Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

Summary

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

Abstract

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency.

Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position.

We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites' viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a systematic screen for the most biological active conformation. This method provides a general, fast, and facile way to synthesize cyclic peptides.

Introduction

Protein-protein interactions (PPIs) play a pivotal role in most biological processes, from intracellular signal transduction to cell death¹. Hence, targeting PPIs is of fundamental importance to basic research and therapeutic applications. PPIs can be regulated by specific and stable antibodies, but antibodies are expensive and difficult to manufacture and have poor bioavailability. Alternatively, PPIs can be targeted by small molecules. Small molecules are easier to synthesize and inexpensive compared to antibodies; however, they are relatively less flexible and fit better to small cavities than to large protein-protein interfaces^2,3. Diverse st....

Protocol

1. Equipment and Reagents Preparation

1. Preparing equipment
   1. Perform all steps inside a fume hood using proper personal protective equipment.
   2. Chemically synthesize peptides on solid support using a Microwave Peptide Synthesizer with an additional module of Discover equipped with a fiber-optic temperature probe for controlling the microwave power delivery in a Teflon reaction vessel (30 ml, with a glass frit) or in a disposable polypropylene cartridge (12 ml, with a coarse frit).
   3. For proper mixing, connect nitrogen supply to the reaction vessel, or alternatively seal both ends of the polypropylene cartridge, and place on a rot....

Results

Here we describe the development of a focused small library of backbone cyclic peptides that specifically target vital PPIs of the Leishmania parasite and act as antiparasitic agents (for review about peptides that target PPIs as antiparasitic agents⁸⁷). Through the synthesis of novel backbone cyclic peptides, pharmacophores are conserved in a scaffold of extendable size. The strength of the focused library proposed here is the ability to vary peptide scaffold sizes while allowing a restricted degree .......

Discussion

The synthesis of a focused library of backbone cyclic peptides derived from the LACK protein of the Leishmania parasite using a fully automated microwave synthesizer is described. A focused library of cyclic peptides was developed with conserved pharmacophores and various linkers. Addition of various linkers such as glutaric anhydride, succinic anhydride, adipic acid, pimelic acid, lysine, ornithine, and other building blocks can be used to increase the variety of the conformational space of the cyclic peptides........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
Solid support, Rink Amide AM resin ML	CBL	BR-1330	loading: 0.49 mmol/g
Fmoc-Ala-OH	Advanced Chemtech	FA2100
Fmoc-Arg(Pbf)-OH	Advanced Chemtech	FR2136
Fmoc-Asn(Trt)-OH	Advanced Chemtech	FN2152
Fmoc-Asp(OBut)-OH	Advanced Chemtech	FD2192
Fmoc-Cys(Trt)-OH	Advanced Chemtech	FC2214
Fmoc-Gln(Trt)-OH	Advanced Chemtech	FQ2251
Fmoc-Glu(OtBu)-OH	Advanced Chemtech	FE2237
Fmoc-Gly-OH	Advanced Chemtech	FG2275
Fmoc-His(Trt)-OH	Advanced Chemtech	FH2316
Fmoc-Ile-OH	Advanced Chemtech	FI2326
Fmoc-Leu-OH	Advanced Chemtech	FL2350
Fmoc-Lys(Boc)-OH	Advanced Chemtech	FK2390
Fmoc-Met-OH	Advanced Chemtech	FM2400
Fmoc-Phe-OH	Advanced Chemtech	FF2425
Fmoc-Pro-OH	Advanced Chemtech	FP2450
Fmoc-Ser-(tBu)-OH	Advanced Chemtech	FS2476
Fmoc-Thr(tBu)-OH	Advanced Chemtech	FT2518
Fmoc-Trp(Boc)-OH	Advanced Chemtech	FW2527
Fmoc-Tyr(But)-OH	Advanced Chemtech	FY2563
Fmoc-Val-OH	Advanced Chemtech	FV2575
1-Methyl-2-pyrrolidinone (NMP)	Sigma	328634	Caution Toxic/Highly flammable/Irritant.
N,N-Dimethylformamide (DMF)	Alfa Aesar	43465	Caution Toxic. Use high quality DMF to eliminate side reactions such as Fmoc removal as a result of the dimethylamine traces from DMF decomposition.
Dichloromethane (DCM)	Sigma	D65100	Caution Harmful
Dibromomethane (DBM)	Sigma	D41868	Caution Harmful
Trifluoroacetic acid (TFA)	Sigma	T62200	Caution Corrosive/Toxic
Trifluoroacetic acid, HPLC grade (TFA)	Sigma	91707	Caution Corrosive/Toxic
Diethylether	Sigma	31690	Caution Highly flammable/Harmful
Triisopropylsilane (TIS)	Sigma	233781	Caution Irritant/Flammable
Water, HPLC grade	Sigma	270733
Acetonitroile, HPLC grade (ACN)	Fisher Scientific	A998-4	Caution Flammable/Irritant/Harmful
N,N-Diisopropylethylamine (DIEA)	Sigma	3440	Caution Corrosive/Highly flammable
Piperidine	Sigma	W290807	Caution Toxic/Highly flammable
Pyridine	Sigma	270970	Caution Highly flammable/Harmful
Ethanol (EtOH)	Sigma	459844	Caution Highly flammable/Irritant
1-Hydroxybenzotriazole hydrate (HOBt)	Sigma	157260	Caution Highly flammable/Irritant/Harmful
O-(Benzotriazol-1-yl)-N,N,N′,N′- tetramethyluronium hexafluorophosphate (HBTU)	Sigma	12804	Caution Irritant/Harmful
Benzotriazole-1-ly-oxy-tris-pyrrolidinophosphonium hexafluorphosphate (PyBOP)	Advanced Chemtech	RC8602	Caution Irritant
Ninhydrin	Sigma	454044	Caution Harmful
Phenol	Sigma	P3653	Caution Corrosive/Toxic
Potassium cyanide (KCN)	Sigma	11813	Caution Very Toxic
Potassium hydroxide (KOH)	Sigma	221473	Caution Toxic
N,N’-Diisopropylcarbodiimide (DIC)	Sigma	38370	Caution Flammable/ Toxic
4-Dimethylaminopyridine (DMAP)	Sigma	522805	Caution Toxic/Irritant
Glutaric anhydride	Sigma	G3806	Caution Flammable/Irritant/Harmful
Succinic anhydride	Sigma	239690	Caution Irritant/Harmful
Adipic acid	Sigma	A26357	Caution Toxic/Irritant
Pimelic acid	Sigma	P45001	Caution Toxic/Irritant
Chloranil	Sigma	23290	Caution Toxic/Irritant
Acetaldehyde	Sigma	402788	Caution Flammable/ Toxic
EQUIPMENT
Centrifuge	Beckman Coulter	Allegra 6R centrifuge
Lyophilizer	Labconco	freezone 4.5
Vacuum pump	Franklin Electric	model 1101101416 with 3/4 HP	Alcatel pump with Franklin Motor
Polypropylene cartridge 12 ml	Applied Separation	2419
Cap plug for 12 ml polypropylene cartridge	Applied Separation	8157
Polypropylene cartridge 3 ml	Applied Separation	2413
Cap plug for 3 ml polypropylene cartridge	Applied Separation	8054
Stop cocks PTFE	Applied Separation	2406
Tubes flat, 50 ml	VWR	21008-240
Extraction manifold, 20 pos, 16 x 100 mm tubes	Waters	WAT200609
Shaker, BD adams nutator mixer	Fisher scientific	22363152
Nalgene HDPE narrow mouth IP2 bottles, 125 ml	Fisher scientific	03-312-8
Erlenmeyer flask	Fisher Scientific	FB-501, 500 ml
Heating block	Thermolyne	1760 dri bath
Disposable borosilicate glass tubes with plain end	Fisher Scientific	14-961-25
Micropipettes and tips Finnpipette	Thermo	20–200 and 100–1,000 μl
HPLC vials - micro vl pp 400 µl PK100	VWR	69400-124
HPLC vial- Blue Snap-It Cap	VWR	66030-600
Analytical HPLC column	Peeke Scientific	U1-5C18Q-JJ	ultro 120 5 µm C18Q, 4.6 mm ID 150 mm
Prep HPLC column, XBridge	Waters	OBD C18 5 µm column	19 mm × 150 mm
Mass spectrometer	Applied Biosystems	Voyager DE-RP
Nitrogen cylinder
Desiccator
Analytical RP-HPLC system Shimadzu LC-20			equipped with: CBM-20A system controller, SPD-20A detector, CTO-20A column oven, 2 x LC-20AD solvent delivery unit, SIL-20AC autosampler, DGU-20A5 degasser (Shimadzu, MD, USA).
Preparative RP-HPLC system Shimadzu LC-20			equipped with: CBM-20A system controller, SPD-20A detector, CTO-20A column oven, 2 x LC-6AD solvent delivery unit and FRC-10A fraction collector (Shimadzu, MD, USA).