Solid-phase Peptide Synthesis Research Articles

Total Syntheses of Cyclomarin and Metamarin Natural Products.

The first total synthesis of the heptapeptide Cyclomarin A (CymA) was achieved via new routes to chiral amino acid building blocks (highlighted) and solid-phase peptide synthesis. A structurally misassigned epimer of CymA (CymA'), Cyclomarin C, and Metamarin were also synthesized. Affirmation of the syntheses was corroborated by observations that the synthetic molecules have antimicrobial activities mirroring those of the natural products. Interestingly, CymA' is more potent than CymA.

Stereorandomized Oncocins with Preserved Ribosome Binding and Antibacterial Activity.

We recently showed that solid-phase peptide synthesis using racemic amino acids yields stereorandomized peptides comprising all possible diastereomers as homogeneous, single-mass products that can be purified by HPLC and that stereorandomization modulates activity, toxicity, and stability of membrane-disruptive cyclic and linear antimicrobial peptides (AMPs) and dendrimers. Here, we tested if stereorandomization might be compatible with target binding peptides with the example of the proline-rich AMP oncocin, which inhibits the bacterial ribosome. Stereorandomization of up to nine C-terminal residues preserved ribosome binding and antibacterial effects including activities against drug-resistant bacteria and protected against serum degradation. Surprisingly, fully stereorandomized oncocin was as active as L-oncocin in dilute growth media stimulating peptide uptake, although it did not bind the ribosome, indicative of an alternative mechanism of action. These experiments show that stereorandomization can be compatible with target binding peptides and can help understand their mechanism of action.

Multiplex antimicrobial activities of the self-assembled amphiphilic polypeptide β nanofiber KF-5 against vaginal pathogens

BackgroundVaginal infections caused by multidrug-resistant pathogens such as Candida albicans and Gardnerella spp. represent a significant health challenge. Current treatments often fail because of resistance and toxicity. This study aimed to synthesize and characterize a novel amphiphilic polypeptide, KF-5, and evaluate its antibacterial and antifungal activities, biocompatibility, and potential mechanisms of action.ResultsThe KF-5 peptide was synthesized via solid-phase peptide synthesis and self-assembled into nanostructures with filamentous and hydrogel-like configurations. Characterization by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) confirmed the unique nanostructural properties of KF-5. KF-5 (125, 250, or 500 µg/ml) demonstrated potent antibacterial and antifungal activities, with significant inhibitory effects on drug-resistant Candida albicans and Gardnerella spp. (P < 0.05). In vitro assays revealed that 500 µg/ml KF-5 disrupted microbial cell membranes, increased membrane permeability, and induced lipid oxidation, leading to cell death (P < 0.05). Cytotoxicity tests revealed minimal toxicity in human vaginal epithelial cells, keratinocytes, and macrophages, with over 95% viability at high concentrations. Molecular dynamics simulations indicated that KF-5 interacts with phospholipid bilayers through electrostatic interactions, causing membrane disruption. In vivo studies using a mouse model of vaginal infection revealed that 0.5, 1, and 2 mg/ml KF-5 significantly reduced fungal burden and inflammation, and histological analysis confirmed the restoration of vaginal mucosal integrity (P < 0.01). Compared with conventional antifungal treatments such as miconazole, KF-5 exhibited superior efficacy (P < 0.01).ConclusionsKF-5 demonstrates significant potential as a safe and effective antimicrobial agent for treating vaginal infections. Its ability to disrupt microbial membranes while maintaining biocompatibility with human cells highlights its potential for clinical application. These findings provide a foundation for further development of KF-5 as a therapeutic option for combating drug-resistant infections.

Scalable synthesis of sulfonium and selenonium peptides for selective methyllysine reader crosslinking

Lysine methylation readers are an important class of proteins that bind site-specifically methylated proteins for downstream regulation. Chemical probes that crosslink lysine methylation readers are highly desired to investigate the proteins from cellular samples. We recently reported NleS+me2 (norleucine-ε-dimethylsulfonium) as dimethyllysine mimic selectively crosslinks corresponding methyllysine readers. Although the sulfonium tools exhibited great promise, the synthetic availability may limit broad applications. In order to incorporate the unnatural amino acid, l-Fmoc-NleSme-OH (Fmoc: fluorenyl methoxycarbonyl) was synthesized as an important building block for solid-phase peptide synthesis (SPPS) in the previous synthetic route. It took six steps with resolution of racemic mixture and radical mediated thiol-ene reaction. As a result, the synthesis was not scalable with only 4.4 % overall yield. Here we report a much-improved synthesis method so that diverse NleS+me2 peptides could be readily prepared. In addition, the new method enables preparation of selenonium peptides for methyllysine reader crosslinking. We thus believe this synthetic method will be widely used to prepare sulfonium and selenonium probes for site-selective crosslinking.

Total Synthesis and Anticancer Evaluation of BZR-cotoxin IV

Anticancer peptides (ACPs) is a potential alternative for future cancer therapy. ACPs specifically inhibit cancer cells through a non-enzymatic membranolysis mechanism. One potential anticancer peptide worthy of investigation is BZR-cotoxin IV. Currently, research on BZR-cotoxin IV is limited to isolation, making it an interesting area for further development. One approach to developing BZR-cotoxin IV is through chemical synthesis techniques. This compound belongs to the group of cyclodepsipeptides that are naturally produced by the fungus Bipolaris zeicola Race 3. The synthesis of BZR cotoxin IV involves 3 steps: (1) Synthesis of the hydroxy acid precursor by conversion of L-valine amino acid to (2R)-hydroxyisovaleric acid, (2) synthesis of the linear depsipeptide using solid-phase peptide synthesis on 2-chlorotrityl chloride resin and (3) cyclization of the linear depsipeptide using solution phase synthesis to produce the BZR cotoxin IV compound with a purity of 11.7 %. BZR-cotoxin IV was characterized using HR-TOF-MS and 1H and 13C-NMR to validate the desired product. Anticancer activity testing was performed using the Resazurin assay on the HeLa cancer cell line, resulting in an IC50 of 187.50 μg/mL (214.014 μM) categorized as moderate. HIGHLIGHTS The chemical synthesis of BZR-cotoxin IV compounds has not been firstly reported. The synthesis of BZR-cotoxin IV was accomplished through a combination of solid-phase and solution-phase peptide synthesis. The objective was to synthesize and evaluate BZR-cotoxin IV for its cancer properties. GRAPHICAL ABSTRACT

Chemically Stable Diazo Peptides as Selective Probes of Cysteine Proteases in Living Cells.

Diazo peptides have been described earlier, however, due to their high reactivity have not been broadly used until today. Here, we report the preparation, properties, and applications of chemically stable internal diazo peptides. Peptidyl phosphoranylidene-esters and amides were found to react with triflyl azide primarily to novel 3,4-disubstituted triazolyl-peptides. Nonaflyl azide instead furnished diazo peptides, which are chemically stable from pH 1-14 as amides and from pH 1-8 as esters. Thus, diazo peptides prepared by solid phase peptide synthesis were stable to final deprotection with 95% trifluoroacetic acid. Diazo peptides with the recognition sequence of caspase-3 were identified as specific, covalent, and irreversible inhibitors of this enzyme at low nanomolar concentrations. A fluorescent diazo peptide entered living cells enabling microscopic imaging and quantification of apoptotic cells via flow cytometry. Thus, internal diazo peptides constitute a novel class of activity-based probes and enzyme inhibitors useful in chemical biology and medicinal chemistry.

Unveiling the Self-assembly and Therapeutic Efficacy of Antimicrobial Peptides SA4 Against Multidrug-Resistant A. baumannii.

Infections linked to Acinetobacter baumannii are one of the main risks of modern medicine. Biofilms formed by A. baumannii due to a protective extracellular polysaccharide matrix make them highly tolerant to conventional antibiotics and raise the possibility of antibiotic resistance. Antimicrobial peptides (AMPs) are gaining popularity due to their broad-spectrum actions and key properties of peptide self-assembly, making them a promising alternative to antibiotics. Here, we demonstrate that 12-residue synthetic self-assembled peptide SA4 nanostructures have enough antibacterial action to prevent the growth of mature bacterial biofilms. The SA4 peptide was successfully synthesized by using the solid-phase peptide synthesis method, and its self-assembly was prepared in water. The self-assembled peptide hydrogel formed nanotube structure was observed under a scanning electron microscope and further characterized to confirm their physical and molecular properties. The resulting hydrogel exhibits significant antibacterial activity against MDR A. baumannii strains (MDR-1 and MDR-2), responsible for many nosocomial infections. In addition, at various gel concentrations, this hydrogel has the potential to inhibit about 30-80% of biofilms formed by MDR strains. Furthermore, under a microscope, it has been observed that the rupture of the bacterial cell membrane and cell wall of A. baumannii cells is caused by peptide nanotubes generated by self-assemblies. Thus, peptide-based nanotubes present intriguing avenues for various biomedical applications. This is the first report of bacterial biofilm removal with SA4 peptide nanotubes, and offering a unique treatment for infections linked to biofilms.

Innovative On-Resin and in Solution Peptidomimetics Synthesis via Metal-Free Photocatalytic Approach.

Nowadays, peptidomimetics are widely studied, being useful tools in drug discovery and medicinal chemistry. The coupling between a carboxylic acid with an amine to form a peptide bond is the most common reaction to obtain peptides/peptidomimetics. In this work, we have investigated an innovative metal-free photoredox-catalyzed carbamoylation to form peptidomimetics thanks to the reaction between dihydropyridines functionalized with amino acids (or peptide sequences) and differently functionalized imines. As the organic photocatalyst, we used 4CzIPN, a donor-acceptor cyanoarene vastly used in photoredox catalysis. By easily modulating the amino acid (or peptide sequence), which is directly attached to the dihydropyridine, we proposed this key-reaction as new valuable method to obtain peptidomimetics, in situ building the not-natural portion of the sequence. Moreover, we successfully employed this methodology in solid phase peptide synthesis, both inserting the new photoredox-generated amino acid at the end or in the middle of the sequence. Peptides with different lengths and secondary structures were prepared, proving the success of this approach, even in sterically hindered environment. Herein, to the best of our knowledge, we describe the first photocatalytic protocol which allows the building of the peptide backbone, with the possibility of simultaneously inserting a non-coded amino acid in the sequence.

Sec-isoamyl Mercaptan (SIT), a Multi-faceted Disulfide Based Protecting Group for Cysteine Thiol

IntroductionThe successful synthesis of a peptide requires the synchronization of several processes, including the efficient execution of protecting group chemistry. For cysteine (Cys)-peptides, this is more crucial because the trifunctional Cys has a free thiol in its side chain. During synthesis, this free thiol function remains protected with suitable protecting groups and can be removed after synthesis using appropriate methods. Sec-isoamyl mercaptan (SIT) is a versatile disulfide-based protecting group for Cys side chain thiol. The removal of SIT from Cys thiol can be achieved using a mild reducing agent (e. g. DTT). This later promotes efficient disulfide bond formation by oxidation. SIT can also direct/activate the Cys thiol for the chemoselective formation of disulfide bonds by thiol-disulfide interchange.MethodsPeptides were synthesized using solid-phase peptide synthesis techniques. The removal of the SIT group was carried out either in the solid phase or in the solution.ResultsIn the present study, we have shown that SIT can be efficiently removed both in solution and on-resin to facilitate disulfide-bridged peptide synthesis. This was exemplified by two syntheses of an atosiban derivative, where the SIT was removed in solution or in solid-phase. Furthermore, a SIT-based facile one-pot synthesis pathway was devised for disulfide-rich peptides. The strategy was faster and greener as it did not involve using an oxidizer. Conotoxin (two S–S) and linaclotide amide (three S–S) were successfully synthesized by adopting the SIT-based strategy. Finally, a racemization study was carried out for SIT, Trt and StBu-protected Cys-peptides. In all cases, SIT-protected peptides showed lesser racemization than StBu-protected peptides. In some instances (synthesis using DMF), SIT-protected peptides showed less racemization compared to the Trt congeners.ConclusionOverall, the multifaceted use of SIT-protection during the synthesis of disulfide-rich peptides has illustrated its versatility as a Cys thiol protecting group.

Application of Sortase-Mediated Ligation for the Synthesis of Block Copolymers and Protein-Polymer Conjugates.

Sortase-mediated ligation (SML) has become a powerful tool for site-specific protein modification. However, sortaseA (SrtA) suffers from low catalytic efficiency and mediates an equilibrium reaction. Therefore, ligations with large macromolecules may be challenging. Here, the synthesis of polymeric building blocks for sortase-mediated ligation constituting peptide-polymers with either the recognition sequence for sortaseA (LPX1TGX2) or its nucleophilic counterpart (Gx) is demonstrated. The peptide-polymers are synthesized by solid-phase peptide synthesis followed by photo-iniferter (PI) reversible addition-fragmentation chain-transfer (RAFT) polymerization of various monomers. The building blocks are subsequently utilized to investigate possibilities and limitations when using macromolecules in SML. In particular, diblock copolymers are obtained even when using the orthogonal building blocks in equimolar ratio by exploiting a technique to shift the reaction equilibrium. However, ligations of two polymers can not be achieved when the degree of polymerization exceeds 100. Subsequently, C-terminal protein-polymer conjugates are synthesized. Several polymers are utilized that can replace the omnipresent polyethylene glycol (PEG) in future therapeutics. The conjugation is exemplified with a nanobody that is known for efficient neutralization of SARS-CoV-2. The study demonstrates a universal approach to polymer-LPX1TGX2 and Gx-polymer building blocks and gives insight into their application in SML.

Development of Donor-acceptor-type Molecules and Their Application as Analytical Reagents

π-Extended donor-acceptor (D-A)-type molecules, which bear both electron-donor and electron-acceptor substituents on the backbone, exhibit unique optical properties, such as bathochromic shifts in absorption and emission, large Stokes shifts, solvatochromic behavior, and fluorescence quenching in polar solvents. These unique properties are attributed to intramolecular charge transfer (ICT) or twisted intramolecular charge transfer (TICT) in the ground and excited states. This review article introduces three types of D-A-type molecules that are used as detection reagents for (1) methanol, (2) amino acids during solid-phase peptide synthesis (SPPS), and (3) amines present in the biological environment. For methanol detection, D-A-type fluorophores with basic guanidine moieties were developed to differentiate between methanol (MeOH) and ethanol (EtOH) based on the small difference in their pKa values (ΔpKa=0.4). Selective protonation of the guanidine moiety in methanol disrupts the D-A structure, allowing emission in the resultant polar environment. Similarly, an acid-base reaction between the hydrogen chloride (HCl) salts of the D-A-type molecules and amines is applied to detect amines during SPPS. In this method, a colorless solution of an HCl salt of the D-A-type molecule is deprotonated by amines, forming a yellow solution. This is the first reported quantitative and non-destructive colorimetric method for detecting amines. Finally, a turn-on-type amine-labeling reagent was developed for the nucleophilic aromatic substitution (SNAr) reaction. This new reagent enables protein staining of living cells with a large Stokes shift and without solvent-polarity-dependent fluorescence quenching.

BracketMaker: Visualization and optimization of chemical protein synthesis.

Chemical protein synthesis (CPS), in which custom peptide segments of ~20-60 aa are produced by solid-phase peptide synthesis and then stitched together through sequential ligation reactions, is an increasingly popular technique. The workflow of CPS is often depicted with a "bracket" style diagram detailing the starting segments and the order of all ligation, desulfurization, and/or deprotection steps to obtain the product protein. Brackets are invaluable tools for comparing multiple possible synthetic approaches and serve as blueprints throughout a synthesis. Drawing CPS brackets by hand or in standard graphics software, however, is a painstaking and error-prone process. Furthermore, the CPS field lacks a standard bracket format, making side-by-side comparisons difficult. To address these problems, we developed BracketMaker, an open-source Python program with built-in graphic user interface (GUI) for the rapid creation and analysis of CPS brackets. BracketMaker contains a custom graphics engine which converts a text string (a protein sequence annotated with reaction steps, introduced herein as a standardized format for brackets) into a high-quality vector or PNG image. To aid with new syntheses, BracketMaker's "AutoBracket" tool automatically performs retrosynthetic analysis on a set of segments to draft and rank all possible ligation orders using standard native chemical ligation, protection, and desulfurization techniques. AutoBracket, in conjunction with an improved version of our previously reported Automated Ligator (Aligator) program, provides a pipeline to rapidly develop synthesis plans for a given protein sequence. We demonstrate the application of both programs to develop a blueprint for 65 proteins of the minimal Escherichia coli ribosome.

Structure and functional studies of Avt1, a novel peptide from the sea anemone Aulactinia veratra

Sea anemones are a rich source of peptide toxins spanning a diverse range of biological activities, typically targeting proteins such as ion channels, receptors and transporters. These peptide toxins and their analogues are usually highly stable and selective for their molecular targets, rendering them of interest as molecular tools, insecticides and therapeutics. Recent transcriptomic and proteomic analyses of the sea anemone Aulactinia veratra identified a novel 28-residue peptide, designated Avt1. Avt1 was produced using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The liquid chromatography-mass spectrometry profile of synthetic Avt1 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds. The solution structure determined by NMR revealed that Avt1 adopts an inhibitor cystine knot (ICK) fold, in which a ring is formed by two disulfide bonds with a third disulfide penetrating the ring to create the pseudo-knot. This structure provides ICK peptides with high structural, thermal and proteolytic stability. Consistent with its ICK structure, Avt1 was resistant to proteolysis by trypsin, chymotrypsin and pepsin, although it was not a trypsin inhibitor. Avt1 at 100 nM showed no activity in patch–clamp electrophysiological assays against several mammalian voltage-gated ion channels, but has structural features similar to toxins targeting insect sodium ion channels. Although sequence homologues of Avt1 are found in a number of sea anemones, this is the first representative of this family to be characterised structurally and functionally.

DRUG PRECURSOR TARGETING THE BOMBESIN RECEPTOR FOR PEPTIDE-RECEPTOR RADIONUCLIDE THERAPY

Cancer is a leading cause of death worldwide. A promising modality for cancer treatment is peptide receptor radionuclide therapy. Therapeutic radionuclide is delivered using peptide-based vectors, which can bind to specific receptors on the cancer cell surface. Bombesin receptors are one of the receptors peculiar to many types of cancer, which can be targeted by peptide vectors. Peptides have a number of advantages, but they also have one serious drawback: low stability in the internal environment. To solve the problem, it is possible to the include a therapeutic peptide in the structure of a highly stable knottin peptide. Objective. The aim of the study is to examine the stability of BBN/C1-C2 structure, created on the basis of U5-scytotoxinSth1a knottin and bombesin tropic to bombesin receptor, and the ability of this structure to bind to target receptors on the cancer cell surface. Materials and Methods. BBN/C1-C2 peptide was obtained by solid-phase peptide synthesis. Then, is underwent chromatography purification under analytical chromatography and mass spectrometry control. Stability was studied by analytical chromatography. Competitive inhibition analysis was carried out using a fluorescently labeled GRP peptide with excess BBN/C1-C2 and fluorescently labeled BBN/C1-C2 with GRP bombesin receptor inhibitor. Cancer cell line PC-3 expressing bombesin receptors and normal cell line CHO-K1 not expressing bombesin receptors were used in the work. Results. The conducted studies have shown that hybrid BBN/C1-C2 peptide based on bombesin peptide inserted into the U5-scytotoxinSth1a knottin framework between the first and second cysteine residues has a greater stability compared to the commercial radiopharmaceutical PSMA-617. BBN/C1-C2 peptide is specific to bombesin receptor: it binds to PC-3 cancer cell line with a target bombesin receptor on its surface, and does not bind to the healthy CHO-K1 cell line, without a target receptor. BBN/C1-C2 peptide shows high affinity for the bombesin receptor, since GRP prevents its binding to the PC-3 cell line.

Synergistic combinations of novel polymyxins and rifampicin with improved eradication of colistin-resistant Pseudomonas aeruginosa biofilms

BackgroundIncreased prevalence of antimicrobial resistance coupled with a lack of new antibiotics against Gram-negative bacteria emphasize the imperative for novel therapeutic strategies. Colistin-resistant Pseudomonas aeruginosa constitutes a challenge, where conventional treatment options lack efficacy, in particular for biofilm-associated infections. Previously, synergy of colistin with other antibiotics was explored as an avenue for the treatment of colistin-resistant infections, and recently we reported our efforts towards colistin analogs capable of combating planktonic colistin-resistant strains. AimsThe aim of the present study was to investigate whether analogs of polymyxin B with improved potency in wild-type and moderate resistant Gram-negative pathogens would retain similarly increased activity in highly colistin-resistant clinical P. aeruginosa isolates (in planktonic and biofilm growth) when applied alone and in combination with rifampicin. Materials and methodsIn this in vitro study, we tested three analogs of polymyxin B prepared by solid-phase peptide synthesis. Antimicrobial susceptibility testing was performed by measurement of minimum inhibitory concentrations via the broth microdilution method. Interactions between two antimicrobials was quantified in a checkerboard broth microdilution assay by calculating the fractional inhibitory concentration index for each combination. For testing of antibiofilm activity a previously described model with alginate beads encapsulating a biofilm culture was applied. The minimum biofilm eradication concentrations (MBECs) were evaluated, and the fractional biofilm eradication concentration indices were calculated. Three recently identified colistin analogs (CEP932, CEP936 and CEP938) were tested against three isogenic pairs of colistin-susceptible and colistin-resistant P. aeruginosa clinical isolates as well as the reference strain PAO1. ResultsFor bacteria in planktonic growth CEP938 retained almost full potency in all three resistant isolates, while exhibiting similar activity as colistin in susceptible isolates. Against biofilms CEP938 was slightly more potent against PAO1 as compared to colistin, while also retaining activity against a biofilm of the colistin-resistant strain 41,782/98. Next, synergy between CEP938 and the antibiotic rifampicin was explored. Interestingly, CEP938 did not exhibit synergy with rifampicin in planktonic cultures. Importantly, for colistin-resistant biofilms the CEP938-rifampicin combination demonstrated activity superior to that found for the colistin-rifampicin combination. ConclusionsThe present study showed in vitro efficacy of CEP938 against both colistin-susceptible and colistin-resistant P. aeruginosa biofilms as well as an ability of CEP938 to synergize with rifampicin in biofilm eradication.

Ultrasound-Assisted Solid-Phase Affibody Synthesis Using ZEGFR:1907 as an Example-Superior to the Conventional Protocol?

Affibody molecules represent a class of highly specific binders of particular interest for the development of highly affine target-specific radiopharmaceuticals. Their chemical synthesis is, however, intricate due to their considerable length of 58 amino acids; thus, approaches to optimize their preparation are constantly being sought. As ultrasound assistance has recently been shown to increase the efficiency of amino acid conjugation during solid-phase peptide synthesis (SPPS), the influence of ultrasonication on the outcome of the SPPS-based preparation of the EGFR-specific affibody ZEGFR:1907 was compared to a common protocol relying on mechanical shaking. After the identification of a suitable solid support for the study, the execution of the systematic comparison of both approaches showed that conventional and ultrasound-assisted syntheses yielded equivalent results with analogous composition of the raw products. Further, both approaches produced the affibody in good isolated yields of >20% when applying the same optimal reagent excesses and coupling times for the conjugation of each amino acid. This indicates that, under optimal reaction conditions, the choice of solid support used has a much stronger influence on the outcome of the preparation of ZEGFR:1907 than the application of ultrasound, which did not further improve the synthesis results. Therefore, for the chemical synthesis of affibodies, great attention should be paid to the choice of a suitable solid support, enabling this highly interesting class of biomolecules to be obtained in good yields and to bring them more into the focus of radiopharmaceutical research.

Development of DuoMYC: a synthetic cell penetrant miniprotein that efficiently inhibits the oncogenic transcription factor MYC.

The master regulator transcription factor MYC is implicated in numerous human cancers, and its targeting is a long-standing challenge in drug development. MYC is a typical 'undruggable' target, with no binding pockets on its DNA binding domain and extensive intrinsically disordered regions. Rather than trying to target MYC directly with classical modalities, here we engineer synthetic miniproteins that can bind to MYC's target DNA, the enhancer box (E-Box), and potently inhibit MYC-driven transcription. We crafted the miniproteins via structure-based design and a combination of solid phase peptide synthesis and site-specific crosslinking. Our lead variant, DuoMYC, binds to E-Box DNA with high affinity (KD ~ 0.1 µM) and is able to enter cells and inhibit MYC-driven transcription with submicromolar potency (IC50 = 464 nM) as shown by reporter gene assay and confirmed by RNA sequencing. Notably, DuoMYC surpasses the efficacy of several other recently developed MYC inhibitors. Our results highlight the potential of engineered synthetic protein therapeutics for addressing challenging intracellular targets.

Development of DuoMYC: a synthetic cell penetrant miniprotein that efficiently inhibits the oncogenic transcription factor MYC

The master regulator transcription factor MYC is implicated in numerous human cancers, and its targeting is a long‐standing challenge in drug development. MYC is a typical ‘undruggable’ target, with no binding pockets on its DNA binding domain and extensive intrinsically disordered regions. Rather than trying to target MYC directly with classical modalities, here we engineer synthetic miniproteins that can bind to MYC’s target DNA, the enhancer box (E‐Box), and potently inhibit MYC‐driven transcription. We crafted the miniproteins via structure‐based design and a combination of solid phase peptide synthesis and site‐specific crosslinking. Our lead variant, DuoMYC, binds to E‐Box DNA with high affinity (KD ~ 0.1 µM) and is able to enter cells and inhibit MYC‐driven transcription with submicromolar potency (IC50 = 464 nM) as shown by reporter gene assay and confirmed by RNA sequencing. Notably, DuoMYC surpasses the efficacy of several other recently developed MYC inhibitors. Our results highlight the potential of engineered synthetic protein therapeutics for addressing challenging intracellular targets.

Application of a new green protocol in solid-phase peptide synthesis: identification of a new green solvent mixture compatible with TBEC/ETT

ABSTRACT Solid Phase Peptide Synthesis (SPPS) is the preferred technique for synthesizing bioactive peptides. However, traditional SPPS generates significant waste and employs hazardous solvents like DMF and DCM. The aim of this research is to investigate solvents and agents of coupling that align with the green chemistry and are suitable for all stages of SPPS. Some solvents, such as p-cymene and anisole, taken into consideration in this work, can be derived from renewable sources like plants and biomass, rendering them environmentally sustainable choices. However, many of these alternative solvents possess different physicochemical properties compared to DMF. To overcome this challenge, solvent mixtures are employed. In this study, we identified a novel green solvent mixture by combining anisole with NOP; its ability to swell different resins and its capability to solubilize all Fmoc protected amino acids was investigated. The same mixture was also assessed with a green coupling agent, TBEC, in combination with ETT as additive. Model peptides Aib-enkephalin and Aib-ACP were synthesized resulting in favorable outcomes in terms of peptide synthesis efficiency, 97.81% and 98.86%, respectively.

Indolizinylalanine Regioisomers: Tryptophan Isosteres with Bathochromic Fluorescence Emission.

We have developed a high yielding synthesis of indolizine and directly elaborated the molecule into three optically active indolizinylalanine regioisomers. The protocols exploit metal catalyzed coupling of indolizinyl-halides with organozinc reagents derived from carbamoylated iodoalanine esters. The scalable protocols provide products in a form amenable to solid-phase peptide synthesis (SPPS). When incorporated into peptides, the indolizine heterocycle is more basic and markedly less nucleophilic than tryptophan. Its protonated vinylpyridinium form is deeply colored in solution while the neutral heterocycle is highly fluorescent. The fluorescence quantum yield of indolizine exceeds that of indole and aza-indoles in water, suggesting that indolizinylalanines could be powerful optical probes of protein structure and dynamics, functioning as true tryptophan isosteres.