Summary
Summary Drug Target Biochemistry and Signaling (XM_0111)
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This document consists of a summary of the course Drug Target Biochemistry and Signaling, corresponding to the Master Drug Discovery Sciences.
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Drug Target Biochemistry and Signaling, Daniëlle BandLecture 1. Introduction (cellular signal transduction)
Cellular signaling
The process from a drug to the desired effects
happens via targets (signaling proteins). Most
drugs interfere with signal transduction.
Signaling is the brain of the cell, and consists
of integration of various stimuli (inputs), and coherent response
(outputs). All the outputs will feedback on the way the cell will
respond to an input. Most of the signaling pathways have
homogenous pathways in different organisms. This conservation
makes it easy because model organisms can be used to learn more about signal pathways in humans.
In signaling (cross-) regulation happens in which there are different pathways that interconnected with
each other. It is important to understand that this depends on the context. This (cross-) regulation is for
example, cell type dependent, cell cycle dependent, nutrient status dependent, and differentiation
status dependent. Not only a single receptor output is looped, but pathways are connected and
changing. Depending on the pathway, the output can be understanded. This can be a nightmare,
because the number of different pathways and components and regulation.
An example is the fact
that post-translational
modifications control
Akt/PKB.
Ubiqutination of the
Pleckstrin homology
domain (PD) causes
activation of Akt, while
acetylation of this
domain causes
inhibition of the activation of Akt. Phosphorylation and SUMOylation of the kinase domain (KD) causes
activation of Akt, while glycosylation of this domain causes phosphorylation inhibition and thus
inhibition of the active Akt. Lastly, phosphorylation of the regulatory domain (RD) causes activation of
Akt. The activation pathway of Akt can be investigated by using inhibitors for posttranslational
modifications (e.g. phosphatase inhibitor).
Signal transduction can happen on different levels: (1) molecular
signaling device, (2) network signaling device, which is a simple
build-up of connections result in sophisticated responses, or (3)
cellular signaling device.
Drug target and therapeutic strategies
The characterization of targets can have a relevance for the industry
and cause the development of (novel) therapeutic tools. Novel
therapeutic strategies include vaccines, repurposed drugs, and novel drugs. Over the past years there
is a shift in the top 200 drugs in the pharmaceutical industry from small molecules towards more
biologically ordered molecules. This is shown by the fact that there is a rise in biologics and antibiotics
in the first 40 of the top 200 drugs.
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,Drug Target Biochemistry and Signaling, Daniëlle Band
Drug targets
Drug targets have a confirmed role in the pathophysiology of a disease and/or is disease-modifying.
Target expression is not evenly distributed throughout the body and the 3D-structure is available to
assess drug-ability. The target is easily ‘assayable’ enabling high-throughput screening. The target also
possesses a promising toxicity profile, and the potential adverse effects can be predicted using
phenotypic data. Lastly, the proposed target has a favorable intellectual property (IP) status which is
relevant for pharmaceutical companies.
Drug development
Drug development is a lengthy process, expensive and always too late. It starts with the discovery in
which gene identification, understanding of the disease mechanism, and target identification takes
place. Than, in the preclinical phase, there is screening and optimization of candidates. In Investigational
New Drug (IND) application, a company submits an IND application to the FDA and the FDA evaluates
the quality of the data and determines if the therapy can be tested in humans. If human studies (clinical
testing) may proceed, the drug enters phase 1, in which there is a focus on monitoring the safety of the
drug. In phase 2 it is evaluated if the new drug is effective in patients.
In phase 3 it is evaluated if the new drug is effective in larger cohorts
of patients. In this course we will talk about only the beginning of the
process.
In the research strategy, knock-in and knock-out can happened
which leads to different protein amounts in a cell. It is used to show
the functionality of a compound. Knock-in adds DNA to a cell which
causes an increase in the amount of protein. Knock-out removes the
RNA from a cell which causes a decrease in the amount of protein.
Lipinski’s rule of 5
A good target is drugable. (1) This means that it is able to be screened (High-
>5 H-bridge donors
Throughput Screening). (2) It also needs to be a small molecule which >10 H-bridge acceptors
adheres Lipinski’s the rule of 5. (3) Lastly it needs to be a biologic, which can >500 Da molecular weight
be an antibody or a peptide. >5 LogP
Target finding can occur in two different directions. (1)
Phenotypical way in which there is a compound and
the target needs to be identified. This gives context
and is (patho)physiological (based on abnormal
changes in the body functions that are the causes,
consequences or concomitants of disease processes),
but there is limited availability of (proper) cells. (2)
Target based in which there is a target and the
compound needs to be identified. In this way the target is known, but there is no context (which is
needed for a disease model) and the toxicology of the target is unknown.
Polypharmacology
Complex diseases (multifactorial) may need complex treatment with multiple drugs at the same time,
and there may be a risk of unwanted effects, because it can lead to drug-drug interactions, resistance
to current therapies, and drug-undesired target interactions. This can be solved by polypharmacology
in which one drug hits multiple targets (e.g. Clozapine (antipsychotic) or Staurosporine pan-kinase
inhibitor).
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,Drug Target Biochemistry and Signaling, Daniëlle Band
Lecture 2. Information transfer across the membrane via signaling
mediators
You can look at the target and signaling pathway at different levels: (1) molecular level, (2) network
level, and (3) cellular level. At each level you get information essential for therapeutic interaction.
Second messengers
are key mediaters
between upstream
and downstream
events. Second
messengers can
covey information
by the
concentration and
distribution. The
smaller the molecule the easier it can diffuse through the cell, but this causes a lower relative
concentration at the site or origin (but higher concentration more distant from the site of origin). They
are highly controlled due to the balance between production and elimination of a second messenger.
Second messengers bind to proteins (readers). You can change second messenger levels in two ways:
(1) more second messenger production, (2) or less degradation.
Many known second messengers are cAMP, cGMP, inositol triphosphate
(𝐼𝑃3 ), diacylglycerol (DAG), 𝐶𝑎2+ , and nitric oxide (NO). NO is highly diffusible
and activates guanylyl cyclase (GC). This causes the conversion of GTP to
cGMP. cGMP activates kinases or ion channels. Deactivation of cGMP
(conversion of cGMP to GMP) is done by phosphodiesterases (PDE). An
inhibitor of PDE was originally developed for treatment of high blood
pressure by relaxation of the bloodvessels, but it turned out to be a solution
for erectile dysfunction.
There are different types of receptors that regulate second messengers: (1)
ionchannels, (2) RTKs, and (3) GPCRs.
G-proteins
G-proteins were discovered because they were the missing link between receptors and downstream
signaling. G-proteins are a GTP-dependent transducer between the receptor and the second
messenger-forming enzyme. G-proteins are a GTPase which causes them to be switched on and off.
They are a heterotrimeric protein complex. GTP binding is catalyzed by the GPCR.
The inactive G-protein consists of all
three subunits and a GDP bound. When a
signal molecule binds to the receptor, the
G-protein binds to the intracellular site of
the receptor which causes GDP to be
exchanged for GTP. This activation of the
G-protein leads to dissociation of the 𝐺𝛼
subunit from the 𝐺𝛽𝛾 complex (stable
dimer). Both the 𝐺𝛼 subunit and the 𝐺𝛽𝛾
complex can activate downstream effects.
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, Drug Target Biochemistry and Signaling, Daniëlle Band
When the G-protein is bound to GDP, the switch
I and switch II are in distant proximity. When
GTP is bound to the G-protein, the extra
phosphor group causes in interaction with
amino acids on switch I and switch II. This leads
to a closer proximity between switch I and
switch II that causes the G-protein the bind to
effector molecules.
There are four different 𝐺𝛼 subunits in the 𝐺𝛼 protein
family. These subunits are 𝐺𝛼,𝑠 , 𝐺𝛼,𝑖 , 𝐺𝛼,𝑞 , and 𝐺𝛼,12/13. Do
to the difference receptor interacting domain, which
causes a change in structure, the 𝐺𝛼 subunits bind to the
GPCR in different ways. For the 𝐺𝛽𝛾 there are also different
subunits. In general, this 𝐺𝛽𝛾 complex stabilizes 𝐺𝛼 for the
receptor interaction.
For the inactivation of the G-protein, GTPase activator proteins (GAPS) are used. These proteins
hydrolysis the GTP to GDP. RGS is an GAP protein that regulates the G-protein signaling. It accelerates
the turn off of the protein, because it increases the GTPase activity. Without RGS there is a prolonged
effect of the G-protein. For the activation of G-proteins, guanine exchange factors (GEFs) exchange GDP
with GTP. For G-proteins, GPCRs act as GEFs. Some inputs cause the activation of G-proteins, while other
inputs cause the inactivation of the G-protein.
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