Sustained inhibition of the NaV1.7 sodium channel by engineered dimers of the domain II binding peptide GpTx-1.

Many efforts are underway to develop selective inhibitors of the voltage-gated sodium channel NaV1.7 as new analgesics. Thus far, however, in vitro selectivity has proved difficult for small molecules, and peptides generally lack appropriate pharmacokinetic properties. We previously identified the NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity via structure-guided analoging. To further understand GpTx-1 binding to NaV1.7, we have mapped the binding site to transmembrane segments 1-4 of the second pseudosubunit internal repeat (commonly referred to as Site 4) using NaV1.5/NaV1.7 chimeric protein constructs. We also report that select GpTx-1 amino acid residues apparently not contacting NaV1.7 can be derivatized with a hydrophilic polymer without adversely affecting peptide potency. Homodimerization of GpTx-1 with a bifunctional polyethylene glycol (PEG) linker resulted in a compound with increased potency and a significantly reduced off-rate, demonstrating the ability to modulate the function and properties of GpTx-1 by linking to additional molecules.

Liver-specific mitochondrial amidoxime-reducing component 1 (Mtarc1) knockdown protects the liver from diet-induced MASH in multiple mouse models.

BACKGROUND: Human genetic studies have identified several mitochondrial amidoxime-reducing component 1 (MTARC1) variants as protective against metabolic dysfunction-associated steatotic liver disease. The MTARC1 variants are associated with decreased plasma lipids and liver enzymes and reduced liver-related mortality. However, the role of mARC1 in fatty liver disease is still unclear. METHODS: Given that mARC1 is mainly expressed in hepatocytes, we developed an N-acetylgalactosamine-conjugated mouse Mtarc1 siRNA, applying it in multiple in vivo models to investigate the role of mARC1 using multiomic techniques. RESULTS: In ob/ob mice, knockdown of Mtarc1 in mouse hepatocytes resulted in decreased serum liver enzymes, LDL-cholesterol, and liver triglycerides. Reduction of mARC1 also reduced liver weight, improved lipid profiles, and attenuated liver pathological changes in 2 diet-induced metabolic dysfunction-associated steatohepatitis mouse models. A comprehensive analysis of mARC1-deficient liver from a metabolic dysfunction-associated steatohepatitis mouse model by metabolomics, proteomics, and lipidomics showed that Mtarc1 knockdown partially restored metabolites and lipids altered by diet. CONCLUSIONS: Taken together, reducing mARC1 expression in hepatocytes protects against metabolic dysfunction-associated steatohepatitis in multiple murine models, suggesting a potential therapeutic approach for this chronic liver disease.

PADLOC: a powerful tool to assign disulfide bond connectivities in peptides and proteins by NMR spectroscopy.

The determination of the disulfide bond connectivity in a peptide or protein represents a significant challenge. It is notoriously difficult to use NMR spectroscopy to assign disulfide connectivities because NMR spectra lack direct evidence for disulfide bonds. These bonds are typically inferred from three-dimensional structure calculations, which can result in ambiguous disulfide assignment. Here, we present a new NMR based methodology, in which the disulfide connectivity is obtained by applying Bayesian rules of inference to the local topology of cysteine residues. We illustrate how this approach successfully predicts the disulfide connectivity in proteins for which crystal structures are available in the protein data bank (PDB). We also demonstrate how this methodology is used with experimental NMR data for peptides with complex disulfide topologies, including hepcidin, Kalata-B1, and µ-Conotoxin KIIIA. In the case of µ-Conotoxin KIIIA, the PADLOC connectivity (1-15,2-9,4-16) differs from previously published results; additional evidence is presented demonstrating unequivocally that this newly proposed connectivity is correct.

Purification of N-acetylgalactosamine-modified-oligonucleotides using orthogonal anion-exchange and mixed-mode chromatography approaches.

N-acetylgalactosamine (GalNAc)-modified small interfering ribonucleic acids (siRNA) have shown promising outcomes for targeted siRNA delivery resulting in gene silencing in vivo; however, their structural complexity requires development of new purification methods to address high purity and recovery requirements. The current study evaluates complementary purification approaches using a mixed-mode Scherzo SS-C18 and anion-exchange (AEX) TSK-gel SuperQ-5PW for a range of single-stranded triantennary GalNAc-oligonucleotides. Initially, the semi-preparative mixed-mode support (10 × 250 mm, 3 µm) was compared against the preparative AEX analogue (21.5 × 300 mm, 13 µm), with the former affording double the recovery and higher purity of 95% over its AEX counterpart displaying 91% for a selected siRNA conjugate. An assortment of GalNAc-modified oligonucleotides was later purified using the mixed-mode resin revealing good recoveries (∼30-60%) and high purities of 90-94% ranging from straightforward to more challenging purifications. High sample loading in the 20 mg range was achieved, which was comparable with the larger preparative TSKgel SuperQ-5PW support. The Scherzo-SS-C18 resin also afforded some degree of resolution between diastereomers containing phosphorothioate functionalities. The TSKgel SuperQ-5PW support was later investigated to provide orthogonal separation selectivity to the Scherzo-SS-C18 column enabling purification of a selected, GalNAc-siRNA conjugate. The developed pH (8.5-11) and salt (0.3-0.7 M) gradients method provided enhanced separation selectivity between the free and conjugated siRNA, while minimizing formation of secondary structures and highlighting a complementary approach to deal with challenging purifications of oligonucleotide-GalNAc conjugates. Together, the use of AEX and mixed-mode columns provide much needed orthogonality to deal with complex GalNAc-modified oligonucleotides and potentially other upcoming modalities.

An antiangiogenic neurokinin-B/thromboxane A2 regulatory axis.

Establishment of angiogenic circuits that orchestrate blood vessel development and remodeling requires an exquisite balance between the activities of pro- and antiangiogenic factors. However, the logic that permits complex signal integration by vascular endothelium is poorly understood. We demonstrate that a "neuropeptide," neurokinin-B (NK-B), reversibly inhibits endothelial cell vascular network assembly and opposes angiogenesis in the chicken chorioallantoic membrane. Disruption of endogenous NK-B signaling promoted angiogenesis. Mechanistic analyses defined a multicomponent pathway in which NK-B signaling converges upon cellular processes essential for angiogenesis. NK-B-mediated ablation of Ca2+ oscillations and elevation of 3'-5' [corrected] cyclic adenosine monophosphate (cAMP) reduced cellular proliferation, migration, and vascular endothelial growth factor receptor expression and induced the antiangiogenic protein calreticulin. Whereas NK-B initiated certain responses, other activities required additional stimuli that increase cAMP. Although NK-B is a neurotransmitter/ neuromodulator and NK-B overexpression characterizes the pregnancy-associated disorder preeclampsia, NK-B had not been linked to vascular remodeling. These results establish a conserved mechanism in which NK-B instigates multiple activities that collectively oppose vascular remodeling.

Identification and Optimization of a Minor Allele-Specific siRNA to Prevent PNPLA3 I148M-Driven Nonalcoholic Fatty Liver Disease.

Human genome wide association studies confirm the association of the rs738409 single nucleotide polymorphism (SNP) in the gene encoding protein patatin like phospholipase domain containing 3 (PNPLA3) with nonalcoholic fatty liver disease (NAFLD); the presence of the resulting mutant PNPLA3 I148M protein is a driver of nonalcoholic steatohepatitis (NASH). While Pnpla3-deficient mice do not display an adverse phenotype, the safety of knocking down endogenous wild type PNPLA3 in humans remains unknown. To expand the scope of a potential targeted NAFLD therapeutic to both homozygous and heterozygous PNPLA3 rs738409 populations, we sought to identify a minor allele-specific small interfering RNA (siRNA). Limiting our search to SNP-spanning triggers, a series of chemically modified siRNA were tested in vitro for activity and selectivity toward PNPLA3 rs738409 mRNA. Conjugation of the siRNA to a triantennary N-acetylgalactosamine (GalNAc) ligand enabled in vivo screening using adeno-associated virus to overexpress human PNPLA3I148M versus human PNPLA3I148I in mouse livers. Structure-activity relationship optimization yielded potent and minor allele-specific compounds that achieved high levels of mRNA and protein knockdown of human PNPLA3I148M but not PNPLA3I148I. Testing of the minor allele-specific siRNA in PNPLA3I148M-expressing mice fed a NASH-inducing diet prevented PNPLA3I148M-driven disease phenotypes, thus demonstrating the potential of a precision medicine approach to treating NAFLD.

Dipeptidyl-quinolone derivatives inhibit hypoxia inducible factor-1α prolyl hydroxylases-1, -2, and -3 with altered selectivity.

Intracellular levels of the hypoxia-inducible transcription factor (HIF) are regulated under normoxic conditions by prolyl hydroxylases (PHD1, 2, and 3). Treatment of cells with PHD inhibitors stabilizes HIF-1α, eliciting an artificial hypoxic response that includes the transcription of genes involved in erythropoiesis, angiogenesis, and glycolysis. The different in vivo roles of the three PHD isoforms are not yet known, making a PHD-selective inhibitor useful as a biological tool. Although several chemical series of PHD inhibitors have been described, significant isoform selectivity has not been reported. Here we report the synthesis and activity of dipeptidyl analogues derived from a potent but non-selective quinolone scaffold. The compounds were prepared by Pd-catalyzed reductive carbonylation of the 6-iodoquinolone derivative to form the aldehyde directly, which was then attached to a solid support via reductive amination. Amino acids were coupled, and the resulting dipeptidyl-quinolone derivatives were screened, revealing retention of PHD inhibitory activity but an altered PHD1, 2, and 3 selectivity profile. The compounds were found to be ∼10-fold more potent against PHD1 and PHD3 than against PHD2, whereas the specific parent compound had shown no appreciable selectivity among the different PHD isoforms.

Purification of guanine-quadruplex using monolithic stationary phase under ion-exchange conditions.

The current study investigates a method for purification of the G-quadruplex secondary structure, naturally formed by a guanine-rich 21-mer oligonucleotide strand using a monolithic convective interaction media-quaternary amine (CIM-QA) column under ion-exchange conditions. The monolithic support was initially evaluated on a preparative scale against a highly efficient TSKgel SuperQ-5PW ion-exchange support designed for oligonucleotide purification. The CIM analogue demonstrated clear advantages over the particle-based support on the basis of rapid separation times, while also affording high purity of the G-quadruplex. Various parameters were investigated including the type of mobile phase anion, cation, pH and injection load to induce and control quadruplex formation, as well as enhance chromatographic separation and final purity. Potassium afforded the most prominent quadruplex formation, yet sodium allowed for the highest resolution and purity to be achieved with a 30 mg injection on an 8 ml CIM-QA monolithic column. This method was applied to purify in excess of 300 mg of the quadruplex, with excellent retention time precision of under 1% RSD. Native mass spectrometry was utilized to confirm the identity of the intact G-quadruplex under non-denaturing conditions, while ion-pairing reversed-phase methods confirmed the presence of the single-stranded oligonucleotide in high purity (92%) under denaturing conditions. The key advantage of the purification method enables isolation of the G-quadruplex in its native state on a milli-gram scale, allowing structural characterization to further our knowledge of its role and function. The G-quadruplex can also be subsequently denaturated at elevated temperature causing single strand formation if additional reactions are to be pursued, such as annealing to form a duplex, and evaluation in in vitro or in vivo studies.

Engineering NaV1.7 Inhibitory JzTx-V Peptides with a Potency and Basicity Profile Suitable for Antibody Conjugation To Enhance Pharmacokinetics.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.

Exploration of structure--activity relationships among foldamer ligands for a specific protein binding site via parallel and split-and-mix library synthesis.

We describe the use of parallel and split-and-mix library synthesis strategies for exploration of structure-activity relationships among peptidic foldamer ligands for the BH3-recognition cleft of the anti-apoptotic protein Bcl-xL. This effort began with a chimeric (alpha/beta+alpha)-peptide oligomer (composed of an alpha/beta-peptide segment and an alpha-peptide segment) that we previously identified to bind tightly to the target cleft on Bcl-xL. The side chains that interact with Bcl-xL were varied in a 1000-member one-bead-one-compound library. Fluorescence polarization (FP) screening identified four new analogues with binding affinities similar to that of the lead compound but no analogues with enhanced affinity. These results suggested that significant improvements in affinity were unlikely in this series. We then used library synthesis to examine backbone variations in the C-terminal alpha-peptide segment of the lead compound. These studies provided an opportunity for direct comparison of parallel and split-and-mix synthesis formats for foldamer libraries with respect to synthetic variability and assay sensitivity. We found that compounds from both the parallel and one-bead-one-compound libraries could be reliably screened in a competition FP assay without purification of library members. Our findings should facilitate the use of combinatorial library synthesis for exploration of foldamers as inhibitors of protein-protein interactions.

Discovery of Tarantula Venom-Derived NaV1.7-Inhibitory JzTx-V Peptide 5-Br-Trp24 Analogue AM-6120 with Systemic Block of Histamine-Induced Pruritis.

Inhibitors of the voltage-gated sodium channel NaV1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified NaV1.7-inhibitory peptides GpTx-1 and JzTx-V from tarantula venom screens. Potency and selectivity were modulated through attribute-based positional scans of native residues via chemical synthesis. Herein, we report JzTx-V lead optimization to identify a pharmacodynamically active peptide variant. Molecular docking of peptide ensembles from NMR into a homology model-derived NaV1.7 structure supported prioritization of key residues clustered on a hydrophobic face of the disulfide-rich folded peptide for derivatization. Replacing Trp24 with 5-Br-Trp24 identified lead peptides with activity in electrophysiology assays in engineered and neuronal cells. 5-Br-Trp24 containing peptide AM-6120 was characterized in X-ray crystallography and pharmacokinetic studies and blocked histamine-induced pruritis in mice after subcutaneous administration, demonstrating systemic NaV1.7-dependent pharmacodynamics. Our data suggests a need for high target coverage based on plasma exposure for impacting in vivo end points with selectivity-optimized peptidic NaV1.7 inhibitors.

Pharmacological characterization of potent and selective NaV1.7 inhibitors engineered from Chilobrachys jingzhao tarantula venom peptide JzTx-V.

Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.

Engineering Antibody Reactivity for Efficient Derivatization to Generate NaV1.7 Inhibitory GpTx-1 Peptide-Antibody Conjugates.

The voltage-gated sodium channel NaV1.7 is a genetically validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain; however functional NaV1.7 targeting antibodies are not known. In this report, we describe NaV1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the NaV1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of 13 potential cysteine conjugation sites in the antibody scaffold was tuned using a model alkylating agent. Subsequent reactions with the peptide identified cysteine locations with the highest conversion to desired conjugates, which blocked NaV1.7 currents in whole cell electrophysiology. Variations in attachment site, linker, and peptide loading established design parameters for potency optimization. Antibody conjugation led to in vivo half-life extension by 130-fold relative to a nonconjugated GpTx-1 peptide and differential biodistribution to nerve fibers in wild-type but not NaV1.7 knockout mice. This study describes the optimization and application of antibody derivatization technology to functionally inhibit NaV1.7 in engineered and neuronal cells.

Single Residue Substitutions That Confer Voltage-Gated Sodium Ion Channel Subtype Selectivity in the NaV1.7 Inhibitory Peptide GpTx-1.

There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit NaV1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with NaV1.7 and related NaV subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on NaV1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a NaV1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work.

Application of microwave irradiation to the synthesis of 14-helical beta-peptides.

[structure: see text] We have evaluated the effects of microwave irradiation on the solid-phase synthesis of beta-peptides. Sequences designed to adopt the 14-helix, especially those containing the structure-promoting residue trans-2-aminocyclohexanecarboxylic acid (ACHC), suffer from poor synthetic efficiency under standard SPPS conditions. A comparison of microwave and conventional heating shows that both provide excellent synthetic results for shorter sequences; however, we identify a clear benefit from microwave irradiation for longer beta-peptides.

Engineering potent and selective analogues of GpTx-1, a tarantula venom peptide antagonist of the Na(V)1.7 sodium channel.

NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20× and 1000× over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp(29), Lys(31), and Phe(34) near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000× selectivity against NaV1.4 and NaV1.5.

Pharmaceutical Optimization of Peptide Toxins for Ion Channel Targets: Potent, Selective, and Long-Lived Antagonists of Kv1.3.

To realize the medicinal potential of peptide toxins, naturally occurring disulfide-rich peptides, as ion channel antagonists, more efficient pharmaceutical optimization technologies must be developed. Here, we show that the therapeutic properties of multiple cysteine toxin peptides can be rapidly and substantially improved by combining direct chemical strategies with high-throughput electrophysiology. We applied whole-molecule, brute-force, structure-activity analoging to ShK, a peptide toxin from the sea anemone Stichodactyla helianthus that inhibits the voltage-gated potassium ion channel Kv1.3, to effectively discover critical structural changes for 15× selectivity against the closely related neuronal ion channel Kv1.1. Subsequent site-specific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000× over Kv1.1) with picomolar functional activity in whole blood and a pharmacokinetic profile suitable for weekly administration in primates. The pharmacological potential of the optimized toxin peptide was demonstrated by potent and sustained inhibition of cytokine secretion from T cells, a therapeutic target for autoimmune diseases, in cynomolgus monkeys.

Solid-phase peptide synthesis using microwave irradiation.

Since the advent of solid-phase peptide synthesis (SPPS) in the late 1950s, numerous advancements in the underlying chemistry (i.e., orthogonal protection strategy, coupling reagents, and solid support matrices) have greatly improved the efficiency of the technique. More recently, application of microwave radiation to SPPS has been found to reduce reaction time and/or increase the initial purity of synthetic peptide products. In this protocol, conditions are described to accomplish rapid peptide coupling and 9-fluorenylmethoxycarbonyl (Fmoc) removal reactions under temperature-controlled conditions in either a manual or automated synthesis format using a microwave reactor. These microwave-assisted peptide synthesis procedures have been used to rapidly prepare a "difficult" peptide sequence from the acyl carrier protein, ACP(65-74), in less than 3 h and the reduced, linear precursor to human hepcidin, in high initial purity.

Targeting protein-protein interactions: lessons from p53/MDM2.

The tremendous challenge of inhibiting therapeutically important protein-protein interactions has created the opportunity to extend traditional medicinal chemistry to a new class of targets and to explore nontraditional strategies. Here we review a widely studied system, the interaction between tumor suppressor p53 and its natural antagonist MDM2, for which both traditional and nontraditional approaches have been reported. This system has been a testing ground for novel proteomimetic scaffold-based strategies, i.e., for attempts to mimic the recognition surface displayed by a folded protein with unnatural oligomers. Retroinverso peptides, peptoids, terphenyls, beta-hairpins, p-oligobenzamides, beta-peptides, and miniproteins have all been explored as inhibitors of the p53/MDM2 interaction, and we focus on these oligomer-based efforts. Traditional approaches have been successful as well, and we briefly review small molecule inhibitors along with other strategies for reactivation of the p53 pathway, for comparison with oligomer- based approaches. We close with comments on an emerging dichotomy among protein-protein interaction targets.

Parallel synthesis of peptide libraries using microwave irradiation.

The application of microwave irradiation to solid-phase peptide synthesis increases product purity and reduces reaction time. Parallel synthesis in 96-well polypropylene filter plates with microwave irradiation is an efficient method for the rapid generation of combinatorial peptide libraries in sufficient purity to assay the products directly for biological activity without HPLC purification. In this protocol, the solid-phase support is arrayed into each well of a 96-well plate, reagents are delivered using a multichannel pipette and a microwave reactor is used to complete peptide coupling reactions in 6 min and Fmoc-removal reactions in 4 min under temperature-controlled conditions. The microwave-assisted parallel peptide synthesis protocol has been used to generate a library of difficult hexa-beta-peptides in 61% average initial purity (50% yield) and has been applied to the preparation of longer alpha- and beta-peptides. Using this protocol, a library of 96 different hexapeptides can be synthesized in 24 h (excluding characterization).