The pH (low) insertion peptide (pHLIP) is a family of peptides that are able to insert into a lipid bilayer at acidic pH. The molecular mechanism of pHLIPs insertion, folding and stability in the membrane at low pH is based on multiple protonation events, which are challenging to study at molecular level. More specifically, the relation between the experimental pK of insertion (pKexp) of pHLIPs and the pKa of the key residues is yet to be clarified. We carried out a computational study, complemented with new experimental data, and established the influence of (de)protonation of key titrable residues on the stability of the peptide membrane-inserted state. Constant-pH molecular dynamics simulations were employed to calculate the pKa values of these residues along the membrane normal. In the wt-pHLIP, we identified Asp14 as the key residue for the stability of the membrane-inserted state, and its pKa value is strongly correlated with the experimental pKexp measured in thermodynamics studies. Also, in order to narrow down the pH range at which pHLIP is stable in the membrane, we designed a new pHLIP variant, L16H, where Leu in the 16th position was replaced by a titrable His residue. Our results showed that the L16H variant undergoes two transitions. The calculated pK and experimentally observed pKexp values are in good agreement. Two distinct pKexp values delimit a pH range where the L16H peptide is stably inserted in the membrane while, outside this range, the membrane-inserted state is destabilized and the peptide exits from the bilayer. pHLIP peptides have been successfully used to target cancer cells for the delivery of diagnostics and therapeutic agents to acidic tumors. The fine tuning of the stability of pHLIP inserted state and its restriction to a narrow well-defined pH range might allow the design of new pHLIPs, able to discriminate between tissues with different extracellular pH values.

A series of cyclic peptides containing a number of tryptophan (W) and glutamic acid (E) residues were synthesized and evaluated as pH-sensitive agents for targeting of acidic tissue and pH-dependent cytoplasmic delivery of molecules. Biophysical studies revealed the molecular mechanism of peptides action and localization within the lipid bilayer of the membrane at high and low pHs. The symmetric, c[(WE)4WC], and asymmetric, c[E4W5C], cyclic peptides translocated amanitin, a polar cargo molecule of similar size, across the lipid bilayer and induced cell death in a pH- and concentration-dependent manner. Fluorescently-labelled peptides were evaluated for targeting of acidic 4T1 mammary tumors in mice. The highest tumor to muscle ratio (5.6) was established for asymmetric cyclic peptide, c[E4W5C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R4W5C], where glutamic acid residues (E) were replaced by positively charged arginine residues (R), did not exhibit tumor targeting. We have introduced a novel class of cyclic peptides, which can be utilized as a new pH-sensitive tool in investigation or targeting of acidic tissue.

To advance mechanistic understanding of membrane-associated peptide folding and insertion, we have studied the kinetics of three single tryptophan pHLIP (pH-Low Insertion Peptide) variants, where tryptophan residues are located near the N terminus, near the middle, and near the inserting C-terminal end of the pHLIP transmembrane helix. Single-tryptophan pHLIP variants allowed us to probe different parts of the peptide in the pathways of peptide insertion into the lipid bilayer (triggered by a pH drop) and peptide exit from the bilayer (triggered by a rise in pH). By using pH jumps of different magnitudes, we slowed down the processes and established the intermediates that helped us to understand the principles of insertion and exit. The obtained results should also aid the applications in medicine that are now entering the clinic.

Acidosis is a key driver for many diseases, including cancer, sepsis, and stroke. The spatiotemporal dynamics of dysregulated pH across disease remain elusive, and current diagnostic strategies do not provide localization of pH alterations. We sought to explore if PET imaging using hydrophobic cyclic peptides that partition into the cellular membrane at low extracellular pH (denoted as pH [low] insertion cycles, or pHLIC) can permit accurate in vivo visualization of acidosis. Methods: Acid-sensitive cyclic peptide c[E 4 W 5 C] pHLIC was conjugated to bifunctional maleimide-NO2A and radiolabeled with 64 Cu (half-life, 12.7 h). C57BL/6J mice were administered lipopolysaccharide (15 mg/kg) or saline (vehicle) and serially imaged with [ 64 Cu]Cu-c[E 4 W 5 C] over 24 h. Ex vivo autoradiography was performed on resected brain slices and subsequently stained with cresyl violet to enable high-resolution spatial analysis of tracer accumulation. A non-pH-sensitive cell-penetrating control peptide (c[R 4 W 5 C]) was used to confirm specificity of [ 64 Cu]Cu-c[E 4 W 5 C]. CD11b (macrophage/microglia) and TMEM119 (microglia) immunostaining was performed to correlate extent of neuroinflammation with [ 64 Cu]Cu-c[E 4 W 5 C] PET signal. Results: [ 64 Cu]Cu-c[E 4 W 5 C] radiochemical yield and purity were more than 95% and more than 99%, respectively, with molar activity of more than 0.925 MBq/ nmol. Significantly increased [ 64 Cu]Cu-c[E 4 W 5 C] uptake was observed in lipopolysaccharide-treated mice (vs. vehicle) within peripheral tissues, including blood, lungs, liver, and small intestines (P , 0.001-0.05). Additionally, there was significantly increased [ 64 Cu]Cu-c[E 4 W 5 C] uptake in the brains of lipopolysaccharide-treated animals. Autoradiography confirmed increased uptake in the cerebellum, cortex, hippocampus, striatum, and hypothalamus of lipopolysaccharide-treated mice (vs. vehicle). Immunohistochemical analysis revealed microglial or macrophage infiltration, suggesting activation in brain regions containing increased tracer uptake. [ 64 Cu] Cu-c[R 4 W 5 C] demonstrated significantly reduced uptake in the brain and periphery of lipopolysaccharide mice compared with the acid-mediated [ 64 Cu]Cu-c[E 4 W 5 C] tracer. Conclusion: Here, we demonstrate that a pH-sensitive PET tracer specifically detects acidosis in regions associated with sepsis-driven proinflammatory responses. This study suggests that [ 64 Cu]Cu-pHLIC is a valuable tool to noninvasively assess acidosis associated with both central and peripheral innate immune activation.

Poly (ADPribose) polymerase inhibitors (PARPi) have shown great promise in the treatment of cancer, however, their current clinical use has been largely limited to homologous recombination-deficient (HRD) tumors. While these drugs are efficacious as monotherapies, durable responses beyond 12 months are uncommon and their activity against HRD-negative tumors is limited. These findings have prompted great interest in combining PARPi’s with chemotherapy to increase the duration of response in HRD-positive tumors and expand the activity of these drugs against HRD-negative tumors which comprise a much greater fraction of the total number of cancer cases each year. While such combinations are highly active against tumors independent of HRD status, they are also extremely cytotoxic against bone marrow cells. As such, dose reductions ranging from 5- to 20-fold are required when PARPi’s are combined with chemotherapy which has resulted in favorable safety profiles but limited efficacy. Tumor-targeting strategies could overcome this efficacy barrier but the technologies developed thus far are limited by numerous issues, including: (a) lack of universal, non-saturating tumor-targeting mechanisms which limit their use to specific tumor types and their corresponding antigens, (b) inability to deliver therapeutically relevant levels of drug(s) directly into tumor cells, and (c) insufficient tumor penetration leading to sub-optimal tumor exposure. Herein, we report the development of alphalex, a novel tumor-targeted drug delivery platform which allows the efficient and selective delivery of PARPi’s into tumors with significant normal-tissue sparing. Tumor-targeting is achieved using a pH- sensitive peptide which forms an alpha-helix in cellular membrane under low pH conditions associated with the tumor microenvironment. Under these conditions, the peptide translocates across cancer cell membranes to deliver the PARPi directly into the cytoplasm. We demonstrate that alphalex peptide-based conjugates can be combined safely and effectively with both cytotoxic chemotherapies and radiation therapy (RT) and that this approach can be used to selectively kill both HRD-positive and –negative tumors with significant sparing of the bone marrow. The safety and efficacy of the approach was demonstrated in both engineered and patient-derived xenografts (PDXs) in vivo using two FDA-approved PARPi’s across two independent laboratories. These data highlight an entirely new approach to apply PARPi’s against solid tumors independent of HRD status. Furthermore, our approach can be applied to a diverse range of DNA repair inhibitors for potent and selective chemo/radio-sensitization in a tissue-agnostic manner. Citation Format: Vishwas Paralkar, Robert J. Aiello, Dan Marshall, Johanna Csengery, Patricia Bourassa, Qing Zhang, Brett S. Robinson, Lori Lopresti-Morrow, Jane Bechtold, Laurie Tylaska, Timothy Paradis, Gregory Slaybaugh, Hanna Visca, Anna Moshnikova, Dhammika Weerakkody, Oleg Andreev, Yana K. Reshetnyak, Donald Engelman, Ranjit Bindra, Peter Glazer, Per Hellsund. Targeting solid tumor acidic microenvironment with an alphalex PARP inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2981.