In silico tumor-targeting technologies for the evasion of acidity-induced multidrug resistance

The physiology of tumors is tied to MDR mechanisms that hamper chemotherapeutic effects, particularly passive membrane crossing compounds, like hydrophobic Lewis base drugs. Although the lysosomal entrapment phenomena remains to be fully understood, this pH-dependent MDR mechanism induces drug sequestration in the acidic lysosomal lumen. Overcoming the MDR requires multi-pronged therapies, which often overlook an ubiquitous tumor trait: the extracellular acidity of the tumor microenvironment (TME). To address this, pHLIP peptides have emerged as an acidity-selective technology for tumor-targeting drug delivery. We focused on refining our protocols with enhanced sampling techniques and tumor-like features to improve the predictive abilities of the CpHMD-L methodology and augment the realism of these biomolecular models, thus bridging the gap to in vivo and cellular conditions. The optimized protocol coupled the CpHMD-L method with a pHRE scheme, providing a robust baseline. Then, we applied the protocol to study the diverging therapeutic efficiency of the wt and an over-performing Var3 peptide. A novel implementation of a pH gradient CpHMD-L method successfully reproduced experimental performances, thus elucidating pivotal residues electrostatic networks that dictate peptides thermodynamic stability in TME conditions. A multi-peptide study highlighted the remarkable effects of permuting arginines in modulating the local vicinity of key aspartates. These findings heavily correlate with their tumor-targeting performance, supporting more rational and in silico-based approaches to peptide design. Finally, the pH-dependent mechanism of lysosomal entrapment was modelled, hinting at the important role of acidity in Lewis base drugs membrane intercalation. Additional pH-dependent permeability calculations, using a novel US-CpHMD method, identified the TME acidity as an additional MDR defense mechanism that impairs clinical efficiency. It also revealed an intrinsic flaw of these compounds, since they preferably target healthy cells. These findings have important implications in rational drug design, especially of conjugated therapies with pHLIP-like drug delivery systems to overcome these challenges

Membrane-Induced p Ka Shifts in wt-pHLIP and Its L16H Variant

The pH (low) insertion peptides (pHLIPs) 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 the 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 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 pKa 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 the pHLIP inserted state and its restriction to a narrow well-defined pH range might allow the design of new peptides, able to discriminate between tissues with different extracellular pH values

Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase

DyP-type peroxidases (DyPs) are microbial enzymes that catalyze the oxidation of a wide range of substrates, including synthetic dyes, lignin-derived compounds, and metals, such as Mn2+ and Fe2+, and have enormous biotechnological potential in biorefineries. However, many questions on the molecular basis of enzyme function and stability remain unanswered. In this work, high-resolution structures of PpDyP wild-type and two engineered variants (6E10 and 29E4) generated by directed evolution were obtained. The X-ray crystal structures revealed the typical ferredoxin-like folds, with three heme access pathways, two tunnels, and one cavity, limited by three long loops including catalytic residues. Variant 6E10 displays significantly increased loops’ flexibility that favors function over stability: despite the considerably higher catalytic efficiency, this variant shows poorer protein stability compared to wild-type and 29E4 variants. Constant-pH MD simulations revealed a more positively charged microenvironment near the heme pocket of variant 6E10, particularly in the neutral to alkaline pH range. This microenvironment affects enzyme activity by modulating the pKa of essential residues in the heme vicinity and should account for variant 6E10 improved activity at pH 7–8 compared to the wild-type and 29E4 that show optimal enzymatic activity close to pH 4. Our findings shed light on the structure–function relationships of DyPs at the molecular level, including their pH-dependent conformational plasticity. These are essential for understanding and engineering the catalytic properties of DyPs for future biotechnological applications

Corrigendum to “Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase” Comput Struct Biotechnol J, 20 (2022), 3899–3910

Funding Information: This work was supported by the Fundação para a Ciência e Tecnologia, Portugal , grants CEECIND/02300/2017 , PTDC/BBBEBB/0122/2014 , PTDC/BII-BBF/29564/2017 , EXPL/BIA-BQM/0473/2021 , MOSTMICRO-ITQB ( UIDB/04612/2020 and UIDP/04612/2020 ), LS4FUTURE Associated Laboratory ( LA/P/0087/2020 ), BioISI ( UIDB/04046/2020 and UIDP/04046/2020 ), and projects UIDB/04326/2020 , UIDP/04326/2020 , LA/P/0101/2020 , and the operational programs CRESC Algarve 2020 and COMPETE through project EMBRC.PT ALG-01–0145-FEDER-022121 . Publisher Copyright: © 2022 The Author(s)The authors would like to add in the Funding section the project reference EXPL/BIA-BQM/0473/2021 in order to be like this: This work was supported by the Fundação para a Ciência e Tecnologia, Portugal, grants CEECIND/02300/2017, PTDC/BBBEBB/0122/2014, PTDC/BII-BBF/29564/2017, EXPL/BIA-BQM/0473/2021, MOSTMICRO-ITQB (UIDB/04612/2020 and UIDP/04612/2020), LS4FUTURE Associated Laboratory (LA/P/0087/2020), BioISI (UIDB/04046/2020 and UIDP/04046/2020), and projects UIDB/04326/2020, UIDP/04326/2020, LA/P/0101/2020, and the operational programs CRESC Algarve 2020 and COMPETE through project EMBRC.PT ALG-01–0145-FEDER-022121. The authors would like to apologise for any inconvenience caused.publishersversionpublishe