Chemometrics in Protein Formulation: Stability Governed by Repulsion and Protein Unfolding.

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization.

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.

Improved Physical Stability of an Antibody-Drug Conjugate Using Host-Guest Chemistry.

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceutical products for oncology, with the cytotoxic pyrrolobenzodiazepine (PBD) family of "warheads" well-established in the clinic. While PBDs offer high potency, they are also characterized by their hydrophobicity, which can make formulation of the ADC challenging. Several approaches have been investigated to improve the physicochemical properties of PBD-containing ADCs, and herein a supramolecular approach was explored using cucurbit[8]uril (CB[8]). The ability of CB[8] to simultaneously encapsulate two guests was exploited to incorporate a 12-mer polyethylene glycol harboring a methyl viologen moiety at one terminus (MV-PEG12), together with a PBD harboring an indole moiety at the C2' position (SG3811). This formulation approach successfully introduced a hydrophilic PEG to mask the hydrophobicity of SG3811, improving the physical stability of the ADC while avoiding any loss of potency related to chemical modification.

Distinct biological responses of metastatic castration resistant prostate cancer cells upon exposure to G-quadruplex interacting naphthalenediimide derivatives.

Small molecules able to bind non-canonical G-quadruplex DNA structures (G4) have been recently tested as novel potential agents for the treatment of prostate cancer thanks to their repression of aberrant androgen receptor gene. However, metastatic castration-resistant prostate cancer (mCRPC), a letal form of prostate cancer, is still incurable. Here we tested two naphthalenediimide derivatives, previously reported as multitarget agents, on a couple of relevant mCRPC cell models (DU145 and PC-3). We showed that these compounds interfere with the RAS/MEK/ERK and PI3K/AKT pathways. Interestingly, both these two biological processes depend upon Epidermal Growth Factor Receptor (EGFR) activation. By means of biological and analytical tools we showed that our compounds are efficient inducers of the structural transition of the EGFR promoter towards a G-quadruplex conformation, ultimately leading to a reduction of the receptor production. The overall result is an interesting cytotoxic profile for these two derivatives. Thanks to their activity at different steps, these compounds can open the way to novel therapeutic approaches for mCRPC that could contribute to escape resistance to selective treatments.

pH-driven conformational switch between non-canonical DNA structures in a C-rich domain of EGFR promoter.

EGFR is an oncogene that encodes for a trans-membrane tyrosine kinase receptor. Its mis-regulation is associated to several human cancers that, consistently, can be treated by selective tyrosine kinase inhibitors. The proximal promoter of EGFR contains a G-rich domain located at 272 bases upstream the transcription start site. We previously proved it folds into two main interchanging G-quadruplex structures, one of parallel and one of hybrid topology. Here we present the first evidences supporting the ability of the complementary C-rich strand (EGFR-272_C) to assume an intramolecular i-Motif (iM) structure that, according to the experimental conditions (pH, presence of co-solvent and salts), can coexist with a different arrangement we referred to as a hairpin. The herein identified iM efficiently competes with the canonical pairing of the two complementary strands, indicating it as a potential novel target for anticancer therapies. A preliminary screening for potential binders identified some phenanthroline derivatives as able to target EGFR-272_C at multiple binding sites when it is folded into an iM.

Naphthalene diimide-polyamine hybrids as antiproliferative agents: Focus on the architecture of the polyamine chains.

Naphthalene diimides (NDIs) have been widely used as scaffold to design DNA-directed agents able to target peculiar DNA secondary arrangements endowed with relevant biochemical roles. Recently, we have reported disubstituted linear- and macrocyclic-NDIs that bind telomeric and non-telomeric G-quadruplex with high degree of affinity and selectivity. Herein, the synthesis, biological evaluation and molecular modelling studies of a series of asymmetrically substituted NDIs are reported. Among these, compound 9 emerges as the most interesting of the series being able to bind telomeric G-quadruplex (ΔTm = 29 °C at 2.5 µM), to inhibit the activity of DNA processing enzymes, such as topoisomerase II and TAQ-polymerase, and to exert antiproliferative effects in the NCI panel of cancer cell lines with GI50 values in the micro-to nanomolar concentration range (i.e. SR cell line, GI50 = 76 nM). Molecular mechanisms of cell death have been investigated and molecular modelling studies have been performed in order to shed light on the antiproliferative and DNA-recognition processes.

Hit Identification of a Novel Dual Binder for h-telo/c-myc G-Quadruplex by a Combination of Pharmacophore Structure-Based Virtual Screening and Docking Refinement.

It is well known that G-quadruplexes are targets of great interest for their roles in crucial biological processes, such as aging and cancer. Hence, a promising strategy for anticancer drug therapy is the stabilization of these structures by small molecules. We report a high-throughput in silico screening of commercial libraries from several different vendors by means of a combined structure-based pharmacophore model approach followed by docking simulations. The compounds selected by the virtual screening procedure were then tested for their ability to interact with human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and fluorescence intercalator displacement. Our approach resulted in the identification of a 13-[(dimethylamino)methyl]-12-hydroxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one derivative as a novel promising stabilizer of G-quadruplex structures within the human telomeric and the c-myc promoter sequences.

An optimized polyamine moiety boosts the potency of human type II topoisomerase poisons as quantified by comparative analysis centered on the clinical candidate F14512.

Combined computational-experimental analyses explain and quantify the spermine-vectorized F14512's boosted potency as a topoII poison. We found that an optimized polyamine moiety boosts drug binding to the topoII/DNA cleavage complex, rather than to the DNA alone. These results provide new structural bases and key reference data for designing new human topoII poisons.

Double stranded promoter region of BRAF undergoes to structural rearrangement in nearly physiological conditions.

The folding of oncogene promoters into non-canonical DNA secondary structures is considered a strategy to control gene expression. Herein, we focused on a 30 bases sequence located upstream of the transcription start site of BRAF (Braf-176) that contains 80% of guanines. We analyzed the structural behavior of the G- and C-rich strands. By the use of spectroscopic and electrophoretic techniques we confirmed that they actually fold into a predominant antiparallel G-quadruplex and into an i-motif, respectively, and that they can coexist at nearly physiological conditions. Finally, the influence of several factors (KCl, pH, PEG200) on the conversion of the double stranded form of the oncogene promoter into the two above mentioned non-canonical structures has been explored.

Aryl ethynyl anthraquinones: a useful platform for targeting telomeric G-quadruplex structures.

Aryl ethynyl anthraquinones have been synthesized by Sonogashira cross-coupling and evaluated as telomeric G-quadruplex ligands, by the FRET melting assay, circular dichroism, the DNA synthesis arrest assay and molecular docking. Both the binding properties and G-quadruplex vs. duplex selectivity are controlled by the structures of the aryl ethynyl moieties.

Further SAR studies on bicyclic basic merbarone analogues as potent antiproliferative agents.

Pyrimidopyrimidine derivatives 1 were prepared as rigid thioanalogues of merbarone (a catalytic topoisomerase II inhibitor) and screened as antiproliferative agents against different tumor cell lines. A number of the synthesized compounds emerged as cytotoxic in cell-based assays (MT-4, HeLa and MCF-7 cells) at low micromolar concentrations. In a National Cancer Institute screening, selected member of the series showed a broad spectrum of antiproliferative activity against various tumours (melanoma, renal, CNS, colon and breast cancers). The acid-base and steric properties of the substituent at position 7 of the pyrimidopyrimidine scaffold deeply affected potency. Enzymatic assays evidenced that a subset of tested derivatives efficiently inhibit topoisomerase IIα accordingly to merbarone mechanism of action. However this property does not fully rationalize the cytotoxicity data of the full ligand panel, suggesting that different target(s) should be additionally involved.