5. Safire
2
™ plate reader (Tecan, M€ annedorf, Switzerland)
6. MicroCal iTC200 (Malvern Pananalytical Ltd., Malvern, UK)
7. MiniSpin
® centrifuge (Eppendorf, Hamburg, Germany)
8. ImageQuant LAS 4000 (GE Healthcare, Chicago, USA)
9. ELISA microplate reader (Molecular Devices, San Jose, USA)
10. ELISA plate shaker
11. CO 2 INCUBATOR REVCO ULTIMA for shaking at 37
C in
a humidified atmosphere of 5% CO 2
12. Sorvall LYNX 6000 Superspeed Centrifuge
3 Methods
Here we describe the step-by-step procedure of the development of
AVLX-144, a potential stroke candidate, from the screening to
in vitro studies. This method section should inspire and provide
the reader an overview of peptide-based drug development.
3.1 Solid-Phase
Peptide Synthesis
Standard Fmoc peptide synthesis is described here to generate the
free carboxyl peptides. While there are two different strategies for
peptide synthesis, Fmoc and tert-butyloxycarbonyl (Boc), the latter
has harsher reaction conditions, especially during the cleavage step
where hydrofluoric acid (HF) must be used. On the other hand,
Fmoc chemistry offers milder conditions and also the chance to
follow up the reaction by spectrophotometric monitoring of the
dibenzofulvene, the removal product of the Fmoc protection
group.
In this method section we are going to tackle the standard
synthesis of peptide fragments able to bind to PDZ domains and
the generation of fluorescent peptide probes with Cy5-maleimide
for fluorescence polarization assays.
3.1.1 Synthesis of PDZ
Peptide Binders
1. For the synthesis of free carboxy PDZ binders we use preloaded Wang resin (see Note 2).
2. In a 10 mL disposable reaction tube, transfer 0.1 mmol of resin
(see Note 3) and wash with 5 mL of dichloromethane (DCM)
followed by 10 mL of DMF. Add 2 mL of DMF and cap the
tube. Allow the resin to swell for 30 min (see Fig. 2, step 1).
3. Drain solvent and resuspend resin with 2 mL of 20% (v/v)
piperidine in DMF. Incubate on a MiniBlock® (500 rpm) at
room temperature for 5 min. Drain solvent and wash resin with
20 mL of DMF. Repeat this procedure one more time.
4. Taking into account the synthesis scale (0.1 mmol g
À1 ), dissolve 4 eq of Fmoc-amino acid in 4 eq HBTU solution (see
Note 4). Vortex the solution until its complete solution.
PDZ Domain Peptide Inhibitors
163
2
™ plate reader (Tecan, M€ annedorf, Switzerland)
6. MicroCal iTC200 (Malvern Pananalytical Ltd., Malvern, UK)
7. MiniSpin
® centrifuge (Eppendorf, Hamburg, Germany)
8. ImageQuant LAS 4000 (GE Healthcare, Chicago, USA)
9. ELISA microplate reader (Molecular Devices, San Jose, USA)
10. ELISA plate shaker
11. CO 2 INCUBATOR REVCO ULTIMA for shaking at 37
C in
a humidified atmosphere of 5% CO 2
12. Sorvall LYNX 6000 Superspeed Centrifuge
3 Methods
Here we describe the step-by-step procedure of the development of
AVLX-144, a potential stroke candidate, from the screening to
in vitro studies. This method section should inspire and provide
the reader an overview of peptide-based drug development.
3.1 Solid-Phase
Peptide Synthesis
Standard Fmoc peptide synthesis is described here to generate the
free carboxyl peptides. While there are two different strategies for
peptide synthesis, Fmoc and tert-butyloxycarbonyl (Boc), the latter
has harsher reaction conditions, especially during the cleavage step
where hydrofluoric acid (HF) must be used. On the other hand,
Fmoc chemistry offers milder conditions and also the chance to
follow up the reaction by spectrophotometric monitoring of the
dibenzofulvene, the removal product of the Fmoc protection
group.
In this method section we are going to tackle the standard
synthesis of peptide fragments able to bind to PDZ domains and
the generation of fluorescent peptide probes with Cy5-maleimide
for fluorescence polarization assays.
3.1.1 Synthesis of PDZ
Peptide Binders
1. For the synthesis of free carboxy PDZ binders we use preloaded Wang resin (see Note 2).
2. In a 10 mL disposable reaction tube, transfer 0.1 mmol of resin
(see Note 3) and wash with 5 mL of dichloromethane (DCM)
followed by 10 mL of DMF. Add 2 mL of DMF and cap the
tube. Allow the resin to swell for 30 min (see Fig. 2, step 1).
3. Drain solvent and resuspend resin with 2 mL of 20% (v/v)
piperidine in DMF. Incubate on a MiniBlock® (500 rpm) at
room temperature for 5 min. Drain solvent and wash resin with
20 mL of DMF. Repeat this procedure one more time.
4. Taking into account the synthesis scale (0.1 mmol g
À1 ), dissolve 4 eq of Fmoc-amino acid in 4 eq HBTU solution (see
Note 4). Vortex the solution until its complete solution.
PDZ Domain Peptide Inhibitors
163