.0 mg/day must be fully characterized

ADME of each metabolite explored. MBI

Often radiolabelled analogs are prepared to tract them.

Toxicology FDA

All preclinical toxicology (like hERG) reported

Must determine how much is toxic. Maximally tolerated dose (MTD). aka dose range finding study. Must do in animals first. What is observed

Typically required more than 100g of product. Non-GLP toxicology batch (single batch to ensure impurity profile is the same)

Every impurity and excipient (like stabilizing components for IVs) is examined for toxicity.

Mutagenicity examined

Ames Test

Mutagen testing. Take salmonella that can’t make its own histidine, put on plates with and without histidine. Add drug. If salmonella grows in non histidine plate, drug helped it grow via mutation.

Clinical trial approval

File IND. Submit Investigator’s Brochure (IB).

IB

information that will be provided to the clinicians who will be studying the compound. Includes all pertinent safety data as well as the drug characterization. Required for every trial.

Clinical trial phases

Phase I - safety study, tens of patients

Phase II and II - efficacy studies, hundreds of patients for II, thousands for III.

Phase IV - undertaken after approval

Each considered a different clinical trial, each have own protocols and IBs.

Phase I trial

First time dosed in humans

Goal is to determine safety and PK profile

Most examine multiple dose PK. FInd MTD (when Grade 3 reached).

Identify the certified GCP clinic where study will be done in IND

Age, gender, race, ethnicity, health of patients must be detailed.

Changes in formulation or administration can only be considered if already accounted for in IND

Grades for toxicity

Clinical trial participants

Participants are voluntary. Careful screening, see if they’re taking other medications, likelihood to adhering to study conditions.

GCp facility where trial is conduction must monitor compliance as well as data.

Clinical trial protocols

Number of patients enrolled, exclusion criteria. Monitor data carefully to…

Toxicity in trials identified how by FDA

Through adverse events list (AE). Classified by Grades 1-5. Mild, Moderate, Severe, Life Threatening/Disabling, Lethal. Protocols in IND predetermine what adverse effects must be achieved to be considered MTD. Obviously, long term toxicity can’t be determined.

Career choices

Medical writing

Biostatistician

Regulatory Control

Clinical research Associate

Clinical Trial Liaison/Medical Science Liaison

Phase II

Does drug work. Multiple GCP facilities

New IND needed, including updated drug date (with human PK), safety data, protocols (ensure safe dosing but accounting for the need to determine effective dosing), IB.

Many IND applications are written as combination Phase I and Phase II trials.

Patient selection/screening more significant. Ethics plays a role (ethical biostatisticians, patients).

Phase II patients typically aware, risking placebo. RDT attempts to overcome this. Some drugs approved at this stage is data is strong enough.

Combined IND app for Phase I and II

Sometimes necessary in order to find an efficient number of Volunteers willing to enroll in risky trial (ex. cancer)

Complicates things because you are already examing efficacy before safety is established

Can cloud the attribution of SAE’s due to drug safety vs disease progression

Phase IIA and IIB

A: How much of drug do I need to give to safely elicit a response

B: Measure of whether a meaningful response can be seen. Do we see statistical efficacy.

Randomized Discontinuation Trial

Random groups of people rotated off of drug and onto something else to better evaluate if drug is working.

Phase III

Compare drug to current standard of care.

New IND needed with new IB. Multiple GCP facilities. Considerable communication between various national (and international) regulating agencies.

Patients dividied into test and control groups. Control not equal to placebo, placebo has no medicinal/biological effect.

Double Blind. Date assigned solely based on patient number. Only when certain conditions as outlined in protocol is the data unblinded and efficacy evaluated.

SPA

Special Protocol Assessment. Demonstrate that the drug is effective compared to standard through RDT. Request automatic approval if results are replicated in Phase III.

Phase IV

After approval.

How much improvement needed in Phase III for approval?

FDA requires 22% improvement over the standard of care.

DSMB

Data Safety Monitoring Board. Independent group approved by FDA that periodically reviews the blind data to evaluate all aspects of the trial. Eval safety, efficacy. ethics.

Can stop trial for control or test group. For good and bad reasons. Protocol outlines the next actions in these cases.

Since patients in Phase II are volunteers and typically aware of their condition, not uncommon for them to figure out which group they are in. So people may drop out.

Tuskegee Syphilis Study

enrolled 600 african american men with the promise of free healthcare. conducted a screen so 2/3 had syphilis. never told them they had it and never treated. continued for 40 years.