Using Chemical Epigenetics to Target Cancer.

Transcription is epigenetically regulated by the orchestrated function of chromatin-binding proteins that tightly control the expression of master transcription factors, effectors, and supportive housekeeping genes required for establishing and propagating the normal and malignant cell state. Rapid advances in chemical biology and functional genomics have facilitated exploration of targeting epigenetic proteins, yielding effective strategies to target transcription while reducing toxicities to untransformed cells. Here, we review recent developments in conventional active site and allosteric inhibitors, peptidomimetics, and novel proteolysis-targeted chimera (PROTAC) technology that have deepened our understanding of transcriptional processes and led to promising preclinical compounds for therapeutic translation, particularly in cancer.

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

Human defensin-inspired discovery of peptidomimetic antibiotics.

SignificanceWe report the development of peptidomimetic antibiotics derived from a natural antimicrobial peptide, human α-defensin 5. By engaging multiple bacterial targets, the lead compound is efficacious in vitro and in vivo against bacteria with highly inducible antibiotic resistance, promising a useful therapeutic agent for the treatment of infections caused by antibiotic-resistant bacteria.

Structural basis of peptidomimetic agonism revealed by small- molecule GLP-1R agonists Boc5 and WB4-24.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in treating type 2 diabetes and obesity with proven cardiovascular benefits. However, most of these agonists are peptides and require subcutaneous injection except for orally available semaglutide. Boc5 was identified as the first orthosteric nonpeptidic agonist of GLP-1R that mimics a broad spectrum of bioactivities of GLP-1 in vitro and in vivo. Here, we report the cryoelectron microscopy structures of Boc5 and its analog WB4-24 in complex with the human GLP-1R and Gs protein. Bound to the extracellular domain, extracellular loop 2, and transmembrane (TM) helices 1, 2, 3, and 7, one arm of both compounds was inserted deeply into the bottom of the orthosteric binding pocket that is usually accessible by peptidic agonists, thereby partially overlapping with the residues A8 to D15 in GLP-1. The other three arms, meanwhile, extended to the TM1-TM7, TM1-TM2, and TM2-TM3 clefts, showing an interaction feature substantially similar to the previously known small-molecule agonist LY3502970. Such a unique binding mode creates a distinct conformation that confers both peptidomimetic agonism and biased signaling induced by nonpeptidic modulators at GLP-1R. Further, the conformational difference between Boc5 and WB4-24, two closed related compounds, provides a structural framework for fine-tuning of pharmacological efficacy in the development of future small-molecule therapeutics targeting GLP-1R.

Scaffold Matcher: A CMA-ES based algorithm for identifying hotspot aligned peptidomimetic scaffolds.

The design of protein interaction inhibitors is a promising approach to address aberrant protein interactions that cause disease. One strategy in designing inhibitors is to use peptidomimetic scaffolds that mimic the natural interaction interface. A central challenge in using peptidomimetics as protein interaction inhibitors, however, is determining how best the molecular scaffold aligns to the residues of the interface it is attempting to mimic. Here we present the Scaffold Matcher algorithm that aligns a given molecular scaffold onto hotspot residues from a protein interaction interface. To optimize the degrees of freedom of the molecular scaffold we implement the covariance matrix adaptation evolution strategy (CMA-ES), a state-of-the-art derivative-free optimization algorithm in Rosetta. To evaluate the performance of the CMA-ES, we used 26 peptides from the FlexPepDock Benchmark and compared with three other algorithms in Rosetta, specifically, Rosetta's default minimizer, a Monte Carlo protocol of small backbone perturbations, and a Genetic algorithm. We test the algorithms' performance on their ability to align a molecular scaffold to a series of hotspot residues (i.e., constraints) along native peptides. Of the 4 methods, CMA-ES was able to find the lowest energy conformation for all 26 benchmark peptides. Additionally, as a proof of concept, we apply the Scaffold Match algorithm with CMA-ES to align a peptidomimetic oligooxopiperazine scaffold to the hotspot residues of the substrate of the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our implementation of CMA-ES into Rosetta allows for an alternative optimization method to be used on macromolecular modeling problems with rough energy landscapes. Finally, our Scaffold Matcher algorithm allows for the identification of initial conformations of interaction inhibitors that can be further designed and optimized as high-affinity reagents.

In-silico assessment of novel peptidomimetics inhibitor targeting STAT3 and STAT4 N-terminal domain dimerization: A comprehensive study using molecular docking, molecular dynamics simulation, and binding free energy analysis.

Dysregulation in Janus kinase-Signal Transducer and Activation of Transcription (JAK-STAT) pathway is closely linked to various cancer types. The N-terminal domain (NTD) of STAT proteins, upon dimerization, assumes a multifaceted role with remarkable adaptability in mediating interactions between proteins. Consequently, the strategic targeting of the N-terminal domain of STATs has emerged as a promising tactic for disrupting dimerization and impeding the translocation of STAT proteins. In this study, we have deployed an integrated in-silico methodology to rationally design Peptidomimetic foldamers as inhibitors of the N-terminal domains of STAT3 and STAT4, with the objective of disrupting protein dimerization. Consequently, we have judiciously designed a series of peptidomimetics that encompass ß3-amino acids, bearing side chains that mimic the residues within interface II of the dimeric structures of the NTDs. Employing molecular docking techniques; we have assessed the binding affinity of these designed peptidomimetics toward both the NTDs. Furthermore, we have conducted an evaluation of the stability and conformational alterations within the docked complexes over an extensive Molecular Dynamics, subsequently computing the binding free energy utilizing MM/PBSA calculations. Our findings unequivocally demonstrate that the peptidomimetic foldamers we have devised (Peptide-A, Peptide-B, and Peptide-C) exhibit a propensity to bind to and impede the dimerization process of the NTDs of both STAT3 and STAT4. These outcomes serve to underscore the potential of these meticulously designed peptidomimetics as potential candidates meriting further exploration in the realm of cancer prevention and management.

Binding Dynamics of a Stapled Peptide Targeting the Transcription Factor NF-Y.

Transcription factors (TFs) play a central role in gene regulation, and their malfunction can result in a plethora of severe diseases. TFs are therefore interesting therapeutic targets, but their involvement in protein-protein interaction networks and the frequent lack of well-defined binding pockets render them challenging targets for classical small molecules. As an alternative, peptide-based scaffolds have proven useful, in particular with an α-helical active conformation. Peptide-based strategies often require extensive structural optimization efforts, which could benefit from a more detailed understanding of the dynamics in inhibitor/protein interactions. In this study, we investigate how truncated stapled α-helical peptides interact with the transcription factor Nuclear Factor-Y (NF-Y). We identified a 13-mer minimal binding core region, for which two crystal structures with an altered C-terminal peptide conformation when bound to NF-Y were obtained. Subsequent molecular dynamics simulations confirmed that the C-terminal part of the stapled peptide is indeed relatively flexible while still showing defined interactions with NF-Y. Our findings highlight the importance of flexibility in the bound state of peptides, which can contribute to overall binding affinity.

Composition and Conformation of Hetero- versus Homo-Fluorinated Triazolamers Influence their Activity on Islet Amyloid Polypeptide Aggregation.

Novel fluorinated foldamers based on aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF2) units were synthesized and their conformational behaviour was studied by NMR and molecular dynamics. Their activity on the aggregation of the human islet amyloid polypeptide (hIAPP) amyloid protein was evaluated by fluorescence spectroscopy and mass spectrometry. The fluorine labelling of these foldamers allowed the analysis of their interaction with the target protein. We demonstrated that the preferred extended conformation of homotriazolamers of 1,4-Tz-CF2 unit increases the aggregation of hIAPP, while the hairpin-like conformation of more flexible heterotriazolamers containing two 1,4-Tz-CF2 units mixed with natural amino acids from the hIAPP sequence reduces it, and more efficiently than the parent natural peptide. The longer heterotriazolamers having three 1,4-Tz-CF2 units adopting more folded hairpin-like and ladder-like structures similar to short multi-stranded ß-sheets have no effect. This work demonstrates that a good balance between the structuring and flexibility of these foldamers is necessary to allow efficient interaction with the target protein.

Exploiting high-energy hydration sites for the discovery of potent peptide aldehyde inhibitors of the SARS-CoV-2 main protease with cellular antiviral activity.

Small-molecule antivirals that prevent the replication of the SARS-CoV-2 virus by blocking the enzymatic activity of its main protease (Mpro) are and will be a tenet of pandemic preparedness. However, the peptidic nature of such compounds often precludes the design of compounds within favorable physical property ranges, limiting cellular activity. Here we describe the discovery of peptide aldehyde Mpro inhibitors with potent enzymatic and cellular antiviral activity. This structure-activity relationship (SAR) exploration was guided by the use of calculated hydration site thermodynamic maps (WaterMap) to drive potency via displacement of waters from high-energy sites. Thousands of diverse compounds were designed to target these high-energy hydration sites and then prioritized for synthesis by physics- and structure-based Free-Energy Perturbation (FEP+) simulations, which accurately predicted biochemical potencies. This approach ultimately led to the rapid discovery of lead compounds with unique SAR that exhibited potent enzymatic and cellular activity with excellent pan-coronavirus coverage.

Design, synthesis and evaluation of bioactivity of peptidomimetics based on chloroalkene dipeptide isosteres.

Ample biologically active peptides have been found, identified and modified for use in drug discovery to date. However, several factors, such as low metabolic stability due to proteolysis and non-specific interactions with multiple off-target molecules, might limit the therapeutic use of peptides. To enhance the stability and/or bioactivity of peptides, the development of "peptidomimetics," which mimick peptide molecules, is considered to be idealistic. Hence, chloroalkene dipeptide isosteres (CADIs) was designed, and their synthetic methods have been developed by us. Briefly, in a CADI an amide bond in peptides is replaced with a chloroalkene structure. CADIs might be superior mimetics of amide bonds because the Van der Waals radii (VDR) and the electronegativity value of a chlorine atom are close to those of the replaced oxygen atom. By a developed method of the "liner synthesis", N-tert-butylsulfonyl protected CADIs can be synthesized via a key reaction involving diastereoselective allylic alkylation using organocopper reagents. On the other hand, by a developed method of the "convergent synthesis", N-fluorenylmethoxycarbonyl (Fmoc)-protected carboxylic acids can be also constructed based on N- and C-terminal analogues from corresponding amino acid starting materials via an Evans syn aldol reaction and the Ichikawa allylcyanate rearrangement reaction involving a [3.3] sigmatropic rearrangement. Notably, CADIs can also be applied for Fmoc-based solid-phase peptide synthesis and therefore introduced into bioactive peptides including as the Arg-Gly-Asp (RGD) peptide and the amyloid ß fragment Lys-Leu-Val-Phe-Phe (KLVFF) peptide, which are correlated with cell attachment and Alzheimer's disease (AD), respectively. These CADI-containing peptidomimetics stabilized the conformation and enhanced the potency of the cyclic RGD peptide and the cyclic KLVFF peptide.

Combination of the amide-to-triazole substitution strategy with alternative structural modifications for the metabolic stabilization of tumor-targeting, radiolabeled peptides.

Radiolabeled peptides play a key role in nuclear medicine to selectively deliver radionuclides to malignancies for diagnosis (imaging) and therapy. Yet, their efficiency is often compromised by low metabolic stability. The use of 1,4-disubstituted 1,2,3-triazoles (1,4-Tzs) as stable amide bond bioisosteres can increase the half-life of peptides in vivo while maintaining their biological properties. Previously, the amide-to-triazole substitution strategy was used for the stabilization of the pansomatostatin radioligand [111In]In-AT2S, resulting in the mono-triazolo-peptidomimetic [111In]In-XG1, a radiotracer with moderately enhanced stability in vivo and retained ability to bind multiple somatostatin receptor (SSTR) subtypes. However, inclusion of additional 1,4-Tz led to a loss of affinity towards SST2R, the receptor overexpressed by most SSTR-positive cancers. To enhance further the stability of [111In]In-XG1, alternative modifications at the enzymatically labile position Thr10-Phe11 were employed. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-peptide conjugates were synthesized with a 1,4-Tz (Asn5-Ψ[Tz]-Phe6) and either a ß-amino acid (ß-Phe11), reduced amide bond (Thr10-Ψ[NH]-Phe11), or N-methylated amino acid (N-Me-Phe11). Two of the new peptidomimetics were more stable in blood plasma in vitro than [111In]In-XG1. Yet none of them retained high affinity towards SST2R. We demonstrate for the first time the combination of the amide-to-triazole substitution strategy with alternative stabilization methods to improve the metabolic stability of tumor-targeting peptides.

A virtual library of small molecules mimicking dipeptides.

Virtual combinatorial libraries are prevalent in drug discovery due to improvements in the prediction of synthetic reactions that can be performed. This has gone hand in hand with the development of virtual screening capabilities to effectively screen the large chemical spaces spanned by exhaustive enumeration of reaction products. In this study, we generated a small-molecule dipeptide mimic library to target proteins binding small peptides. The library was created based on the general idea of peptide synthesis, that is, amino acid mimics were reacted in silico to form the dipeptide mimics, yielding 2,036,819 unique compounds. After docking calculations, two compounds from the library were synthesized and tested against WD repeat-containing protein 5 (WDR5) and histamine receptors H1-H4 to evaluate whether these molecules are viable in assays. The compounds showed the highest potency at the histamine H3 receptor, with Ki values in the two-digit micromolar range.

Cyclobutanone Inhibitors of Diaminopimelate Desuccinylase (DapE) as Potential New Antibiotics.

Based on our previous success in using cyclobutanone derivatives as enzyme inhibitors, we have designed and prepared a 37-member library of α-aminocyclobutanone amides and sulfonamides, screened for inhibition of the bacterial enzyme diaminopimelate desuccinylase (DapE), which is a promising antibiotic target, and identified several inhibitors with micromolar inhibitory potency. Molecular docking suggests binding of the deprotonated hydrate of the strained cyclobutanone, and thermal shift analysis with the most potent inhibitor (3y, IC50 = 23.1 µM) enabled determination of a Ki value of 10.2 +/- 0.26 µM and observed two separate Tm values for H. influenzae DapE (HiDapE).

Ligand-Based Design of Selective Peptidomimetic uPA and TMPRSS2 Inhibitors with Arg Bioisosteres.

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases.

Exploring Peptido-Nanocomposites in the Context of Amyloid Diseases.

Therapeutic peptides are a major class of pharmaceutical drugs owing to their target-binding specificity as well as their versatility in inhibiting aberrant protein-protein interactions associated with human pathologies. Within the realm of amyloid diseases, the use of peptides and peptidomimetics tailor-designed to overcome amyloidogenesis has been an active research endeavor since the late 90s. In more recent years, incorporating nanoparticles for enhancing the biocirculation and delivery of peptide drugs has emerged as a frontier in nanomedicine, and nanoparticles have further demonstrated a potency against amyloid aggregation and cellular inflammation to rival strategies employing small molecules, peptides, and antibodies. Despite these efforts, however, a fundamental understanding of the chemistry, characteristics and function of peptido-nanocomposites is lacking, and a systematic analysis of such strategy for combating a range of amyloid pathogeneses is missing. Here we review the history, principles and evolving chemistry of constructing peptido-nanocomposites from bottom up and discuss their future application against amyloid diseases that debilitate a significant portion of the global population.

Structure-Based Design of Bicyclic Helical Peptides That Target the Oncogene ß-Catenin.

The inhibition of intracellular protein-protein interactions is challenging, in particular, when involved interfaces lack pronounced cavities. The transcriptional co-activator protein and oncogene ß­catenin is a prime example of such a challenging target. Despite extensive targeting efforts, available high-affinity binders comprise only large molecular weight Inhibitors. This hampers the further development of therapeutically useful compounds. Herein, we report the design of a considerably smaller peptidomimetic scaffold derived from the α-helical ß­catenin-binding motif of Axin. Sequence maturation and bicyclization provided a stitched peptide with an unprecedented crosslink architecture. The binding mode and site were confirmed by a crystal structure. Further derivatization yielded a ß-catenin inhibitor with single-digit micromolar activity in a cell-based assay. This study sheds a light on how to design helix mimetics with reduced molecular weight thereby improving their biological activity.

Conventional Understanding of SARS-CoV-2 Mpro and Common Strategies for Developing Its Inhibitors.

The Coronavirus Disease 2019 (COVID-19) pandemic has brought a widespread influence on the world, especially in the face of sudden coronavirus infections, and there is still an urgent need for specific small molecule therapies to cope with possible future pandemics. The pathogen responsible for this pandemic is Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and understanding its structure and lifecycle is beneficial for designing specific drugs of treatment for COVID-19. The main protease (Mpro ) which has conservative and specific advantages is essential for viral replication and transcription. It is regarded as one of the most potential targets for anti-SARS-CoV-2 drug development. This review introduces the popular knowledge of SARS-CoV-2 Mpro in drug development and lists a series of design principles and relevant activities of advanced Mpro inhibitors, hoping to provide some new directions and ideas for researchers.

Design, synthesis and biological evaluation of covalent peptidomimetic 3CL protease inhibitors containing nitrile moiety.

In this paper, a series of peptidomimetic SARS-CoV-2 3CL protease inhibitors with new P2 and P4 positions were synthesized and evaluated. Among these compounds, 1a and 2b exhibited obvious 3CLpro inhibitory activities with IC50 of 18.06 nM and 22.42 nM, respectively. 1a and 2b also showed excellent antiviral activities against SARS-CoV-2 in vitro with EC50 of 313.0 nM and 170.2 nM, respectively, the antiviral activities of 1a and 2b were 2- and 4-fold better than that of nirmatrelvir, respectively. In vitro studies revealed that these two compounds had no significant cytotoxicity. Further metabolic stability tests and pharmacokinetic studies showed that the metabolic stability of 1a and 2b in liver microsomes was significantly improved, and 2b had similar pharmacokinetic parameters to that of nirmatrelvir in mice.

Synthesis and Conformational Behaviors of Unnatural Peptides Alternating Chiral and Achiral α,α-Disubstituted α-Amino Acid Units.

The development of peptidomimetics to modulate the conformational profile of peptides has been extensively studied in the fields of biological and medicinal chemistry. However, large-scale synthesis of peptidomimetics with both an ordered sequence and a controlled secondary structure is highly challenging. In this paper, the framework of peptidomimetics has been designed to be alternating an achiral α,α-disubstituted α-amino acid unit and a chiral α-methylphenylalanine unit. The polymers are synthesized via invented Ugi reaction-based polycondensation technique. The chiral higher-order structures of the alternating peptides are evaluated mainly through circular dichroism (CD) spectroscopy. The UV-Vis and CD spectra of the polymers in three solvents are systematically measured at various temperatures. The anisotropic factors of CD (gCD ) values are calculated to know the chiroptical response. The results indicate the characteristic conformational behaviors. In a polar solvent, the hydrogen bonds between the N-H group of MePhe unit and the C=O of α,α-diphenylglycine unit outweigh the intraresidue hydrogen bonds in α,α-diphenylglycine unit, leading to the formation of a prevailing preferred-handed 310 -helical conformation. On the other hand, in a less polar solvent, the intrachain hydrogen bonds switch to intraresidue hydrogen bonds in α,α-diphenylglycine unit, which make the polymer adopting a prevailing extended planar C5 -conformation.

Design and synthesis of broad-spectrum antimicrobial amphiphilic peptidomimetics to combat drug-resistance.

The gradual depletion of antibiotic discovery pipeline makes the antibiotic resistance a difficult clinical problem and a global health emergency. The membrane-active antimicrobial peptides (AMPs) attracted much attention due to a lower tendency to bacterial resistance than traditional antibiotics. However, some immanent drawbacks of AMPs may hamper their application in combating antibiotic resistance in the long run, such as susceptible to enzymatic degradation and low cell permeability. Herein, we report the design and synthesis of a novel series of amphiphilic peptidomimetics, from which we identified compounds that exhibited potent antimicrobial activity against a panel of clinically relevant Gram-positive and Gram-negative bacteria strains. The most potent compound 20 (SD-110-12) is able to kill intracellular bacterial pathogens and prevent the development of bacterial resistance under the tested conditions by targeting cell membranes. Additionally, compound 20 (SD-110-12) obtains good in vivo efficacy that is comparative to vancomycin by eradicating MRSA and suppressing inflammation in a mice infected skin wound model, demonstrating its promising therapeutic potential.