Peptide-Platinum(IV) Conjugation Minimizes the Negative Impact of Current Anticancer Chemotherapy on Nonmalignant Cells.

The relative success of platinum (Pt)-based chemotherapy comes at the cost of severe adverse side effects and is associated with a high risk of pro-oncogenic activation in the tumor microenvironment. Here, we report the synthesis of C-POC, a novel Pt(IV) cell-penetrating peptide conjugate showing a reduced impact against nonmalignant cells. In vitro and in vivo evaluation using patient-derived tumor organoids and laser ablation inductively coupled plasma mass spectrometry indicates that C-POC maintains robust anticancer efficacy while displaying diminished accumulation in healthy organs and reduced adverse toxicity compared to the standard Pt-based therapy. Likewise, C-POC uptake is significantly lowered in the noncancerous cells populating the tumor microenvironment. This results in the downregulation of versican, a biomarker of metastatic spreading and chemoresistance that we found upregulated in patients treated with standard Pt-based therapy. Altogether, our findings underscore the importance of considering the off-target impact of anticancer treatment on normal cells to improve drug development and patient care.

Long-term platinum-based drug accumulation in cancer-associated fibroblasts promotes colorectal cancer progression and resistance to therapy.

A substantial proportion of cancer patients do not benefit from platinum-based chemotherapy (CT) due to the emergence of drug resistance. Here, we apply elemental imaging to the mapping of CT biodistribution after therapy in residual colorectal cancer and achieve a comprehensive analysis of the genetic program induced by oxaliplatin-based CT in the tumor microenvironment. We show that oxaliplatin is largely retained by cancer-associated fibroblasts (CAFs) long time after the treatment ceased. We determine that CT accumulation in CAFs intensifies TGF-beta activity, leading to the production of multiple factors enhancing cancer aggressiveness. We establish periostin as a stromal marker of chemotherapeutic activity intrinsically upregulated in consensus molecular subtype 4 (CMS4) tumors and highly expressed before and/or after treatment in patients unresponsive to therapy. Collectively, our study underscores the ability of CT-retaining CAFs to support cancer progression and resistance to treatment.

Target-templated de novo design of macrocyclic d-/l-peptides: discovery of drug-like inhibitors of PD-1.

Peptides are a rapidly growing class of therapeutics with various advantages over traditional small molecules, especially for targeting difficult protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing bioactive cyclic topologies that go beyond natural l-amino acids. Here, we report a generalizable framework that exploits the computational power of Rosetta, in terms of large-scale backbone sampling, side-chain composition and energy scoring, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we developed two new inhibitors (PD-i3 and PD-i6) of programmed cell death 1 (PD-1), a key immune checkpoint in oncology. A comprehensive biophysical evaluation was performed to assess their binding to PD-1 as well as their blocking effect on the endogenous PD-1/PD-L1 interaction. Finally, NMR elucidation of their in-solution structures confirmed our de novo design approach.

Probing the Kinetic and Thermodynamic Fingerprints of Anti-EGF Nanobodies by Surface Plasmon Resonance.

Despite the widespread use of antibodies in clinical applications, the precise molecular mechanisms underlying antibody-antigen (Ab-Ag) interactions are often poorly understood. In this study, we exploit the technical features of a typical surface plasmon resonance (SPR) biosensor to dissect the kinetic and thermodynamic components that govern the binding of single-domain Ab or nanobodies to their target antigen, epidermal growth factor (EGF), a key oncogenic protein that is involved in tumour progression. By carefully tuning the experimental conditions and transforming the kinetic data into equilibrium constants, we reveal the complete picture of binding thermodynamics, including the energetics of the complex-formation transition state. This approach, performed using an experimentally simple and high-throughput setup, is expected to facilitate mechanistic studies of Ab-based therapies and, importantly, promote the rational development of new biological drugs with suitable properties.

A Third Shot at EGFR: New Opportunities in Cancer Therapy.

Epidermal growth factor receptor (EGFR) inhibitors were among the first type of targeted agents discovered in cancer and currently constitute the standard of care for a wide range of lung and colon malignancies. However, the therapeutic progress achieved with these drugs has been accompanied by the identification of an ever-increasing number of acquired resistance mechanisms that inevitably appear in nearly all patients. Increased knowledge on EGFR biochemistry, cellular crosstalk, and resistance pathways provides an opportunity to establish effective combination therapies and discover novel-acting inhibitors that prevent or overcome therapeutic resistance. One such strategy is the selective blockade of circulating growth factors such as EGF. In this review, we address the uses and limitations of approved EGFR inhibitors and explore the potential of drug combinations and new third avenues to block the activation of the EGFR.

Protein Chemical Synthesis Combined with Mirror-Image Phage Display Yields d-Peptide EGF Ligands that Block the EGF-EGFR Interaction.

The epidermal growth factor (EGF) pathway, being overactive in a number of cancers, is a good target for clinical therapy. Although several drugs targeting the EGF receptor (EGFR) are on the market, tumours acquire resistance very rapidly. As an alternative, small molecules and peptides targeting EGF have been developed, although with moderate success. Herein, we report the use of mirror-image phage display technology to discover protease-resistant peptides with the capacity to inhibit the EGF-EGFR interaction. After the chemical synthesis of the enantiomeric protein d-EGF, two phage-display peptide libraries were used to select binding sequences. The d versions of these peptides bound to natural EGF, as confirmed by surface acoustic waves (SAWs). High-field NMR spectroscopy showed that the best EGF binder, d-PI_4, interacts preferentially with an EGF region that partially overlaps with the receptor binding interface. Importantly, we also show that d-PI_4 efficiently disrupts the EGF-EGFR interaction. This methodology represents a straightforward approach to find new protease-resistant peptides with potential applications in cancer therapy.

Enthalpy- versus Entropy-Driven Molecular Recognition in the Era of Biologics.

Our laboratory has recently identified two nanobodies (small antibodies produced by camelids)-Nb1 and Nb6-that bind efficiently to epithelial growth factor (EGF) and inhibit its ability to activate its receptor (EGFR). Because of the relevance of the EGF/EGFR axis as a target in oncology, these new nanobodies have promising therapeutic potential. This article, however, is focused on another feature of these nanobodies: their distinct thermodynamic signatures. Nb1 binds to EGF through an entropy-driven mechanism whereas Nb6 binds to this factor under enthalpic control. We discuss the advantages and disadvantages of each mechanism in the contexts of traditional medical chemistry (small-molecule drugs) and also of biological drugs. In this latter case, the implications in terms of selectivity are far from being clearly established and further experimental data are required. Their monomeric natures, high stability, and ease of recombinant production make nanobodies ideally suited for thermodynamic studies. Moreover, nanobodies, thanks to their simpler structures in comparison with conventional antibodies, might provide better understanding of the structural basis of the thermodynamic parameters of antigen recognition.

Targeted Nanoswitchable Inhibitors of Protein-Protein Interactions Involved in Apoptosis.

Progress in drug delivery is hampered by a lack of efficient strategies to target drugs with high specificity and precise spatiotemporal regulation. The remote control of nanoparticles and drugs with light allows regulation of their action site and dosage. Peptide-based drugs are highly specific, non-immunogenic, and can be designed to cross the plasma membrane. In order to combine target specificity and remote control of drug action, here we describe a versatile strategy based on a generalized template to design nanoswitchable peptides that modulate protein-protein interactions upon light activation. This approach is demonstrated to promote photomodulation of two important targets involved in apoptosis (the interactions Bcl-xL-Bak and MDM2-p53), but can be also applied to a large pool of therapeutically relevant protein-protein interactions mediated by α-helical motifs. The template can be adjusted using readily available information about hot spots (residues contributing most to the binding energy) at the protein-protein interface of interest.

Blocking EGFR Activation with Anti-EGF Nanobodies via Two Distinct Molecular Recognition Mechanisms.

One of the hallmarks of cancer is the overproduction of growth factors such as EGF. Despite the clinical success achieved by EGFR-targeted therapies, their long-term efficacy is compromised by the onset of drug-resistant mutations. To address this issue, a family of camelid-derived single-domain antibodies (Nbs) were generated, obtaining the first direct EGF inhibitors that prevent EGFR phosphorylation and pathway activation through this new mechanism of action. The two best Nbs were subjected to a detailed investigation of their interaction mechanism that revealed important differences in their binding kinetics and equilibrium thermodynamics. These distinct behaviors at the biophysical level translate into an equally efficient inhibition of the cellular EGFR phosphorylation, thus proving the efficacy of these Nbs to turn off the initiation of this key oncogenic pathway in cancer cells.

Toward a Novel Drug To Target the EGF-EGFR Interaction: Design of Metabolically Stable Bicyclic Peptides.

In cancer, proliferation of malignant cells is driven by overactivation of growth-signalling mechanisms, such as the epidermal growth factor receptor (EGFR) pathway. Despite its therapeutic relevance, the EGF-EGFR interaction has remained elusive to inhibition by synthetic molecules, mostly as a result of its large size and lack of binding pockets and cavities. Designed peptides, featuring cyclic motifs and other structural constraints, have the potential to modulate such challenging protein-protein interactions (PPIs). Herein, we present the structure-based design of a series of bicyclic constrained peptides that mimic an interface domain of EGFR and inhibit the EGF-EGFR interaction by targeting the smaller partner (i.e., EGF). This design process was guided by the integrated use of in silico methods and biophysical techniques, such as NMR spectroscopy and surface acoustic wave. The best analogues were able to reduce selectively the viability of EGFR+ human cancer cells. In addition to their efficacy, these bicyclic peptides are endowed with exceptional stability and metabolic resistance-two features that make them suitable candidates for in vivo applications.

Combating virulence of Gram-negative bacilli by OmpA inhibition.

Preventing the adhesion of pathogens to host cells provides an innovative approach to tackling multidrug-resistant bacteria. In this regard, the identification of outer membrane protein A (OmpA) as a key bacterial virulence factor has been a major breakthrough. The use of virtual screening helped us to identify a cyclic hexapeptide AOA-2 that inhibits the adhesion of Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli to host cells and the formation of biofilm, thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. Inhibition of OmpA offers a strategy as monotherapy to address the urgent need for treatments for infections caused by Gram-negative bacilli.

Combined Use of Oligopeptides, Fragment Libraries, and Natural Compounds: A Comprehensive Approach To Sample the Druggability of Vascular Endothelial Growth Factor.

The modulation of protein-protein interactions (PPIs) is emerging as a highly promising tool to fight diseases. However, whereas an increasing number of compounds are able to disrupt peptide-mediated PPIs efficiently, the inhibition of domain-domain PPIs appears to be much more challenging. Herein, we report our results related to the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR). The VEGF-VEGFR interaction is a typical domain-domain PPI that is highly relevant for the treatment of cancer and some retinopathies. Our final goal was to identify ligands able to bind VEGF at the region used by the growth factor to interact with its receptor. We undertook an extensive study, combining a variety of experimental approaches, including NMR-spectroscopy-based screening of small organic fragments, peptide libraries, and medicinal plant extracts. The key feature of the successful ligands that emerged from this study was their capacity to expose hydrophobic functional groups able to interact with the hydrophobic hot spots at the interacting VEGF surface patch.