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Revision as of 20:48, 30 September 2024

PNC-27

Overview

Aiming to address the problem of complications presented due to conventional breast adenocarcinoma therapy methods, we are proposing an innovative solution - bacteriotherapy, using non-pathogenic chassis E.coli to synthesize anticancer peptide. Bacteria will serve the synthesis purpose, therefore cannot directly interact with cancer cells. That is why the E.coli will be enclosed in the hydrogel network. The specificity of the treatment is ensured by using lactate sensor and peptide specificity itself. In order to prevent spreading out of the bacteria inside the body, the kill switch was designed.

Anticancer peptide

PNC-27 is a 32-residue peptide composed of an HDM2 binding domain of p53 (residues 12–26) and CPP leader sequence. The peptide is synthetic in nature, meaning that it was initially produced through protein engineering methods.

Penetratin sequence

CPP leader sequence represents part essential for binding and entrance into target cells. The fragment is also known as Penetratin. It was essentially derived from a leader sequence of the antennapedia protein [1]. Penetratin contains a high density of positively charged residues that stabilize an α-helix when present on its carboxyl terminal end [1]. Because of this property, aside from the main function Penetratin is essential for proper folding of PNC-27.

HDM2 binding domain

PNC-27 has been shown to eradicate cancer cells with higher specificity due to the nature of its binding partner, indicating that normal cells are typically not affected by it [2,3]. Human Double Minute Homolog 2 or HDM-2, is known to be overexpressed in cancerous cells [3]. Through binding to HDM2, PNC-27 becomes cytotoxic for cancer cells as this interaction leads to the formation of pores on cell membranes [4]. Direct binding to HDM-2 is conducted via α-helical conformation of the protein [1]. HDM-2 is overexpressed in the membranes of both solid and non-solid tissue tumors [3]. The experimental results suggest that early developing tumor cells exhibit high concentrations of HDM-2 in their membranes [5,6]. In addition, HDM-2 was reported to be a marker of rapidly growing tumors. Its elevated levels correlate with metastatic properties of primary tumor cell cultures obtained from breast cancer patients [7]. Cancer cells obtain these motility properties due to co-localization of peptide with E-cadherin in the cancer cells plasma membranes, which leads to the ubiquitination and degradation of the latter.

Treatment efficiency

PNC-27 demonstrated its efficiency in a wide variety of cancer cell lines. For instance, PNC-27 induced rapid total cell necrosis (within 1 hr) of several breast cancer cell lines [8]. The results of another study show that PNC-27 is cytotoxic to cells from long-established and chemotherapy-resistant human ovarian cancer cell lines [9]. Necrosis of cells was confirmed as elevated concentrations of lactate dehydrogenase (LDH) were released from the samples treated with the peptide [3]. IC50 values of the peptide range from 75 ug/ml (18.6 uM) to 200 ug/ml (50 uM) [3]. Notably, the studies reported that PNC-27 induced pores in the membranes of cancer cells, but cell membrane lysis was not observed after treatment of untransformed cells [10, 7, 11]. Lastly, for the in vivo experiments, PNC-27 was tested on human pancreatic cancer cells (MIA-PaCa-2) and a melanoma cell line (A2058) in nude mice. While efficient tumor eradication was observed, no evidence of toxic side effects was documented [3].

Modelling

Introduction

By adjusting the model's parameters, the system can be adjusted to a PNC-27 concentration within the therapeutic window. It is possible to reduce the number of experiments and enhance the experiment configurations by adjusting the concentrations and values of sensitive parameters. These elements make the experimental work effective. This is what we do to help other researchers who are using the modified bacteria in a hydrogel system, as well as our ongoing research. An Ordinary Differential Equation (ODE) is used to predict the mechanism of action of PNC-27 against cancer cells. Figure 1 is an illustration of the simulation. The relationship between a function and its derivatives is explained by an ODE, a type of mathematical equation. Our goal is to gain further insight into the behaviour of the modified plasmid in the hydrogel system. Transcribing equations allows an ODE model to mimic our signalling route and predict the system's behaviour across time.

We have projected that our modified plasmid will produce PNC-27 and related proteins in response to lactate by using Matlab simbiology. It made it easier for us to estimate how much PNC-27 would be applied to the tumour site. As a result, we can effectively control the concentration to treat cancer.

Methods

Model structure

The model was created in MATLAB Simbiology Entension with the usage of Ordinary Differential Equations (ODEs) with the basic principle to modulate the level of expression of PNC-27 protein to the tumour site. Generally, the model relies on the central dogma of the molecular biology mechanisms - DNA transcription and protein translation, supplied by additional processes such as transcriptional regulation by promoter-repressor system and binding affinity of transcription factors.

Model description

After the transcription, PNC-27 mRNA is translated into PNC-27 protein, which is further transported outside the bacterial cells. As soon as PNC-27 is exported, it binds to the HDM-2 receptor on the surface of cancer cells and includes pore formation in the cancer cell membrane, leading to cell lysis and apoptosis. 100% of the cancer cells undergo apoptosis within 90 minutes of induction with PNC-27.

Model construction methods

ODE15s is the solver type used in the system, with the reactions being mostly Mass-Action kinetics and a few being set to Unknown laws as an exception for more accurate results. All the concentrations and values in the system of the PNC-27 synthesis are identified per 1 transformed bacterial cell. In total, the model contains 10 reactions for PNC-27 synthesis

The model is comprised of three main components:

1. Tumor site - applying the average volume of cancer, this compartment is responsible for identifying the PNC-27 binding to HDM-2.
2. Environment (hydrogel) - this compartment is responsible for identifying the overall volume of the system, in which PNC-27 is exported.
3. E.coli BL21(DE3) - the main compartment of the system, in which the synthesis of PNC-27 is regulated.

Assumptions and Limitations

1. The concentration of lactate. The literature states that the concentration of lactate in the tumour varies from 10 mM to 30 mM.[11] Taking the average value, we assume that the lactate concentration equals 20 mM in our system.
2. The copy number of plasmid in the E.coli BL21 (DE3) cell. To construct the plasmid, a pET9a, defined as a low-copy plasmid, the copy number is approximately 10 per cell.
3. The model contains several reactions, the rate for which was neither found in the literature nor studied yet in vitro. In such cases, the values for the reaction rates were either calculated manually by combining different sources and simple algebraic equations or were assumed to equal 1 if no data were available in the literature. All the values for compartment properties and parameters are represented in Table 1 and Table 2, respectively.
4. The major part of the equations in the model works on the Mass Action kinetics, which is not the most precise equation type for modelling biomolecular synthesis processes. Although some values for Michaelis-Menten kinetics for receptor-ligand binding are present in the literature, they are not enough to fully convert the system into another type of kinetics.
5. PNC-27 is not a native protein for either E.coli BL21 (DE3) or any other living organism. Additionally, no one tried to synthesize it in vivo using transformed bacteria or transfected cells. Based on this, the behaviour of the PNC-27 production in E.coli BL21 (DE3) cells is hardly predictable.

Table 1. Initial conditions in the modelled system

Abbreviation	Full name	Value	Units	Source
E.coli	E.coli BL21 (DE3)	0.001	mL	[12]
Tumor	Tumor environment	0.0042	mL	[16]
Hydrogel	Hydrogel environment	0.0018	mL	Derived from hydrogel modelling
TF	Transcription factors	75,000	molecules	[13]
RNAP	RNA polymerase	4,600	molecules	[14]
Ribosomes	Ribosomes	26,100	molecules	[12]
P9 promoter	P9 promoter	10	molecules	*(plasmid copy number)
ALPaGA promoter	ALPaGA promoter	10	molecules	*(plasmid copy number)

Parameters

The following parameters are included in the system to model the expression of PNC-27 protein in a single bacterial cell. The values that were not found are assumed to equal 1, with the prescription “assumption” in the source column. Additionally, some of the values were manually calculated from the combinations of different sources via using simple mathematical operations such as multiplication and division; in the table, those values are labelled as “manually calculated”.

Table 2. Parameters for expression model of PNC-27

Abbreviation	Full name	Value	Units	Source
Translation per mRNA	The average number of translations per 1 mRNA molecule	40	dimensionless	[17]
P9-TF binding	The rate of the transcription factor binding to the P9 promoter	1	1/(molecule*second)	assumption
LldR transcription	The rate of the LldR gene transcription under the P9 promoter	0.0644	1/(molecule*second)	[16,17] manually calculated
LldR translation rate	The rate of the LldR mRNA translation	0.0581	1/(molecule*second)	[19] manually calculated
kf	The rate of LldR mRNA degradation	0.0022	1/second	[20] manually calculated
ALPaGA-LldR-Lactate association rate	The affinity of the LldR unbinding from the ALPaGA promoter in the presence of lactate	1	1/(second*molecule)	assumption
LldR degradation rate	The rate of the LldR protein degradation	0.0010	1/second	[20] manually calculated
PNC-27 transcription rate	The rate of PNC-27 gene transcription	3.180	1/(second*molecule)	[18] manually calculated
PNC-27 mRNA degradation rate	The rate of PNC-27 mRNA degradation	0.0033	1/second	[21] manually calculated
PNC-27 translation rate	The rate of PNC-27 mRNA translation	0.2885	1/(second*molecule)	[19] manually calculated
PNC-27 degradation rate	The rate of PNC-27 protein degradation	5.56×10^(-4)	1/second	[22] manually calculated

ODEs

References

[1] Kanovsky, M., Raffo, A., Drew, L., Rosal, R., Do, T., Friedman, F. K., Rubinstein, P., Visser, J., Robinson, R., Brandt-Rauf, P. W., Michl, J., Fine, R. L., & Pincus, M. R. (2001b). Peptides from the amino terminal mdm-2-binding domain of p53, designed from conformational analysis, are selectively cytotoxic to transformed cells. Proceedings of the National Academy of Sciences, 98(22), 12438–12443. https://doi.org/10.1073/pnas.211280698

[2] Sarafraz-Yazdi, E., Mumin, S., Cheung, D., Fridman, D., Lin, B., Wong, L., Rosal, R., Rudolph, R., Frenkel, M., Thadi, A., Morano, W. F., Bowne, W. B., Pincus, M. R., & Michl, J. (2022). PNC-27, a Chimeric p53-Penetratin Peptide Binds to HDM-2 in a p53 Peptide-like Structure, Induces Selective Membrane-Pore Formation and Leads to Cancer Cell Lysis. Biomedicines, 10(5), 945. https://doi.org/10.3390/biomedicines10050945

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Sequence and Features