Current Pharmaceutical Design - Volume 20, Issue 8, 2014

Metal ions that leach into biotherapeutic drug product solution during manufacturing and storage, result in contamination that can cause physico-chemical degradation of the active molecule. In this review, we describe various mechanisms by which metal ion leachates can interact with therapeutic proteins and antibodies. Site-specific modifications due to metal catalyzed oxidation (MCO) of the therapeutic proteins cause them to become destabilized and potentially increasingly aggregation prone. We have examined the molecular sequences and structures for three case studies, human relaxin (hRlx), human growth hormone (hGH) and an IgG2 mAb to rationalize the experimental findings related to their MCO. The analysis indicates that metal-binding sites lie in close spatial proximities to predicted aggregation prone regions in these molecules. From the perspective of pharmaceutical development of biotherapeutic drugs, this link between molecular origins of MCO and subsequent aggregation is undesirable. This article further suggests molecular design strategies involving disruption of APRs that may also help mitigate the impact of metal ion leachates on biotherapeutic drug products as well as improving their solubility.

Increasing amounts of evidence suggest that Alzheimer's disease (AD) and type 2 diabetes (T2D) are linked to each other. We have recently identified in vitro a high affinity interaction between β-amyloid peptide (Aβ) of AD and islet amyloid polypeptide (IAPP) of T2D which results in the formation of non-fibrillar and non-cytotoxic Aβ-IAPP hetero-oligomers. The Aβ-IAPP interaction delays cytotoxic self-association of both polypeptides albeit it is unable to block it. In this context, IAPP-GI, a soluble conformationally constrained mimic of a non-amyloidogenic and non-toxic IAPP conformer, completely blocks Aβ amyloidogenesis and cytotoxicity. Here we studied the hetero-association pathways of Aβ with IAPP and with IAPP-GI. We found that preformed Aβ or IAPP fibrils and cytotoxic assemblies are able to seed amyloidogenesis and cytotoxicity in Aβ-IAPP but not in Aβ-IAPP-GI solutions. Initially non-fibrillar and non-toxic Aβ-IAPP but not Aβ-IAPP-GI hetero-oligomers were found to further aggregate into hetero-fibrils and cytotoxic assemblies in a process strongly enhanced under Aβ or IAPP self-assembly promoting conditions. Importantly, our studies provided evidence that initially non-fibrillar and non-toxic Aβ-IAPP hetero-oligomers are able to misfold into hetero-fibrils and indicated a crucial role of the strong amyloidogenic character of IAPP in this process. These results uncover a novel molecular property of the Aβ and IAPP sequences, i.e. their ability to form hetero-fibrils, and offer mechanistic support to a model linking Aβ and IAPP hetero-association to their cytotoxic self-association pathways and thus likely to the pathogenesis of AD and T2D.

Rational design for chemical compounds targeting protein-protein interactions has grown from a dream to reality after a decade of efforts. There are an increasing number of successful examples, though major challenges remain in the field. In this paper, we will first give a brief review of the available methods that can be used to analyze protein-protein interface and predict hot spots for chemical ligand design. New developments of binding sites detection, ligandability and hot spots prediction from the author’s group will also be described. Pocket V.3 is an improved program for identifying hot spots in protein-protein interface using only an apo protein structure. It has been developed based on Pocket V.2 that can derive receptor-based pharmacophore model for ligand binding cavity. Given similarities and differences between the essence of pharmacophore and hot spots for guiding design of chemical compounds, not only energetic but also spatial properties of protein-protein interface are used in Pocket V.3 for dealing with protein-protein interface. In order to illustrate the capability of Pocket V.3, two datasets have been used. One is taken from ASEdb and BID having experimental alanine scanning results for testing hot spots prediction. The other is taken from the 2P2I database containing complex structures of protein-ligand binding at the original protein-protein interface for testing hot spots application in ligand design.

The cellular network and its environment govern cell and organism behavior and are fundamental to the comprehension of function, misfunction and drug discovery. Over the last few years, drugs were observed to often bind to more than one target; thus, polypharmacology approaches can be advantageous, complementing the “one drug - one target” strategy. Targeting drug discovery from the systems biology standpoint can help in studies of network effects of mono- and poly-pharmacology. In this mini-review, we provide an overview of the usefulness of network description and tools for mono- and poly-pharmacology, and the ways through which protein interactions can help single- and multi-target drug discovery efforts. We further describe how, when combined with experimental data, modeled structural networks which can predict which proteins interact and provide the structures of their interfaces, can model the cellular pathways, and suggest which specific pathways are likely to be affected. Such structural networks may facilitate structure-based drug design; forecast side effects of drugs; and suggest how the effects of drug binding can propagate in multi-molecular complexes and pathways.

Computing volumes and surface areas of molecular structures is generally considered to be a solved problem, however, comparisons presented in this review show that different ways of computing surface areas and volumes can yield dramatically different values. Volumes and surface areas are the most basic geometric properties of structures, and estimating these becomes especially important for large scale simulations when individual components are being assembled in protein complexes or drugs being fitted into proteins. Good approximations of volumes and surfaces are derived from Delaunay tessellations, but these values can differ significantly from those from the rolling ball approach of Lee and Richards (3V webserver). The origin of these differences lies in the extended parts and the less well packed parts of the proteins, which are ignored in some approaches. Even though surface areas and volumes from the two approaches differ significantly, their correlations are high. Atomic models have been compared, and the poorly packed regions of proteins are found to be most different between the two approaches. The Delaunay complexes have been explored for both fully atomic and for coarse-grained representations of proteins based on only Cα atoms. The scaling relationships between the fully atomic models and the coarse-grained model representations of proteins are reported, and the lines fit yield simple relationships for the surface areas and volumes as a function of the number of protein residues and the number of heavy atoms. Further, the atomic and coarse-grained values are strongly correlated and simple relationships are reported.

The assembly of naturally occurring amyloid peptides into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in over 25 human diseases. Blocking of or interfering with the aggregation of amyloid peptides such as amyloid-β (Aβ) using small organic molecules, peptides and peptidomimetics, and nanoparticles that selectively bind or inhibit Aβ aggregates is a promising strategy for the development of novel pharmaceutical approaches and agents to treat Alzheimer's disease (AD). In a broad sense, considering many common features in structure, kinetics, and biological activity of amyloid peptides, potent inhibitors and associated inhibition strategies that are developed for targeting Aβ aggregation could also be generally applied to other amyloid-forming peptides in “protein-aggregation diseases”. Due to the complex nature of Aβ self-assembly process, increasing knowledge in high-resolution structures of Aβ oligomers, atomic-level Aβ-inhibitor binding information, and cost-effective high-throughput screening method will improve our fundamental understanding of amyloid formation and inhibition mechanisms, as well as practical design of pharmaceutical strategies and drugs to treat AD. This review summarizes major findings, recent advances, and future challenges for the development of new Aβ-aggregation inhibitors, mainly focusing on three major classes of Aβ inhibitors with associated inhibition mechanisms and practical examples.

The ability of a single protein fold to make multi-modal interactions with itself and others for transmitting biological signals across multiple receptor families is a recurring theme in signal transduction. The Toll/IL-1 receptor (TIR) domain represents an evolutionarily conserved alpha-beta Flavodoxin-like protein fold [1], which has evolved complex multifaceted molecular interactions capable of transmitting a variety of developmental and immunological signals. In mammals, TIR domains are found on both interleukin-1 receptors (IL-1Rs) and Toll-like receptors (TLRs), as well as in cytoplasmic signaling adaptors and endogenous regulatory proteins. Appropriate TIR-TIR mediated immune interactions result in cytokine responses, pathogen clearance and host immune protection, while inappropriate signaling can lead to autoimmunity, inflammation and death. In the past decade, a number of three-dimensional structures of individual TIR domains have been elucidated. When coupled with the wealth of information from mutagenesis, genetic and peptide studies, this structural data provides additional insight to the molecular mechanisms underlying signal transduction mediated by interactions between TIR domains. Owing to their ability to regulate both innate and adaptive immune responses in a variety of organisms including humans, TIR domain-mediated molecular interactions are of intense interest for therapies targeting autoimmunity, cancer and emerging host-pathogen interactions. Here, we review progress in the development of peptides, peptidomimetics and small molecules designed to regulate TIR-dependent signaling in the context of recent advances in structural and molecular studies of TIR domain proteins and their interactions.

Mutant p53 could have either a dominant negative effect or a gain of function to interfere with p53’s ability to maintain genomic stability. In the present study, we screened for TP53 mutations in hepatocellular carcinoma (HCC) samples from 202 Chinese patients, followed by analysis of transcriptional and apoptotic activities of 21 p53 mutants with or without wild-type p53 present. We identified a new point mutation p.P72A, and a mutation (p.E294SfsX51) with a record long frameshift. We found TP53 mutations in HCC bear mutants without a dominant wild-type p53 inhibition on p21 transcription at a higher frequency. We found an anti-correlation for p53 WT/mutant heterotetramer to activate p21 and BAX transcription, i.e., at given p53 WT/mutant concentration, the fold increase p21 transcription is proportional to the fold of decreasing BAX transcription. Our kinetic model reproduced the trend in the experimental observation and confirmed that the p53 WT-dimer/mutant- heterotetramer is the major species to confer the differential activation of p21 and BAX transcription. p53 may have different binding modes on p21 and BAX, most likely resulting from the combinational effects of core domain binding and C-terminal mediation. Our study demonstrated that p53 mutants interfere with the ability of WT p53 to maintain genomic stability.

We briefly discuss recent progress in computational characterization of the sequence and structural properties of β-barrel membrane properties. We discuss the emerging concept of weakly stable regions in β-barrel membrane proteins, computational methods to identify these regions and mechanisms adopted by β-barrel membrane proteins in nature to stabilize them. We further discuss computational methods to identify protein-protein interactions in β-barrel membrane proteins and recent experimental studies that aim at altering the biophysical properties including oligomerization state and stability of β-barrel membrane proteins based on the emerging organization principles of these proteins from recent computational studies.

Intrinsically disordered proteins (IDPs) and proteins with long intrinsically disordered protein regions (IDPRs) lack ordered structure but are involved in a multitude of biological processes, where they often serve as major regulators and controllers of various functions of their binding partners. Furthermore, IDPs/IDPRs are often related to the pathogenesis of various diseases, including cancer. Intrinsic disorder confers multiple functional advantages to its carriers. As a result, due to their functional versatility and structural plasticity, IDPs and IDPRs are common in various proteomes, including proteomes of different pathological organisms. Viruses are “welleducated” users of various aspects of intrinsic disorder for their advantage. These small but highly efficient invaders broadly use intrinsic disorder to overrun the host organism’s defense system, as well as to seize and overrun host systems and pathways forcing them to work for the virus needs, to ensure accommodation of viruses to their variable and often hostile habitats, and to promote and support the economic usage of the viral genetic material. Human papillomaviruses (HPVs), with their tiny proteomes (the entire HPV genome includes just eight open reading frames), intricate life cycle, and ability to either cause benign papillomas/warts or promote the development of carcinomas of the genital tract, head and neck and epidermis, attracted considerable attention of researchers. This review analyzes the plentitude and demeanor of intrinsic disorder in proteins from HPVs and their cellular targets.

Drug designing targeting protein-protein interactions is challenging. Because structural elucidation and computational analysis have revealed the importance of hot spot residues in stabilizing these interactions, there have been on-going efforts to develop drugs which bind the hot spots and out-compete the native protein partners. The question arises as to what are the key 'druggable' properties of hot spots in protein-protein interactions and whether these mimic the general hot spot definition. Identification of orthosteric (at the protein- protein interaction site) and allosteric (elsewhere) druggable hot spots is expected to help in discovering compounds that can more effectively modulate protein-protein interactions. For example, are there any other significant features beyond their location in pockets in the interface? The interactions of protein-protein hot spots are coupled with conformational dynamics of protein complexes. Currently increasing efforts focus on the allosteric drug discovery. Allosteric drugs bind away from the native binding site and can modulate the native interactions. We propose that identification of allosteric hot spots could similarly help in more effective allosteric drug discovery. While detection of allosteric hot spots is challenging, targeting drugs to these residues has the potential of greatly increasing the hot spot and protein druggability.