Category Archives: Chemical Biology

ELISA-based High Throughput Screening

 
17 APPROXIMATE MODEL -ELISA

In a follow-up to our post introducing ELISA, we wanted to discuss a common application of this technique: small molecule inhibitor screening. The set up is relatively simple (1) Coat a 96-well plate with your two proteins of interest and and a detector antibody (2) add a library of 96 molecules per plate (3) Inspect which molecules inhibit the protein-protein interaction (resulting in a color loss).

Once you have have narrowed down your library to a list of inhibitors, you can rank those inhibitors by their potency/IC50 by running dilution series with the exact same plate set-up:

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What do “PEG-linkers” do to drugs?

06 PEG Linker - ALL MASTER

Where synthetic chemistry has given us many molecules that bind (and inhibit) many different proteins, chemical biology endeavors to “attach” new function to these “classical” drugs. Examples of chemical biological applications include: (1) attaching toxins or imaging agents for targeted deliver or (2) using multivalency to improve a drug’s potency. Unfortunately in order to “attach” new function to a drug you need to use a “linker” which is long and inert so it doesn’t interfer with “binding” or the new “function”. The most common linker material used in chemical biology and pharmacology is polyethylene glycol or PEG (pictured above) which is both long and inert but still impacts the properties of the drugs it is attached to(see figure above):

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Engineering Molecular Electronics with Substituents

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Often, if you are trying to design a molecule which has function (e.g. catalysts, fluorophores, switches, etc.) you have to tweak the electronics of that molecule. Generally, the most important electronic energy levels are the HOMO’s and LUMO’s which can donate and accept electrons, respectively. The figure above summarizes the substituents that are most used to raise the energy (electron donating groups), lower the energy(electron withdrawing groups) or do both (extra conjugation groups).

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Introduction to Fluorescent Probes

10 Fluorescence Sensitivity

In a follow-up to our introduction to fluorescence, we wanted to discuss why fluorescent probes have proven so useful in chemistry and biology. One of the main reasons is that, unlike most spectroscopic techniques which rely on a loss-of-signal or light-absorption, fluorescence is a “gain of signal” technique. As a result, the near-zero baseline/background translates into a very high signal to noise ratio for fluorescent probes. Indeed fluorescent probes have some of the greatest sensitivities of all sensors (radiation is better but has other draw backs)!

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Introduction to Fluorescence

07 Fluorescence Introduction

Fluorescence (i.e. the emission of light from a electronically excited substance) is one of the most utilized physical phenomena in chemistry and biology. Though humans have been aware of fluorescence for thousands of years (e.g. fireflies), it wasn’t until the discovery of quinine in 1845 that we really started to understand its chemical basis(see figure above). Interestingly, during World War II, the study of quinine’s anti-malarial properties led to the development of the first spectrofluorometers which enabled true quantitative study of fluorescence. Finally, in the 1980-1990’s, new tools in synthetic chemistry and molecular biology allowed rational engineering of chemical fluorophores into a critical class of probes in biophysics(microscopy), molecular biology(sequencing), cell-biology(flow cytometry) and even anatomy (histology)).

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Understanding Multivalency (aka Avidity)

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Making a Ligand or Drug multivalent is a common method to try to improve the potency or EC50 of that drug from that predicted by the Hill Equation. Below we summarize the full spectrum of multivalent enhancement for the n = 2 case (n being the degree of multivalency) but these rules are easily extendable to the n-valent case aswell.

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The Thermodynamic Limits on Small Molecule Drug Affinity

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Not too long ago, some really cool papers1,3 sought to examine “The Maximal Affinity of Ligands” by compiling a list of known small-molecule drugs and comparing their affinities (in kcal/mol or Kd‘s see post on the Hill Equation) with various parameters such as molecular weight (see figure above).1

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Kinetic Limits on Engineering Agonist Drugs

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There are many drugs that act as agonists ligands (L) which means they “turn on” their target receptor (RL) so that it induces its normal down-stream signalling. Examples of such drugs include: growth hormones, insulin, steroids and G-protein coupled Recetor(GCPR) ligands such as morphine (opiods), neurotransmitters and scent/aroma compounds. In general you can improve the potency (EC50) of these drugs by improve their binding dissociation constant (Kd) for their receptor (for more detail see post on the Hill Equation).

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