We demonstrate our results with the synthesis of inhibitors of Protein Arginine Deaminase 4 (PAD4), a key regulator of neutrophil elastase extracellular traps (NETs), as potential therapeutics for chronic obstructive pulmonary disease (COPD). Furthermore, we report an automated method for solid phase synthesis of macrocyclic stapled peptides with chloroacetamidine guanidine mimetic function as PAD4 inhibitors. This approach utilizes the 2,4,6-trimethylbenzenesulfenyl protective group for cysteine, followed by reaction with various halogen derivatives upon deprotection under mild conditions. Furthermore, derivatization of the amino group with ethyl chloroacetamidate hydrochloride after its deprotection, performed on solid phase, offers a new, efficient method for rapid and robust synthesis of stapled peptide derivatives. This process notably reduces time, energy, and solvent use compared to traditional in-solution methods.

Although antibody-drug conjugates (ADCs) have made substantial progress as targeted therapies, the range of suitable ADC payloads remains limited. In this study, the highly N-methylated cyclodepsipeptide [MeAla3-MeAla6]-coibamide (CA) was selected as a novel toxin for ADC construction due to its potent toxicity and unique mechanism of action. Using a quaternary ammonium salt approach, two linker-payload variants, MC-VA-PAB-CA and MC-GGFG-PAB-CA, with distinct cathepsin B (CTB)-cleavable linkers, were synthesized and assessed. Among them, MC-GGFG-PAB-CA demonstrated higher enzyme-responsive cleavage efficiency and superior plasma stability and was selected for conjugation with the epidermal growth factor receptor (EGFR) antibody cetuximab (Ctx), resulting in the formation of Ctx-CA. This conjugate exhibited EGFR-dependent antitumor activity, a pronounced "bystander killing effect", and a significant tumor suppression effect in mouse models. Furthermore, the applicability of this conjugation strategy was confirmed through validation with the HER2 antibody. These findings suggest that CA is a promising weapon for next-generation ADCs.

The principle of structure dictating properties is illustrated by the direct correlation between cyclic peptide conformation and their biological efficacy. Plecanatide, a synthetic analog of uroguanylin, has received FDA approval for the treatment of chronic idiopathic constipation and irritable bowel syndrome with constipation. Nevertheless, our investigation has revealed that plecanatide undergoes slow conformational interconversion in slightly acidic conditions. In response, propargylglycine is strategically incorporated at the carboxyl terminal of plecanatide, a modification that not only facilitates additional functionalization and derivatizationbut also confers exceptional conformational stability. Remarkably, the resulting isomers not only maintained long-term conformational stability but also exhibited either preserved or slightly enhanced agonistic activity. This discovery represents a contribution to drug research focused on plecanatide, particularly in elucidating the relationship between its conformational properties and biological activity.

Due to the heterogeneity of tumors and the complex regulatory mechanisms governing PD-L1 expression, immunotherapy that employs PD-1/PD-L1 inhibitors has shown limited efficacy and relatively low response rates. In this work, we propose that a Degrader-Drug Conjugate (DDC), comprising a PD-L1 degrader linked to a cytotoxic agent, may enhance antitumor efficacy by synergizing immune activation with direct cell killing. Mechanistically, the DDC promotes PD-L1 degradation via the endosomal-lysosomal pathway to alleviate immunosuppression, while simultaneously releasing a cytotoxic agent inside tumor cells to induce apoptosis. Among the two DDCs developed in this study, BMS-RGD-MMAE exhibits superior toxin release efficacy, enhanced plasma stability, and increased cytotoxicity compared to BMS-RGD-DXd. Moreover, BMS-RGD-MMAE significantly inhibits tumor growth in vivo through synergistic PD-L1 degradation and toxin-mediated cell death, underscoring its substantial potential for application in tumor immunochemotherapy. Overall, this DDC platform provides new opportunities for the advancement of antitumor therapeutics.

Hela cells were treated with or without 10 μM PIP3 for 72 hours and then labeled by Propidium iodide and Annexin V-FITC for detecting early and late apoptotic signal.

(a and b) A549 cells were treated with different concentrations of PIP2 or PIP3. After 72 h of treatment, cells were incubated with 10 ng/ml TNF-α for additional 12 h, and then harvested for IL6 (a) and IL8 (b) qRT-PCR analysis.

Peptides and peptide drug conjugates are emerging modalities to treat pulmonary diseases. Peptides are sus-ceptible to proteolytic cleavage. Expression levels of specific proteases in the lung can be significantly increased in disease state and may lead to exaggerated peptide proteolysis. To support optimization of peptides for inhaled administration, we have recently reported a streamlined high-throughput LC-HRMS protocol to determine enzymatic protease stability of peptides. This method has now been complemented with profiling of peptide metabolic stability in two respiratory fluids, a lung supernatant (lung S9) and a bronchioalveolar lavage fluid (BALF) taken from rats. We have tested a set of 28 peptides with high structural diversity, analyzed the whole data set for formed metabolites, and identified the differences of cleavage pattern in the two test fluids. Com-parison of our experimental results and literature-derived cleavage site estimates based on e.g. MEROPS show significant differences for a number of peptides. This indicates the need for an experimental workflow using both protease panels and testing of metabolic stability in lung fluid (BALF) to guide peptide optimization and selection of peptides for inhaled in vivo PK/PD studies in our drug discovery projects.

(a) Representative images of mice from vehicle group and PIP3-treated group on the sacrifice day. Yellow arrows indicate tumor locations. (b) Images of isolated tumors from vehicle group or PIP3-treated group. (c) In vivo cellular localization of PIP3. Frozen sections of tumors from PIP3-treated group were imaged under microscopy to visualize the cellular uptake of PIP3 in vivo. The nuclei were stained by Topro3. Scale bar, 100 μm.

(a) Hela cells or (b) A549 cells stably expressing histone H2B-RFP were incubated with 10 μM of PIP3. At different time points, cells were harvested, stained, and imaged. Scale bar, 50 μm. (c) Nuclear localization of PIP3 in A549 cells. Scale bar, 10 μm.

Non-natural amino acids are increasingly used as building blocks in the development of peptide-based drugs as they expand the available chemical space to tailor function, half-life and other key properties. However, while the chemical space of modified amino acids (mAAs) such as residues containing post-translational modifications (PTMs) is potentially vast, experimental methods for measuring the developability properties of mAA-containing peptides are expensive and time consuming. To facilitate developability programs through computational methods, we present CamSol-PTM, a method that enables the fast and reliable sequence-based prediction of the intrinsic solubility of mAA-containing peptides in aqueous solution at room temperature. From a computational screening of 50,000 mAA-containing variants of three peptides, we selected five different small-size mAAs for a total number of 37 peptide variants for experimental validation. We demonstrate the accuracy of the predictions by comparing the calculated and experimental solubility values. Our results indicate that the computational screening of mAA-containing peptides can extend by over four orders of magnitude the ability to explore the solubility chemical space of peptides and confirm that our method can accurately assess the solubility of peptides containing mAAs. This method is available as a web server at https://www-cohsoftware.ch.cam.ac.uk/index.php/camsolptm .

Hela cells or A549 cells stably expressing histone H2B-RFP were incubated with 10 μM of different PIPs for 24 h. Cells were then subjected to live-imaging using confocal microscopy. Scale bar, 10 μm.

Live imaging of mitotic progression in control A549 cells

(a and b) Plk1 mRNA levels (a) and Plk1 protein levels (b) in various cell lines. (c-e) Pearson correlation co-efficiency was calculated between Plk1 mRNA level, Plk1 protein expression level, and PIP3 sensitivity (represented by IC50 value).

(a and b) The hTERT-RPE1 cells and HUVEC cells were synchronized, PIP3 treated, and analyzed as in Figure 4a and 4b. Plk1 inhibition efficiency examined by western blotting was shown in the top. (c and d) Percentages of mitotic cells at different phases were quantified in these treated cells (n.s, not significant; n=3). (e) Time-lapse microscopy of nontransformed cells with or without PIP3 treatment. hTERT-RPE1 cells and HUVEC cells stably expressing histone H2B-RFP were treated with 0.1% DMSO or 20 μM of PIP3 for 72 h and analyzed by time-lapse imaging. (f and g) The duration of total mitosis (f) and time spent in each sub-stage of mitosis (g) were further quantified (n.s, not significant; n{greater than or equal to}25).

(a) Comparison of the cellular uptake of PIP3 and Hoechst 33258. Hela cells were incubated with different concentrations of PIP3 or Hoechst 33258 for 24 hours. Fluorescent microscopy images were acquired and as shown. (b) PIP3 but not Hoechst 33258 shows Plk1 suppression ability. Hela cells were treated with different concentrations of PIP3 or Hoechst 33258 for 72 hours and then collected for western blotting analysis to examine the Plk1 expression. (c) Evaluation of PIP3 stability in vitro. Hela cells were treated with 10 μM PIP3 for 72 hours, then cells were collected and lysed. Released PIP3 was monitored by RP-HPLC analysis. PIP3 dissolved in water was served as control.

Understanding the conformational ensembles of intrinsically disordered proteins and peptides (IDPs) in their various biological environments is essential for understanding their mechanisms and functional roles in the proteome, leading to a greater knowledge of, and potential treatments for, a broad range of diseases. To determine whether molecular simulation is able to generate accurate conformational ensembles of IDPs, we explore the structural landscape of the PLP peptide (an intrinsically disordered region of the proteolipid membrane protein) in aqueous and membrane-mimicking solvents, using replica exchange with solute scaling (REST2), and examine the ability of four force fields (ff14SB, ff14IDPSFF, CHARMM36 and CHARMM36m) to reproduce literature circular dichroism (CD) data. Results from variable temperature (VT) 1H and Rotating frame Overhauser Effect SpectroscopY (ROESY) nuclear magnetic resonance (NMR) experiments are also presented and are consistent with the structural observations obtained from the simulations and CD. We also apply the optimum simulation protocol to TP2 and ONEG (a cell-penetrating peptide (CPP) and a negative control peptide, respectively) to gain insight into the structural differences that may account for the observed difference in their membrane-penetrating abilities. Of the tested force fields, we find that CHARMM36 and CHARMM36m are best suited to the study of IDPs, and accurately predict a disordered to helical conformational transition of the PLP peptide accompanying the change from aqueous to membrane-mimicking solvents. We also identify an α-helical structure of TP2 in the membrane-mimicking solvents and provide a discussion of the mechanistic implications of this observation with reference to the previous literature on the peptide. From these results, we recommend the use of CHARMM36m with the REST2 protocol for the study of environment-specific IDP conformations. We believe that the simulation protocol will allow the study of a broad range of IDPs that undergo conformational transitions in different biological environments.

The inhalation of peptides comes with the advantage of directly targeting the lung as tissue of interest. However, peptides are often rapidly metabolized in lung tissue through proteolytic cleavage. We have developed an assay workflow to obtain half-life and metabolite ID data for peptides incubated with four proteases abundant in lungs of asthma and COPD patients. The assay system has been validated using 28 structurally diverse linear and cyclic peptides with a molecular weight between 708 and 5808 Da. Experimental conditions for incubation, sample preparation, chromatography, data acquisition and analysis are compatible with the required throughput in early stage peptide projects. Together with co-crystal structures and Ala scans, we are using the described assay workflow to guide the first chemical modifications of peptide hits in early respiratory drug discovery projects.

Inhalation of small molecule drugs has proven very efficacious for the treatment of respiratory diseases due to enhanced efficacy and a favourable therapeutic index compared with other dosing routes. It enables targeted delivery to the lung with rapid onset of therapeutic action, low systemic drug exposure, and thereby reduced systemic side effects. An increasing number of pharmaceutical companies and biotechs are investing in new modalities-for this review defined as therapeutic molecules with a molecular weight >800Da and therefore beyond usual inhaled small molecule drug-like space. However, our experience with inhaled administration of PROTACs, peptides, oligonucleotides (antisense oligonucleotides, siRNAs, miRs and antagomirs), diverse protein scaffolds, antibodies and antibody fragments is still limited. Investigating the retention and metabolism of these types of molecules in lung tissue and fluid will contribute to understanding which are best suited for inhalation. Nonetheless, the first such therapeutic molecules have already reached the clinic. This review will provide information on the physiology of healthy and diseased lungs and their capacity for drug metabolism. It will outline the stability, aggregation and immunogenicity aspects of new modalities, as well as recap on formulation and delivery aspects. It concludes by summarising clinical trial outcomes with inhaled new modalities based on information available at the end of 2021.

Although nanomaterials have shown promising biomedical application potential, incomplete understanding of their molecular interactions with biological systems prevents their inclusion into mainstream clinical applications. Here we show that black phosphorus (BP) nanomaterials directly affect the cell cycle’s centrosome machinery. BP destabilizes mitotic centrosomes by attenuating the cohesion of pericentriolar material and consequently leads to centrosome fragmentation within mitosis. As a result, BP-treated cells exhibit multipolar spindles and mitotic delay, and ultimately undergo apoptosis. Mechanistically, BP compromises centrosome integrity by deactivating the centrosome kinase polo-like kinase 1 (PLK1). BP directly binds to PLK1, inducing its aggregation, decreasing its cytosolic mobility and eventually restricting its recruitment to centrosomes for activation. With this mechanism, BP nanomaterials show great anticancer potential in tumour xenografted mice. Together, our study reveals a molecular mechanism for the tumoricidal properties of BP and proposes a direction for biomedical application of nanomaterials by exploring their intrinsic bioactivities.