Application of artificial intelligence and machine learning techniques to the analysis of dynamic protein sequences.

We apply methods of Artificial Intelligence and Machine Learning to protein dynamic bioinformatics. We rewrite the sequences of a large protein data set, containing both folded and intrinsically disordered molecules, using a representation developed previously, which encodes the intrinsic dynamic properties of the naturally occurring amino acids. We Fourier analyze the resulting sequences. It is demonstrated that classification models built using several different supervised learning methods are able to successfully distinguish folded from intrinsically disordered proteins from sequence alone. It is further shown that the most important sequence property for this discrimination is the sequence mobility, which is the sequence averaged value of the residue-specific average alpha carbon B factor. This is in agreement with previous work, in which we have demonstrated the central role played by the sequence mobility in protein dynamic bioinformatics and biophysics. This finding opens a path to the application of dynamic bioinformatics, in combination with machine learning algorithms, to a range of significant biomedical problems.

Global Survey of Protein Dynamic Properties.

Using tools developed to study the dynamic bioinformatics of proteins, we are able to study the dynamic characteristics of very large numbers of protein sequences simultaneously. We study herein the distribution of protein sequences in a space determined by sequence mobility. It is shown that there are statistically significant differences in mobility distribution between folded sequences of different structural classes and between those and sequences of intrinsically disordered proteins. It is also shown that the several regions of mobility space differ significantly with respect to structural makeup. Helical proteins are shown to have distinctive dynamic characteristics at both extremes of the mobility spectrum.

The dynamic basis of structural order in proteins.

We compare the sequences of folded and intrinsically disordered proteins (IDPs), using bioinformatic methods recently developed to study protein dynamic properties. We demonstrate that the two classes of sequences are organized in diametrically opposite ways with respect to long-length-scale dynamic properties. We further demonstrate a statistically significant difference between the amino acid compositions of folded and disordered proteins, which is expressed in dynamic properties. Our results indicate that the long-length-scale properties of sequences are critical in determining whether proteins are able to fold, and, more generally, that they are central to an understanding of protein physics. They further provide a physical basis for the empirically observed differences in amino acid composition between folded and IDPs.

X-Ray fluorescence microscopy reveals that rhenium(i) tricarbonyl isonitrile complexes remain intact in vitro.

The complex fac-[Re(CO)3(dmphen)(para-tolylisonitrile)]+ (TRIP), where dmphen = 2,9-dimethyl-1,10-phenanthroline, is an endoplasmic reticulum stress-inducing anticancer agent (A. P. King, S. C. Marker, R. V. Swanda, J. J. Woods, S.-B. Qian and J. J. Wilson, Chem. - Eur. J., 2019, 25, 9206-9210). A second-generation compound fac-[Re(CO)3(dmphen)(para-iodobenzeneisonitrile)]+ (I-TRIP) was synthesized, and its intracellular distribution was investigated using X-ray fluorescence microscopy to show that these complexes are highly stable in vitro.

Systematically altering the lipophilicity of rhenium(I) tricarbonyl anticancer agents to tune the rate at which they induce cell death.

Rhenium-based anticancer agents have arisen as promising alternatives to conventional platinum-based drugs. Based on previous studies demonstrating how increasing lipophilicity improves drug uptake within the cell, we sought to investigate the effects of lipophilicity on the anticancer activity of a series of six rhenium(i) tricarbonyl complexes. These six rhenium(i) tricarbonyl structures, called Re-Chains, bear pyridyl imine ligands with different alkyl chains ranging in length from two to twelve carbons. The cytotoxicities of these compounds were measured in HeLa cells. At long timepoints (48 h), all compounds are equally cytotoxic. At shorter time points, however, the compounds with longer alkyl chains are significantly more active than those with smaller chains. Cellular uptake studies of these compounds show that they are taken up via both passive and active pathways. Collectively, these studies show how lipophilicity affects the rate at which these Re compounds induce their biological activities.

In Vivo Anticancer Activity of a Rhenium(I) Tricarbonyl Complex.

The rhenium(I) complex fac-[Re(CO)3(2,9-dimethyl-1,10-phenanthroline)(OH2)]+ (1) was previously shown to exhibit potent in vitro anticancer activity in a manner distinct from conventional platinum-based drugs (J. Am. Chem. Soc. 2017, 139, 14302-14314). In this study, we report further efforts to explore its aqueous speciation and antitumor activity. The cellular uptake of 1 was measured in A2780 and cisplatin-resistant A2780CP70 ovarian cancer cells by inductively coupled plasma mass spectrometry, revealing similar uptake efficiency in both cell lines. High accumulation in the mitochondria was observed, contradicting prior fluorescence microscopy studies. The luminescence of 1 is highly dependent on pH and coordination environment, making fluorescence microscopy somewhat unreliable for determining compound localization. The in vivo anticancer activity of 1 was evaluated in mice bearing patient-derived ovarian cancer tumor xenografts. These studies conclusively show that 1 is capable of inhibiting tumor growth, providing further credibility for the use of these compounds as anticancer agents.

Combinatorial Synthesis to Identify a Potent, Necrosis-Inducing Rhenium Anticancer Agent.

Combinatorial synthesis can be applied for developing a library of compounds that can be rapidly screened for biological activity. Here, we report the application of microwave-assisted combinatorial chemistry for the synthesis of 80 rhenium(I) tricarbonyl complexes bearing diimine ligands. This library was evaluated for anticancer activity in three different cancer cell lines, enabling the identification of three lead compounds with cancer cell growth-inhibitory activities of less than 10 µM. These three lead structures, Re-9B, Re-9C, and Re-9D, were synthesized independently and fully characterized by NMR spectroscopy, mass spectrometry, elemental analysis, and X-ray crystallography. The most potent of these three complexes, Re-9D, was further explored to understand its mechanism of action. Complex Re-9D is equally effective in both wild-type and cisplatin-resistant A2780 ovarian cancer cells, indicating that it circumvents cisplatin resistance. This compound was also shown to possess promising activity against ovarian cancer tumor spheroids. Additionally, flow cytometry showed that Re-9D does not induce cell cycle arrest or flipping of phosphatidylserine to the outer cell membrane. Analysis of the morphological changes of cancer cells treated with Re-9D revealed that this compound gives rise to rapid plasma membrane rupture. Collectively, these data suggest that Re-9D induces necrosis in cancer cells. To assess the in vivo biodistribution and stability of this compound, a radioactive 99mTc analogue of Re-9D, 99mTc-9D(H2O), was synthesized and administered to naïve BALB/c mice. Results of these studies indicate that 99mTc-9D(H2O) exhibits high metabolic stability and a distinct biodistribution profile. This research demonstrates that combinatorial synthesis is an effective approach for the development of new rhenium anticancer agents with advantageous biological properties.

Anticancer activity of complexes of the third row transition metals, rhenium, osmium, and iridium.

The clinical success of the platinum-based chemotherapeutic agents has prompted the investigation of coordination and organometallic complexes of alternative metal centers for use as anticancer agents. Among these alternatives, the third row transition metal neighbors of platinum on the periodic table have only recently been explored for their potential to yield anticancer-active complexes. In this Perspective, we summarize developments within the last six years on the application of rhenium, osmium, and iridium complexes as anticancer drug candidates. This review focuses on studies that discuss the potential mechanisms of action of these complexes. As reflected in this Perspective, complexes of these metal ions induce cancer cell death via a diverse range of mechanisms. Notably, small structural changes can significantly alter the mode of cell death, hindering efforts to elucidate structure-activity relationships. This property may both benefit and hinder the clinical development of these compounds.