Medicinal Chemistry C9115Inhibition vs. DepletionParuch
Weiss, W. A.; Taylor, S. T.; Shokat, K. M. Nat.Chem. Biol. 2007, 3, 739.
• Inhibition:
– fast & time-flexible modulation of the activity of the target of interest
– the protein is still present
• Several methods of molecular/cell biology rely on depletion of the protein of interest
 prediction of the effect of inhibitors
Medicinal Chemistry C9115Western Blot
1 Burnette, W. N. Analytical Biochemistry 1981, 112, 195..
2 Towbin, H.; Staehelin, T.; Gordon, J. Proceedings of the National Academy of Sciences 1979, 76, 4350.
Western Blotting is an analytical technique used in molecular biology for the qualitative detection
of single proteins from a tissue sample or cells’ extract.
• Facts:
 Name given by English biologist Edwin Southern in 19801
 Method originated in the laboratory of Harry Towbin in 19792
(Friedrich Miescher Institute, Basel, Switzerland)
• Western blots can be used to:
 identify a specific single protein within a complex mixture of proteins
 evaluate the size of a protein of interest
 measure the amount of protein expression
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Paruch Medicinal Chemistry C9115Western Blot
Picture taken from : https://www.antibodies.com/es/western-blotting
Western blotting is achieved in 4 main steps:
• Preparation of the sample by mixing it with a
detergent called sodium dodecyl sulfate,
which makes the proteins unfold into linear
chains and coats them with a negative charge
• Separation of proteins by size and charge
using gel electrophoresis
• Transfer of separated proteins to a solid
support called a blotting membrane
• Indirect visualization of the desired protein
using a specific primary antibody (binding to
the desired protein) and then a secondary
antibody (binding to the primary antibody).
The secondary antibody is visualized through
different methods (e.g., staining,
immunofluorescence & radioactivity)
Medicinal Chemistry C9115Western Blot
• What is the result of gel electrophoresis before membrane transfer ?
A gel that looks like that:
Pink, Ponceau S staining; blue, Coomassie brilliant blue staining
• What is the result of membrane transfer and indirect visualization of secondary antibody ?
A gel that looks like that:
70
55
40
35
MW: 42 kDa
100
130
170
MM
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Paruch Medicinal Chemistry C9115RNAi
Educational links :
https://www.youtube.com/watch?v=cK-OGB1_ELE
• RNAi stands for RNA interference, also known as post-transcriptional gene silencing.
• RNAi is an RNA-dependent gene silencing process = biotechnological tool capable of targeted
gene inactivation.
• Fire and Mello were awarded the Nobel Prize in Medicine in 2006 for their work on the
nematode worm Caenorhabditis elegans published in 19981 reporting that double-stranded
RNA (dsRNA) is effective in gene silencing, driving the destruction of messenger RNA
(mRNA) with sequences matching the dsRNA and thus blocking the translation of mRNA into
protein.
• the first report of siRNA-mediated gene silencing in mammalian cells in 20012
• Technologies used for RNAi :
 miRNA = micro RNA
 siRNA = small interfering RNA or silencing RNA
 shRNA = short hairpin RNA
1Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Nature 1998, 391, 806.
2Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Nature 2001, 411, 494.
Paruch Medicinal Chemistry C9115RNAi
The miRNA biogenesis pathway in vertebrate cells.
Figure taken from Cullen, B. R. Nature Genetics 2005, 37, 1163.
Artificial siRNAs can enter this pathway as
synthetic siRNA duplexes, as shRNAs
transcribed by Pol III or as artificial primiRNAs
(shRNA-mir).
For simplicity, not all factors involved in
miRNA biogenesis are shown.
Ago2, Argonaute-2; Exp5, Exportin-5.
Recommended literature :
Cullen, B. R. Nature Genetics 2005, 37, 1163.
Paruch Medicinal Chemistry C9115RNAi
siRNA: dsRNA (either transcribed or
artificially introduced) is processed by Dicer
into siRNA which is loaded into the RISC.
AGO2, which is a component of RISC, cleaves
the passenger strand of siRNA. The guide
strand then guides the active RISC to the
target mRNA. The full complementary binding
between the guide strand of siRNA and the
target mRNA leads to the cleavage of mRNA.
miRNA: Transcription of miRNA gene is
carried out by RNA polymerase II in the
nucleus to give pri-miRNA, which is then
cleaved by Drosha to form pre-miRNA. The
pre-miRNA is transported by Exportin 5 to
the cytoplasm where it is processed by Dicer
into miRNA. The miRNA is loaded into the
RISC where the passenger strand is
discarded, and the miRISC is guided by the
remaining guide strand to the target mRNA
through partially complementary binding. The
target mRNA is inhibited via translational
repression, degradation or cleavage.
Gene silencing mechanisms of siRNA and miRNA.
Figure taken from Mol Ther Nucleic Acids 2015, 4, e252.
Paruch Medicinal Chemistry C9115RNAi
• Long dsRNA is cleaved by an endo-ribonuclease
called Dicer. Dicer cuts the long dsRNA to form
siRNA.
• Once siRNA enters the cell, it gets recruited
with other proteins to form the RNA-Induced
Silencing Complex (RISC).
• Once part of the RISC complex, the siRNA is
unwound to form single stranded siRNA.
• The single stranded siRNA-RISC complex can
scan and find a complementary mRNA.
• Once the single stranded siRNA (part of the
RISC complex) binds to its target mRNA, it
induces mRNA cleavage.
• The mRNA is now cut and recognized as abnormal
by the cell. This causes degradation of the mRNA
that is not translated into proteins.
• The gene that encodes that mRNA has been
silenced.
siRNA mechanism
Figure taken from https://en.wikipedia.org/wiki/Small_interfering_RNA#Mechanism
Paruch Medicinal Chemistry C9115RNAi
Mechanism :
Paruch Medicinal Chemistry C9115RNAi
• depletion vs. inhibition
(see e.g. Nature 2016x538x477 – MCL1 knockout lethal, MCL1 inhibition OK)
Recommended literature :
Hannon, G. J. Nature 2002, 418, 244.
Lam, J. K.; Chow, M. Y.; Zhang, Y.; Leung, S. W. Mol Ther Nucleic Acids 2015, 4, e252.
Medicinal Chemistry C9115PROTACs
• PROTACs are currently a VERY HOT TOPIC in pharmaceutical R&D
• PROTAC = acronym for PROteolysis Targeting Chimera
• A PROTAC is a tool amongst others for Targeted Protein Degradation (TPD)
• PROTAC molecules are known as Active Degraders of targeted proteins
• First publication in 2001: Protacs: Chimeric molecules that target proteins to the Skp1–
Cullin–F box complex for ubiquitination and degradation, Sakamoto, K. M.; Kim, K. B.;
Kumagai, A.; Mercurio, F.; Crews, C. M.; Deshaies, R. J. Proceedings of the National
Academy of Sciences of the United States of America 2001, 98, 8554.
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Medicinal Chemistry C9115PROTACs
Neklesa, T. K.; Winkler, J. D.; Crews, C. M. Pharmacology & Therapeutics 2017, 174, 138.
• Upon binding of both moieties, a ternary complex between target protein and E3 ligase is formed,
leading to polyubiquitination of the target protein (on exposed lysine residues).
• Upon dissociation of the complex, the polyubiquitinated target protein is recognized by the
proteasome and subsequently degraded.
• PROTACS are heterobifunctional small molecules (700 to 1000 Da). They consist of :
 a binding moiety for the targeted protein to degrade
 a binding moiety for an E3 ubiquitin ligase
 and a linker joining the two binding moieties
• The PROTAC molecule is then free to repeat the
process (catalytic mode of action => less drug
needed => less off-target toxicity).
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Medicinal Chemistry C9115PROTACs
• Why PROTACs?
• PROs
 Catalytic mode of action.
 The binding to the target protein can occur anywhere on the protein.
 No requirement to target catalytic active sites of proteins or to block protein-protein
interactions.
 Target undruggable proteome.
 Behave like traditional small molecules (exposures are dose proportional, PROTACs show good
tissue distribution, optimized PROTACs show low hepatic clearance).
 Can be very selective (ubiquitin ligase system has to transfer the ubiquitin to the exposed lysine
residue on the target protein => tuning achievable even on closely related proteins depending on
the positioning of the exposed lysine with respect to the ligase).
1 Yang, J., et al.; Journal of Medicinal Chemistry 2019, 62, 9471.
2 Bemis, T. A., et al.; Journal of Medicinal Chemistry 2021, 64, 8042.
3 Jiang, B., et al.; Nature Chemical Biology 2021, 17, 675
• CONs
 Large molecules.
 Linker design can affect the mode of action of the PROTAC (Molecular Glue1, 2), SAR required.
 Acquired resistance by point mutation in targeted protein3.
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Medicinal Chemistry C9115PROTACs
• Design of PROTACS:
 The Cullin 4A E3 ligase complex and the Cullin 2 E3 ligase complex are
two powerful E3 ligase complexes for the design of highly potent and
effective PROTAC molecules, because of the availability of potent and
drug-like small-molecule ligands to recruit these degradation complexes
 immunomodulatory imide drugs (IMiDs) = potent small-molecule ligands for
cereblon, a receptor protein for the Cullin 4A complex:
lenalidomide thalidomide
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Medicinal Chemistry C9115
From DNA to RNA to Protein
Educational links :
• https://www.nature.com/scitable/topicpage/translation-dna-to-mrna-to-protein-393/
A gene is expressed through the processes
of transcription and translation.
During transcription, the enzyme RNA
polymerase (green) uses DNA as a template
to produce a pre-mRNA transcript (primary
transcript, pink).
The pre-mRNA is processed (by 5' capping,
3' polyadenylation, and alternative splicing
processes) to form a mature mRNA molecule
that can be translated by the ribosome to
build the protein molecule (polypeptide)
encoded by the original gene.
Paruch CRISPR-Cas: Introduction
Medicinal Chemistry C9115CRISPR-Cas
Biotechnological tool capable of targeted gene inactivation & targeted gene editing.
Biotechnological tool revolutionizing genetic engineering.
Doudna and Charpentier were awarded the Noble Prize in Chemistry in 2020 for the development
of a method for genome editing.
Origins of CRISPR:
• Discovered by basic research on how a bacteria fights a viral infection1,2
• CRISPR is an RNA-mediated adapted immune system in bacteria and archaea that protects
them from viruses and plasmids
• Origin of CRIPSR name in 20053
• First description of what is now known as CRIPSR locus by Mojica in 19934
Educational links :
• https://media.hhmi.org/biointeractive/click/CRISPR/
• https://www.youtube.com/watch?v=4YKFw2KZA5o
• https://www.youtube.com/watch?v=TdBAHexVYzc
• https://www.youtube.com/watch?v=KSrSIErIxMQ
1 Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. Science 2012, 337, 816.
2 Mojica, F. J.; Díez-Villaseñor, C.; García-Martínez, J.; Soria, E. J Mol Evol 2005, 60, 174
3 Jansen, R.; Embden, J. D.; Gaastra, W.; Schouls, L. M. Mol Microbiol 2002, 43, 1565
4 Mojica, F. J.; Juez, G.; Rodríguez-Valera, F. Mol Microbiol 1993, 9, 613
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Medicinal Chemistry C9115CRISPR-Cas9
• CRIPSR = acronym for Clustered Regularly Interspaced Short Palindromic Repeats
• Technology consists of a Cas9-guide RNA complex (known as Cas9 complex) that binds to
PAMs:
 Cas9 that stands for CRISPR associated protein 9. It is an endonuclease (enzyme that
cuts DNA from within = molecular scissor). Cas9 activity results in DNA double-strand
breaks (DSBs).
 Guide RNA, (sgRNA, single guide RNA) synthetic sequence of RNA (about 20
nucleotides) that matches a target sequence of interest within a particular gene.
 PAM stands for Protospacer Adjacent Motif. Commonly consists of a three-nucleotide
sequence in the genome (5’-NGG-3’ where N represents any nucleotide (A, C, G or T)).
Cas9 recognizes and binds PAM motifs. The presence of PAM close to the targeted
DNA site is a necessity.
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Medicinal Chemistry C9115CRISPR-Cas9
How does it work ?
• Cas9 complex recognizes and binds to PAM motifs (that occurs about every 50 bases)
until the guide RNA finds its perfectly matching sequence along the DNA strands (via
Watson-Crick base pairing). Upon matching, Cas9 unwinds and pulls-apart the DNA
double helix (upstream of PAM) that allows formation of a DNA-RNA helix.
• Only then the DNA-cutting activity of Cas9 is activated. Two specific cuts (on each
strand of the DNA) are made, three nucleotides upstream of the PAM motif. It results
in a DNA double-strand break.
• The break is repaired by naturally occurring DNA repair processes. Two main repair
pathways exist to join the broken DNA strands:
 NHEJ: Non-Homologous End Joining, an error prone repair process leading to
mutation at the site of cleavage. If the targeted cleavage happens within a gene’s
coding region, the repair by NHEJ might inactivate the gene due to the
frameshift in the coding sequence.
 HR: Homologous Repair or Homology-directed Repair, an error free repair process
leading to the original DNA strands. Cas9 (acting as a catalytic machinery) will
keep on repeating its tasks until a mutation arise at the targeted site.
Recommended literature about CRISPR structures and mechanisms :
Lander, E. S. Cell 2016, 164, 18.
Jiang, F.; Doudna, J. A. Annu Rev Biophys 2017, 46, 505.
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Medicinal Chemistry C9115CRISPR-Cas9
Recommended literature about CRISPR structures and mechanisms :
Lander, E. S. Cell 2016, 164, 18.
Jiang, F.; Doudna, J. A. Annu Rev Biophys 2017, 46, 505.
Figure taken from Annu Rev Biophys 2017, 46, 505.
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Medicinal Chemistry C9115CRISPR-Cas
Recommended literature:
Anzalone, A. V.; Koblan, L. W.; Liu, D. R. Nat Biotechnol 2020, 38, 824.
Common targeted edits:
• conversion of DNA base pairs (e.g., point mutation = gene inactivation)
• deletion of DNA base pairs
• insertion of DNA base pairs
• a combination of the above changes (e.g., replacement of DNA base pairs = gene correction)
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Medicinal Chemistry C9115CRISPR-Cas
CRISPR-Cas-derived genome editing agents :
• CRIPSR-Cas nucleases = nuclease variants with broaden targeting scope and specificity. 2 main
nucleases Cas9 and Cas12. Engineered nuclease variants can bind to different PAM motifs.
• CRIPSR-Cas base editors = tool precisely installing point mutations without DSBs = tool to repair
disease related mutations. CRISPR-Cas is engineered with a catalytically impaired Cas nuclease
which is fused with a deaminase enzyme able to convert bases.
• CRIPSR-Cas transposases/recombinases = tool mediating rearrangements of large segments of
DNA. Approaches with many limitations. Emerging editing technology.
• CRIPSR-Cas prime editors = tool changing an original DNA sequence by inserting a new sequence.
CRISPR-Cas is engineered with a catalytically impaired Cas nuclease which is fused with a
reverse transcriptase domain able to direct the synthesis of the edited DNA strand onto the 3’
end of the target DNA strand. The non-edited DNA strand will then be repaired using the
edited strand as a template.
• CRISPRi = CRISPR interference = CRISPR-Cas gene repression Cell 2013, 152, 1173 specifically
interfere with transcriptional elongation, RNA polymerase binding, or transcription factor
binding; effects are reversible
• CRISPRa = CRISPR-Cas gene activators Nat Commun 2018, 9, 2489 transcriptional activation
domains fused to Cas nuclease
Recommended literature:
Anzalone, A. V.; Koblan, L. W.; Liu, D. R. Nat Biotechnol 2020, 38, 824.
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Medicinal Chemistry C9115CRISPR-Cas libraries
CRISPR-Cas screening can be used as a tool for genetic screens :
 to validate novel drug targets.
 to study the underlying causes of diseases.
 CRISPR-Cas libraries are generated to target thousands of human genes with a large number
of sgRNAs per gene.
 3 different mechanisms : the most common is CRISPR KO (knockout) which completely
inactivates a gene. The other two either activate (CRISPRa) or inhibit (CRISPRi) the
expression of a gene without altering the DNA. CRISPR-Cas libraries are used to find out
which genes are providing a positive (cell enrichment) or a negative (cell viability) effect.
 CRISPR-Cas screening can be done in the presence of a specific enzyme inhibitor to
determine which genes promote or reduce its activity or without the presence of an inhibitor.
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Medicinal Chemistry C9115CRISPR-Cas
Cons :
 CRISPR-Cas specificity
 Delivery of CRISPR-Cas therapeutics in vivo
 Ethical issues (e.g., human genetic enhancements)
Pros :
 Accelerates research in basic science
 Potential therapeutics to treat diseases such as:
cancers, Alzheimer’s disease & diabetes, Duchene muscular dystrophy, cystic fibrosis, etc.
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Medicinal Chemistry C9115CRISPR-Cas9 biotech
Picture taken from http://www.crisprtx.com/gene-editing/therapeutic-approach
CRISPR Therapeutics aim to develop transformative gene-based medicines based on
CRISP-Cas gene editing.
Cell therapy approaches in human.
Cells can either be edited ex vivo (outside the body) or in vivo (inside the body)
Another educational link: http://www.crisprtx.com/gene-editing/crispr-cas9
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Paruch Medicinal Chemistry C9115CETSA
Recommended literature :
1Molina et al., Science 2013, 341, 84.
• CETSA = acronym for CEllular Thermal Shift Assay
• CETSA method first described by Nordlund in 2013 published in Science.1
• CETSA based on the biophysical principle of ligand-induced thermal stabilization of target
proteins. Ligand binding can stabilize the protein and prolong its half-life.
• This thermal stability shift phenomenon can be employed to screen for small molecule probes
and to validate target engagement.
• CETSA constitutes the first broadly applicable method to assess direct drug binding in cells
and tissues.
• CETSA has become a valuable tool for the validation and optimization of drug target
engagement in drug discovery projects (industry & academia).
Medicinal Chemistry C9115CETSA
What does Thermal Shift Assay (TSA) measure ?
• TSA measures the thermal melting curves of a protein (corresponding its unfolding).
• TSA are applied only to purified proteins.
• The thermal shifts of a protein exposed to a ligand at different concentrations correlate
with the IC50s.
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Medicinal Chemistry C9115CETSA
Figure taken from Annu Rev Pharmacol Toxicol 2016, 56, 141.
Schematic illustration of a thermal shift ΔTm corresponding to target engagement.
• When the protein binds to a ligand, the melting temperature Tm is typically shifted
to a higher temperature (producing a thermal shift ΔTm).
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Medicinal Chemistry C9115CETSA
What does CETSA measure ?
• CETSA measures the thermal melting curves of a protein in cells or tissues.
How ?
• Multiple aliquots of cell lysate are heated to different temperatures.
• After cooling, the samples are centrifuged to separate soluble fractions from precipitated
proteins.
• The target protein present in the soluble fraction is quantified by Western Blotting.
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Medicinal Chemistry C9115CETSA
Figure taken from Science 2013, 341, 84.
Schematic illustration of CETSA melt curve and ITDRFCETSA
procedure.
ITDRF = IsoThermal Dose Response Fingerprint
Investigation of drug concentrations effects can be
done:
• on purified proteins (in vitro)
• on lysate aliquots or intact cells (ex vivo)
• on animals (in vivo)
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Medicinal Chemistry C9115CETSA
Recommended literature :
Molina et al., Annu Rev Pharmacol Toxicol 2016, 56, 141
Figure taken from Annu Rev Pharmacol Toxicol 2016,
56, 141.
Illustrative examples of CETSA melting curves and
ITDRFs.
When intact cells are assessed via CETSA, factors
contributing to target engagement (e.g., membrane
transport, accumulation, specific activation events) are
detectable and distinguishable from the lysate results.
Dihydrofolate reductase (DHFR) and the drug
methotrexate serve here as examples.
Methotrexate is imported by cells via specific transport
mechanisms and is subsequently polyglutamated in the cell;
this modification hinders it from exiting the cell, resulting
in its accumulation and apparently increased concentration.
In contrast, in a cellular extract (right), no accumulation
occurs, and methotrexate and DHFR exist in a more dilute
environment and are less likely to engage with each other.
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Medicinal Chemistry C9115Mass Spectometry-CETSA
Recommended literature :
1Savitski et al., Science 2014, 346, 1255784.
2Werner et al., Anal Chem 2012, 84, 7188
• First published proteome-wide MS-CETSA study in 2014 in Science.1
• MS-CETSA assay uses the tandem mass tag (TMT)-based isobaric labelling strategy.2
Isobaric mass tags have identical overall mass but vary in terms of the distribution of heavy
isotopes around their structure. The most common isobaric tag is amine-reactive (TMT
which modifies lysine residues), but tags that react with cysteine residues and carbonyl
groups in proteins are also available.
• MS-CETSA assay allows the relative quantification of proteins at different temperatures
and different drug concentrations. Such experiment enables parallel binding studies of
drugs to many proteins in the lysate, cell or tissue context.
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