The Effect of Biomarker Use on the Speed and Duration of Clinical Trials for Cancer Drugs.

BACKGROUND: The purpose of this study was to explore the effects biomarkers have on the duration and speed of clinical trials in oncology. MATERIALS AND METHODS: Clinical trial data was pooled from www.clinicaltrials.gov within the 4 cancer indications of non-small cell lung cancer, breast cancer, melanoma, and colorectal cancer. Heatmaps of clinical timelines were used to display differences in the frequency and timing of clinical trials across trials that used or did not use biomarkers, for all 4 indications. RESULTS: Screening of 8630 clinical trials across the 4 indications yielded 671 unique drugs corresponding to 1224 eligible trials used in our analysis. The constructed heatmaps visually represented that biomarkers did not have an effect on the time gap between trial phases for non-small cell lung cancer and melanoma but did for colorectal and breast cancer trials, reducing the speed of trial timelines. It was also observed that biomarker trials were more often concurrent over shorter periods of time and began later in the timeline for non-small cell lung and colorectal cancers. CONCLUSION: The novel visualization method revealed longer gaps between trial phases, later clinical trial start times, and shorter periods of concurrently run trials for drugs that used biomarkers. The study highlights that biomarker-driven trials might impact drug approval timelines and need to be considered carefully in clinical development plan.

Modulating Transmembrane α-Helix Interactions through pH-Sensitive Boundary Residues.

Changes in pH can alter the structure and activity of proteins and may be used by the cell to control molecular function. This coupling can also be used in non-native applications through the design of pH-sensitive biomolecules. For example, the pH (low) insertion peptide (pHLIP) can spontaneously insert into a lipid bilayer when the pH decreases. We have previously shown that the α-helicity and helix-helix interactions of the TM2 α-helix of the proteolipid protein (PLP) are sensitive to the local hydrophobicity at its C-terminus. Given that there is an ionizable residue (Glu-88) at the C-terminus of this transmembrane (TM) segment, we hypothesized that changing the ionization state of this residue through pH may alter the local hydrophobicity of the peptide enough to affect both its secondary structure and helix-helix interactions. To examine this phenomenon, we synthesized peptide analogues of the PLP TM2 α-helix (wild-type sequence (66)AFQYVIYGTASFFFLYGALLLAEGF(90)). Using circular dichroism and Förster resonance energy transfer in the membrane-mimetic detergent sodium dodecyl sulfate, we found that a decrease in pH increases both peptide α-helicity and the extent of self-association. This pH-dependent effect is due specifically to the presence of Glu-88 at the C-terminus. Additional experiments in which Phe-90 was mutated to residues of varying hydrophobicities indicated that the strength of this effect is dependent on the local hydrophobicity near Glu-88. Our results have implications for the design of TM peptide switches and improve our understanding of how membrane protein structure and activity can be regulated through local molecular environmental changes.

Terminal residue hydrophobicity modulates transmembrane helix-helix interactions.

Central to the formation of tertiary structure in membrane protein folding is the presence of amino acid sequence motifs (such as "small-XXX-small" segments) in the TM segments that promote interaction-compatible surfaces through which the TM α-helices interact. Here, we sought to elucidate additional factors that may work in tandem to dictate the ultimate interaction fate of TM-embedded segments. In this context, we used proteolipid protein (PLP), the major protein from central nervous system myelin for which mutant-dependent non-native oligomerization has been implicated in neurological disorders, to explore the specific effects of TM boundary residues (the membrane entry and exit points), keying on the secondary structure and self-association of peptides corresponding to the PLP TM2 α-helix (wild-type sequence 66AFQYVIYGTASFFFLYGALLLAEGF9°). Using gel electrophoresis, circular dichroism, and Förster resonance energy transfer in the membrane-mimetic detergent sodium dodecyl sulfate (SDS), we found that mutation of F90 to residues such as A, I, L, or V maintains the onset of TM2-TM2 dimerization, whereas mutation to E, G, Q, N, S, or T abrogates dimer formation. We attribute this sensitivity to changes in local hydrophobicity, viz., a decrease in hydrophobicity reduces local lipid-peptide interactions, which in turn disrupts peptide α-helicity and hence the effectiveness of an incipient interaction-compatible surface. Our results show that the secondary structure and oligomeric state of PLP TM2 Lys-tagged peptides are significantly modulated by the specific nature of their C-terminal boundary residue, thus providing insight as to how point mutations, particularly where they produce disease states, can compromise the folding process.

Membrane protein misassembly in disease.

Helix-helix interactions play a central role in the folding and assembly of integral α-helical membrane proteins and are fundamentally dictated by the amino acid sequence of the TM domain. It is not surprising then that missense mutations that target these residues are often linked to disease. In this review, we focus on the molecular mechanisms through which missense mutations lead to aberrant folding and/or assembly of these proteins, and then discuss pharmacological approaches that may potentially mitigate or reverse the negative effects of these mutations. Improving our understanding of how missense mutations affect the interactions between TM α-helices will increase our capability to develop effective therapeutic approaches to counter the misassembly of these proteins and, ultimately, disease. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Modulation of the oligomerization of myelin proteolipid protein by transmembrane helix interaction motifs.

Proteolipid protein (PLP) is a highly hydrophobic 276-residue integral membrane protein that constitutes more than 50% of the total protein in central nervous system myelin. Previous studies have shown that this protein exists in myelin as an oligomer rather than as a monomer, and mutations in PLP that lead to neurological disorders such as Pelizaeus-Merzbacher disease and spastic paraplegia type 2 have been reported to affect its normal oligomerization. Here we employ peptide-based and in vivo approaches to examine the role of the TM domain in the formation of PLP quaternary structure through homo-oligomeric helix-helix interactions. Focusing on the TM4 alpha-helix (sequence (239)FIAAFVGAAATLVSLLTFMIAATY(262)), the site of several disease-causing point mutations that involve putative small residue helix-helix interaction motifs in the TM4 sequence, we used SDS-PAGE, fluorescence resonance energy transfer, size-exclusion chromatography, and TOXCAT assays in an Escherichia coli membrane to show that the PLP TM4 helix readily assembles into varying oligomeric states. In addition, through targeted studies of the PLP TM4 alpha-helix with point mutations that selectively eliminate these small residue motifs via substitution of Gly, Ala, or Ser residues with Ile residues, we describe a potential mechanism through which disease-causing point mutations can lead to aberrant PLP assembly. The overall results suggest that TM segments in misfolded PLP monomers that expose and/or create surface-exposed helix-helix interaction sites that are normally masked may have consequences for disease.

Novel hydrophobic standards for membrane protein molecular weight determinations via sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally employed technique that separates proteins on the basis of molecular weight (MW). However, membrane proteins are known to size anomalously on SDS-PAGE calibrated with conventional standards, an issue that complicates interpretation of protein identity, purity, degradation, and/or stoichiometry. Here we describe the preparation of novel polyleucine hydrophobic standards for SDS-PAGE that reduce the average deviation of the apparent MW from the formula MW of natural membrane proteins to 7% versus 20% with commercially available standards. Our results suggest that gel calibration with hydrophobic standards may facilitate the interpretation of membrane protein SDS-PAGE experiments.

Deletion of a terminal residue disrupts oligomerization of a transmembrane alpha-helix.

In studies of the structural biology of membrane proteins, the success of strategies based on the "divide and conquer" approach, where peptides are used to model the individual transmembrane (TM) alpha-helices of membrane proteins, depends on the correct identification of the membrane-embedded TM alpha-helix amino acid sequence within the full-length protein. In the present work, we examine the effects of excluding or including TM boundary residues on the intrinsic properties of a Lys-tagged TM2 alpha-helix of myelin proteolipid protein (PLP), of parent sequence KKKK-66AFQYVIYGTASFFFLYGALLLAEG89-KKKK along with analogs containing an additional wild type Phe-90, Phe-90 and Tyr-91, and of a hydrophobic mutant Leu-90. Using protein gel electrophoresis, circular dichroism, and fluorescence resonance energy transfer in the membrane-mimetic detergent sodium dodecylsulfate (SDS), we demonstrate that the removal of a single amino acid from the C-terminus of this TM segment is enough to change its intrinsic properties, with TM2 66-89 displaying only a monomeric form, but with dimers arising for the other 3 peptides. A novel use of trifluoroethanol (TFE) as a maximal helix-supporting solvent demonstrated that peptides containing residues at positions 90 and (or) 90-91 displayed significantly increased helical content vs. the TM2 parent peptide. The findings suggest that deletion of critical C-terminal residue(s) tends to reposition the helix terminus toward the membrane-aqueous interface. Our overall results emphasize the potential influence of boundary residues on TM properties when using peptides as models for TM alpha-helices, and may implicate a role for these residues in membrane protein folding and assembly.

Rethinking enzyme kinetics: Designing and developing a biomolecular interactive tutorial (BIOMINT) learning tool for undergraduate students.

Enzyme kinetics is the study of enzymatic catalytic rates in biochemical reactions. This topic is commonly taught to life science students in introductory biochemistry courses during their undergraduate education. Unlike most other biochemistry topics, which focus on visual structures of biomolecules and their processes, enzyme kinetics is explained primarily through abstract mathematical and two-dimensional graphical plots. However, these abstract/symbolic representations often make it difficult for students to relate the kinetic parameters to the underlying molecular system that is being described. In this article, we present the design and development of a web-based multimedia interactive learning tool, biomolecular interactive tutorials (BIOMINT) to help students better bridge the relationships between these abstract mathematical models and the molecular behaviors, interactions, and dynamics that produce kinetic phenomena. This learning tool can be accessed at https://bit.ly/biomint. © 2019 International Union of Biochemistry and Molecular Biology, 48(1):74-79, 2020.

Developing a three-dimensional animation for deeper molecular understanding of michaelis-menten enzyme kinetics.

The mathematical models that describe enzyme kinetics are invaluable predictive tools in numerous scientific fields. However, the daunting mathematical language used to describe kinetic behavior can be confusing for life science students; they often struggle to conceptualize and relate the mathematical representations to the molecular phenomena occurring at both macroscopic and microscopic levels. Students with less developed abstract and mathematical thinking skills may benefit from a visual learning approach. The paucity of visual resources for enzyme kinetics makes this a fertile field for developing novel learning media. We discuss developing a three-dimensional animation aimed at introducing key concepts of Michaelis-Menten enzyme kinetics to undergraduate life science students. This animation uses both realistic and metaphoric depictions of the underlying molecular players, environments, and interactions in enzyme kinetics to contextualize and explain the relationship between the mathematical models and underlying molecular systems. The animation can be viewed at bit.ly/michaelis-menten. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):561-565, 2018.

Helix-helix interactions: is the medium the message?

In this issue of Structure, Zhang and colleagues compare the helix-helix interaction spaces of an extensive database of soluble and membrane proteins. Intriguingly, the resultant clusters show similar helix interaction geometries between the protein classes, differing in detail only by patterns of local interactions and inter-helical distances.

Characterization of an Escherichia coli sulfite oxidase homologue reveals the role of a conserved active site cysteine in assembly and function.

We report the biochemical and biophysical characterization of YedYZ, a sulfite oxidase homologue from Escherichia coli. YedY is a soluble catalytic subunit with a twin arginine leader sequence for export to the periplasm by the Tat translocation system. YedY is the only molybdoenzyme so far isolated from E. coli with the Mo-MPT form of the molybdenum cofactor. The electron paramagnetic resonance (EPR) signal of the YedY molybdenum is similar to that of known Mo-MPT containing enzymes, with the exception that only the Mo(IV) --> Mo(V) transition is observed, with a midpoint potential of 132 mV. YedZ is a membrane-intrinsic cytochrome b with six putative transmembrane helices. The single heme b of YedZ has a midpoint potential of -8 mV, determined by EPR spectroscopy of YedZ-enriched membrane preparations. YedY does not associate strongly with YedZ on the cytoplasmic membrane. However, mutation of the YedY active site Cys102 to Ser results in very efficient targeting of YedY to YedZ in the membrane, demonstrating a clear role for YedZ as the membrane anchor for YedY. Together, YedYZ comprise a well-conserved bacterial heme-molybdoenzyme found in a variety of bacteria that can be assigned to the sulfite oxidase class of enzyme.