PPI Modulators-Toward next generation therapeutics-

Literature Seminar
2014.5.22 (Thu.)
Yusuke Shimizu (M1)1

§0 Protein-protein interactions

Protein-protein interactions (PPI)Many cellular process depend upon enzymatic reactions
However, proteins rarely act alone and protein-protein interactions (PPIs)
mediate a large number of important regulatory pathwayse.g.
• signal transduction• transport across membranes• cell metabolism• muscle contraction2§0 Protein-protein interactions

Definition of PPI
Protein-protein interactions is physical contacts established between two or more proteins
We have to consider that…
• The interaction interface is intentional and not accidental
i.e., the result of specific selected biomolecular events/forces• The interaction interface is non-generic, evolved for a specific purpose
distinct from totally generic functions such as protein production and degradation3§0 Protein-protein interactions

PPI in signal transduction

PPI is central to biological processes
Complete mapping of PPI is one of main scopes of current biological research=Interactome

Nature Reviews Cancer, 2014, 14, 1734

§1 Interactome

Interactome
Finding interaction partners for a protein can reveal its function
Building entire PPI network is the next step after the “Human Genome Project”

Network is represented as protein “nodes”
linked by interaction “edges”

5§1 Interactome

PPI Determination
Yeast two-hybrid (Y2H)

6§1 Interactome

PPI Determination
Co-immunoprecipitation (CoIP)

7

§1 Interactome

PPI Determination

False positives
8§1 Interactome

PPI databases

Large scale identification of PPIs generated hundreds of thousands interactions,and they are collected together in specialized biological databases
9§2.0 PPI modulatorsTraditional inhibitor of protein function

Inhibitors bind to enzyme active site which is• well-defined• deep amd less accessible to bulk solvent• relatively small (100 – 600 Å2)10

§2.0 PPI modulators

PPI inhibitors

PPIs are difficult, unconventional drug target due to• Large binding interface areas(600-1300 Å2)• Shallow solvent-exposed surface• Surfaces differ from small-molecule binding site
in shapes and amino acid residue composition
Traditional medicinal chemistry is less effective for PPIs
11

§2.0 PPI modulators

PPI inhibitorsPPI inhibitors approved for clinical applications today are usually based onhumanized monoclonal antibodies (e.g., Tocilizmab, Bevacizumab)

However, antibodies are expensive and often seriously hindered by solubility,
route of administration, distribution, and stability problems
as well as by the possibility of a strong immune response

Small-molecule inhibitors are the ideal drug for targeting PPIs

12

§2.0 PPI modulators

Constrained Peptides

It was envisioned to use peptides directly derived from binfing epitopes of target PPIHowever...
• These peptides adopt only random coil or become structually less defined• Increased susceptibility to proteolytic degradation• Reduction in cell wall permeability

Research has focused on generating conformationally robust peptide13

§2.1 PPI inhibitors

Constrained secondary structures
Disulfide

J. Am. Chem. Soc. 1991, 113, 9391

LactamCovalent link between i, i+3, i+7
Stabilize α-helix
J. Am. Chem. Soc. 2000, 122, 3007
14

§2.1 PPI inhibitors

Stabilized α-helix
HIV-1 entry inhibitor

IC50 = 35 μM

C14linkmid successfully disrupted the assembly of gp41 core

PNAS. 2002, 99, 14644
15§2.1 PPI inhibitors

Hydrocarbon stapling
while disulfide and lactam bridges are effective in stabilizing α-helix,
such mimetics are not always stable in cells and are generally susceptible to degradation
Stabilized alpha-helix of BCL-2 domains (SAHBs)

KD = 38.8 nm

All hydrocarbon cross-links was introduced via ring-closing metathesis
Metabolically stable α-helix worked as Bcl-2 protein inhibitor

Science, 2004, 305, 1466
16

§2.1 PPI inhibitors

Metallopeptide
Metal ions play an important role in stabilizing α-helices in naturee.g. Zinc finger

Pd2+ clip stabilized α-helix in DMF but low helicity (<40%) in water

J. Am. Chem. Soc. 2004, 126, 4828
17

§2.1 PPI inhibitors

Hydrogen bond surrogate
gp41-mediated cell fusion inhibitor

KD < 5μM

Replacing hydrogen bond into covalent linkage stabilized α-helix

Angew. Chem. Int. Ed. 2008, 47, 187918

§2.1 PPI Inhibitors

Secondary structure mimetics

First non-peptidyl peptidomimetics
Mimetic work as Somatostatin receptor agonist,
although with reduce activityrelative to natural hormone

J. Am. Chem. Soc. 1992, 114, 9217
19

§2.1 PPI Inhibitors

α-helix mimetic
Terphenyl scaffold reasonably mimic the surface of α-helical peptide

The mimetic inhibited calmodulin-smMLCK interaction
IC50 = 9 nM

J. Am. Chem. Soc. 2001, 123, 5382
20

§2.1 PPI inhibitors

Protein grafting

KD = 52 ± 5 μM

Grafting Bak residues to stable aPP gave miniature protein inhibitor
Angew. Chem. Int. Ed. 2001, 40, 3806

21§2.1 PPI inhibitors

Cyclotide
Plant-derived disulfide-rich miniprotein with an intringuing circular cysteine knot (CCK)
Enhanced metabolic stability
Cell penetrating properties
Tolerance to substantial sequence varidation

Good scaffold for peptide grafting

p53-Hdm2/HdmX inhibitor
KD_Hdm2 = 2.3 ± 0.1 nM
KD_HdmX = 9.7 ± 0.9 nM
J. Am. Chem. Soc. 2013, 135, 11623
22

§2.2 Small-molecule PPI inhibitors

Approach to small-molecule PPI inhibitors
Enzyme-linked immunosorbent assay (ELISA)

PPI inhibition

No inhibition
Surface Plasmon Resonance (SPR)

PPI inhibition23§2.2 Small-molecule PPI inhibitors

Approach to small-molecule PPI inhibitors
Fluorescent Polarization (FP)

Fluorescence Resonance Energy Transfer (FRET)24

§2.2 Small-molecule PPI inhibitors

Hot spot theory

Alanine scanning

Only a few strong interactions are important for the binding free energy

Hot spot

A mimic of the smaller functional epitope may suffice for modulators
Science, 1995, 267, 383
25

§2.2 Small-molecule PPI inhibitors

Small-molecule PPI inhibitor
p53

MDM2

Crystal structure of p53-MDM2 complex revealed that• MDM2 possesses a relatively deep hydrophobic pocket• 15-residue α-helical transactivation domain of p53 insert into the hydrophobic cleft• In particular, a triad of p53 amino acids (Phe19, Trp23, and Leu26) is important
These facts raised expectation for small-molecule PPI inhibitor
Science, 1996, 274, 948
26

§2.2 Small-molecule PPI Inhibitors

High throughput screening (HTS)
Discovery of Nutlins (MDM2 inhibitor) through HTS (Roche)

cis-imidazoline

Science, 2004, 303, 844
27

§2.2 Small-molecule PPI inhibitors

Fragment based drug discovery (FBDD)

Structure-Activity Relationship (SAR) by NMRSignificant change is observed
15N-heteronuclear singlequantum correlation (15N-HSQC)

Science, 1996, 274, 1531
28

§2.2 Small-molecule PPI inhibitors

Fragment based drug design
Discovery of Bcl-2 family protein inhibitor ABT-737 (Abbott)
Kd = 4.3 ± 1.6 mM

Kd = 0.30 ± 0.03 mM

Ki ≤ 1 nM
Narure, 2005, 435, 677
Nat. Rev. Drug. Discov. 2008, 7, 989
29

§2.2 Small-molecule PPI inhibitors

Allosteric inhibitors

Pyrimidine-imidazole compound allosterically inhibits dimerization of iNOS

Disrupted helix 7 leads to disorder in helix 8Helix 8 is part of the dimer interface

PNAS, 2000, 97, 1506
30

§2.2 Small-molecule PPI inhibitors

Allosteric inhibitors
Maraviroc (Selzentry®, Pfizer): HIV-1 entry inhibitor
A marketed small-molecule PPI inhibitor

HTS

Maraviroc allosterically inhibits CCR5Mol. Pharmacol. 2008, 73, 789
31

§2.3 PPI stabilizers

PPI stabilizers

FK506 and Cyclosporin A (CsA) are known as immunosuppressive natural products
Their binding proteins were identified and named FKBP and Cyclophilin A (CyPA)
Science, 1984, 226, 544
Nature, 1989, 341, 755

Crystal structure of complex revealed they occupy enzyme active sites
Science, 1991, 252, 839
Nature, 1993, 361, 91

However, inhibition of enzymatic activity alone couldn’t explain immunosuppression32§2.3 PPI stabilizers

PPI stabilizers
CyPA-CsA (FKBP-FK506) binds to Calcineurin subunits CnA and CnBCsA and FK506 stabilize PPI and inhibit Cn phosphatase activity

Cell, 1991, 66, 807
33

§2.3 PPI stabilizers

Natural PPI stabilizer
Paclitaxel: isolated from Taxus brevifolia

Paclitaxel allosterically stabilize tubulin heterodimer
Suppressed dynamicity of tubulins induces
abnormal mitosis and leads to cell death
Paclitaxel works as anticancer drug

PNAS, 2006, 103, 1016634

§2.3 PPI stabilizers

Small-molecule PPI stabilizer
Tafamidis (Vyndaqel®, Pfizer): first-in-class transthyretin stabilizer to treat ATTR
Approved by the European Medicines Agency in 2011 ( Japan in 2013)

PNAS, 2012, 109, 9629
35

§3 PPI as anticancer drug target

PPIs in oncogenic signaling networks

PPIs play essential roles in relaying oncogenic signals
Trends. Pharmacol. Sci. 2013, 34, 39336

§3 PPI as anticancer drug target

Rising interest in targeting PPIs

Interest in targeting PPIs as anticancer strategies has increased
PPI is a higly promising target for anticancer therapeutics

Trends. Pharmacol. Sci. 2013, 34, 393

37§3 PPI as anticancer drug target

Approaches toward anticancer therapeutics

Trends. Pharmacol. Sci. 2013, 34, 393

38§3 PPI as anticancer drug target

Clinical anticancer PPI modulators

XIAP inhibitor

GDC-0152
Completed PhaseⅠ
safety/pharmacokinetic evaluation

Bcl-2 proteins inhibitor

NavitoclaxPhaseⅡ for CLL treatment
MDM2 inhibitor

RG-7112PhaseⅠ for both solid and hematologic malignancies
39§4 Future Outlook

Progress in PPI researches

The last 10-15 years has been significant progress in PPIM development40

§4 Future Outlook

Bio ventures focused on PPIM development

http://www.interprotein.com/

http://www.prismbiolab.sakura.ne.jp/41

§4 Future Outlook

Bio ventures focused on PPIM development42§4 Future Outlook

Bio ventures focused on PPIM development

http://www.interprotein.com/
43§4 Future Outlook

Future Outlook
Effort aimed at developing PPIM will be accelerated by a number of recent advances
e.g., HTS, PPI-focused library, fragment discovery, in silico screening

Further understanding of PPI networks will lead to therapeutics
for at present undruggable targets
e.g., Creutzfeldt-Jakob disease, Alzheimer’s disease44