by Trinity College Dublin
Fig. 1: Tumoricidal activity of two non-homologous alpha-helical peptide–oleate complexes. a Ribbon representation of the crystallographically determined three-dimensional structure of human α-lactalbumin (PDB ID: 1B9O), indicating the alpha1 (blue), beta (green), and alpha2 (gray) domains. The calcium ion is not shown. b Far-UV circular dichroism spectra of synthetic alpha1 peptide, beta peptide, and their respective peptide–oleate complexes. c, d Death response in human lung (A549), kidney (A498), and murine bladder (MB49) carcinoma cells, quantified as a reduction in ATP levels (c, P = 3.26E−5 for A549, 0.013 for A498 and 0.005 for MB49, alpha1–oleate compared to beta–oleate) or PrestoBlue fluorescence (d, P = 0.007 for A549, 0.003 for A498 and 0.002 for MB49, alpha1–oleate compared to beta–oleate). Cells were treated with the alpha1–oleate complex (blue) or the beta–oleate complex (green), (3 h, 35 μM, cell death compared to PBS controls). For controls exposed to the naked peptides or oleate alone, see Supplementary Fig. 1d. e Colony assay showing dose-dependent long-term effects of alpha1-oleate. A representative image is shown from two independent experiments. Scale bar = 5 mm. f Alpha1–oleate triggers rapid membrane blebbing in A549 lung carcinoma cells (35 μM, 10 min). Scale bar = 10 μm. A representative image is shown from three independent experiments. g K+ efflux in A549 lung carcinoma cells exposed to alpha1–oleate and inhibition with BaCl2. h Inhibition of cell death by the ion flux inhibitors Amiloride and BaCl2 (100 μM), measured by PrestoBlue fluorescence (P = 0.031 for 21 μM + BaCl2, 0.005 for 21 μM + Amiloride, 0.028 for 35 μM + BaCl2, and 0.014 for 35 μM + Amiloride, compared to no inhibitor). i DNA strand breaks detected by TUNEL staining in alpha1–oleate-treated A549 lung carcinoma cells (n = 50 cells per group). Scale bar = 20 μm. j AlexaFluor568-labeled alpha1–oleate (red) is internalized by A549 lung carcinoma cells. Nuclei are counterstained with DAPI (blue) (n = 52 cells per group). Scale bar = 10 μm. Data are presented as mean ± SEM from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001, analyzed by two-tailed unpaired t-test (c, d, h, j) and 2-way ANOVA using Dunnett’s correction (i). From: Bladder cancer therapy using a conformationally fluid tumoricidal peptide complex
A new approach to molecular drug design has yielded a highly promising bladder cancer drug, which induced rapid shedding of tumor cells and resulted in a significant reduction in tumor size when used in clinical trials.
These potent effects were seen in patients with non-muscle invasive bladder cancer (NMIBC) and the treatment was shown to be safe, as no drug-related side effects were observed.
The exciting research involved a collaborative group of scientists from Trinity College Dublin, Charles University and Motol Hospital (Prague), Lund University, and startup company Hamlet Pharma. The study has just been published in the leading journal Nature Communications.
Bladder cancer—a global killer
Bladder cancer is the fifth most common malignancy in Europe (and the fourth most common in the US). It is associated with the highest lifetime treatment costs per patient of all cancers, and more than 80% of patients recur after complete surgical removal of the first tumor.
Over the past three decades, few drugs have been approved for non–muscle-invasive disease, and—compounding the problem—access to these drugs is limited by insufficient supply, including BCG immuno-therapy and common chemotherapeutics such as Mitomycin and Epirubicin.
The new approach
The new approach involved designing a drug candidate using the “intrinsically disordered proteins” (IDP) concept, which relates to the recent understanding that a large segment of proteins in the body does not have a fixed 3D structure (they each typically instead take shape in a variety of ways/structures that change depending on a range of factors).
This contrasts with the more common drug design direction taken, which is based on the “lock and key” concept. This relates to the idea that proteins have fixed, well-organized 3D structures, allowing drugs to be designed to target very specific regions.
The new approach involved the use of an IDP complex known as HAMLET—a component of human milk—which, when partially unfolded, possesses tremendous cancer-killing abilities.
Dr. Ken H Mok, Associate Professor in Trinity’s School of Biochemistry and Immunology and the Trinity Biomedical Sciences Institute led the structural element of the work. He said: “Targeted cancer therapies have made significant advances in recent years but the lack of tumor specificity remains a significant concern. Few current therapies kill cancer cells without harming healthy tissues, and severe side effects have become accepted as a necessary price to pay for survival or cure. This research is therefore extremely exciting as the clinical trials show great impact in reducing tumor size in people with this form of bladder cancer without any side effects.
“From a scientific perspective—and with a nod to the great potential for other therapeutic discoveries—it is also extremely exciting to have contributed to an entirely new approach to molecular drug design. Intrinsically disordered proteins compose over 50% of the human proteome and their malleability to adapt towards binding a variety of surfaces may, in some cases such as this, result in a gain of function. One motif may have a ‘targetable weakness’ that others don’t have.
“This concept may help people understand why drugs designed using “lock and key” principles often fail in clinical trials if they encounter different structural forms of the protein they were made to attack. Once the blueprints change, a promising drug may not have the desired impact.
“In a way, we are witnessing an analogous rapid variation in protein structure during this COVID-19 pandemic, albeit through the sampling of mutation space. Although not an intrinsically disordered protein, the spike protein (S-protein) of the virus is continuously varying its thermodynamic stability or allowing it to be processed more readily by our cells—in other words, it is practicing a kind of conformational fluidity.”
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