Molecular Basis of c-MET Inhibition by Approved Small Molecule Drugs: A Structural Perspective

The Kirsten rat sarcoma viral oncoprotein homolog (KRAS) is currently a primary focus of oncologists and translational scientists, driven by exciting results with KRAS-targeted therapies for non-small cell lung cancer (NSCLC) patients. While KRAS mutations continue to drive high cancer diagnosis and death, researchers have developed unique strategies to target KRAS variations. Having been investigated over the past 40 years and considered "undruggable" due to the lack of pharmacological binding pockets, recent breakthroughs and accelerated FDA approval of the first covalent inhibitors targeting KRASG12C, have largely sparked further drug development. Small molecule development has targeted the previously identified primary location alterations such as G12, G13, Q61, and expanded to address the emerging secondary mutations and acquired resistance. Of interest, the non-covalent KRASG12D targeting inhibitor MRTX-1133 has shown promising results in humanized pancreatic cancer mouse models and is seemingly making its way from bench to bedside. While this manuscript was under review a novel class of first covalent inhibitors specific for G12D was published, These so-called malolactones can crosslink both GDP and GTP bound forms of G12D. Inhibition of the latter state suppressed downstream signaling and cancer cell proliferation in vitro and in mouse xenografts. Moreover, a non-covalent pan-KRAS inhibitor, BI-2865, reduced tumor proliferation in cell lines and mouse models. Finally, the next generation of KRAS mutant-specific and pan-RAS tri-complex inhibitors have revolutionized RAS drug discovery. This review will give a structural biology perspective on the current generation of KRAS inhibitors through the lens of emerging secondary mutations and acquired resistance.

Advancements in small molecule drug design: A structural perspective

The cellular metabolism of host tRNAs and life cycle of HIV-1 cross paths at several key virus–host interfaces. Emerging data suggest a multi-faceted interplay between host tRNAs and HIV-1 that plays essential roles, both structural and regulatory, in viral genome replication, genome packaging, and virion biogenesis. HIV-1 not only hijacks host tRNAs and transforms them into obligatory reverse transcription primers but further commandeers tRNAs to regulate the localization of its major structural protein, Gag, via a specific interface. This review highlights recent advances in understanding tRNA–HIV-1 interactions, primarily from a structural perspective, which start to elucidate their underlying molecular mechanisms, intrinsic specificities, and biological significances. Such understanding may provide new avenues toward developing HIV/AIDS treatments and therapeutics including small molecules and RNA biologics that target these host–virus interfaces.

Structural perspectives on HCV humoral immune evasion mechanisms

The structural and functional diversity of proteins can be enhanced by numerous post-translational modifications. C-mannosylation is a rare form of glycosylation consisting of a single alpha or beta D-mannopyranose forming a carbon-carbon bond with the pyrrole ring of a tryptophan residue. Despite first being discovered in 1994, C-mannosylation is still poorly understood and 3D structures are available for only a fraction of the total predicted C-mannosylated proteins. Here, we present the first comprehensive review of C-mannosylated protein structures by analysing the data for all 10 proteins with C-mannosylation/s deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). We analysed in detail the WXXW/WXXWXXW consensus motif and the highly conserved pair of arginine residues in thrombospondin type 1 repeat C-mannosylation sites or homologous arginine residues in other domains. Furthermore, we identified a conserved PXP sequence C-terminal of the C-mannosylation site. The PXP motif forms a tight turn region in the polypeptide chain and its universal conservation in C-mannosylated protein is worthy of further experimental study. The stabilization of C-mannopyranosyl groups was demonstrated through hydrogen bonding with arginine and other charged or polar amino acids. Where possible, the structural findings were linked to other functional studies demonstrating the role of C-mannosylation in protein stability, secretion or function. With the current technological advances in structural biology, we hope to see more progress in the study of C-mannosylation that may correspond to discoveries of novel C-mannosylation pathways and functions with implications for human health and biotechnology.

Dormancy in mycobacteria is defined as a stable but reversible non-replicating state in response to stresses. In Mycobacterium tuberculosis, an important human pathogen, this state is responsible for latent Tuberculosis. The current chemotherapy to defeat Tuberculosis while effective in killing growing tubercle bacilli is largely ineffective in killing dormant bacilli. For this reason there is a recent interest to develop new drugs against this disease in the latent form. To this aim, the knowledge of the molecular basis of bacterial resuscitation from dormancy is necessary and of paramount importance. This review summarizes the current knowledge on the complex mechanism of exit from mycobacterial dormancy; the main molecular players responsible for mycobacterial resuscitation from dormancy are described and their role is discussed from a structural perspective.

Escherichia coli HTH-type transcriptional dual regulator CecR belongs to TetR family regulators (TFRs), which regulate the expression of genes enabling bacteria to survive under stress conditions. Previous studies (Yamanaka etal., Microbiology 2016; 162: 1253-1264) showed that CecR senses the presence of antibiotics, cephalosporins and chloramphenicol, in the cell and activates the expression of a putative drug efflux pump. Although CecR is present in many pathogenic strains of Escherichia and Salmonella genera, this regulator is poorly characterized. Here, we report the first crystal structure of E. coli CecR. Each protomer of the CecR homodimer is composed of an N-terminal DNA-binding and a C-terminal ligand-binding domain. In addition to nine canonical TetR α-helices, CecR contains structural elements characteristic of TetR subfamily D. The ligand-binding cavity of CecR has a tunnel-like shape, not common in TFRs. Unexpectedly, the CecR-ligand-binding cavity contained polyethylene glycol (PEG) fragments, originating from crystallization solution, and suggesting a potential site for effector binding. Additionally, the affinity of CecR to various antibiotics was determined. The strongest interactions were observed for CecR and cefepime, a representative of the fourth-generation cephalosporins. Molecular docking of the analyzed antibiotics into the ligand-binding tunnel of CecR indicated the amino acid residues important for ligand recognition. The CecR structure reported here provides the first structural information on the ligand-binding cavity and ligand recognition by CecR. As CecR is an important regulator, widespread among pathogenic bacteria belonging to the Enterobacteriales order, the results of our study are an important contribution to the understanding of the CecR-related mechanisms underlying antimicrobial resistance.

Simple SummaryUnravelling the molecular basis of ribosomal inhibition by small molecules is crucial to characterise the function of potential anticancer drugs. After approval of the ribosome inhibitor homoharringtonine for treatment of CML, it became clear that acting on the rate of protein synthesis can be a valuable way to prevent indefinite growth of cancers. The present review discusses the state-of-the-art structural knowledge of the binding modes of inhibitors targeting the cytosolic ribosome, with the ambition of providing not only an overview of what has been achieved so far, but to stimulate further investigations to yield more potent and specific anticancer drugs.Protein biosynthesis is a vital process for all kingdoms of life. The ribosome is the massive ribonucleoprotein machinery that reads the genetic code, in the form of messenger RNA (mRNA), to produce proteins. The mechanism of translation is tightly regulated to ensure that cell growth is well sustained. Because of the central role fulfilled by the ribosome, it is not surprising that halting its function can be detrimental and incompatible with life. In bacteria, the ribosome is a major target of inhibitors, as demonstrated by the high number of small molecules identified to bind to it. In eukaryotes, the design of ribosome inhibitors may be used as a therapy to treat cancer cells, which exhibit higher proliferation rates compared to healthy ones. Exciting experimental achievements gathered during the last few years confirmed that the ribosome indeed represents a relevant platform for the development of anticancer drugs. We provide herein an overview of the latest structural data that helped to unveil the molecular bases of inhibition of the eukaryotic ribosome triggered by small molecules.

Protein-Protein Interactions (PPIs) are the key components in many cellular processes including signaling pathways, enzymatic reactions and epigenetic regulation. Abnormal interactions of some proteins may be pathogenic and cause various disorders including cancer and neurodegenerative diseases. Although inhibiting PPIs with small molecules is a challenging task, it gained an increasing interest because of its strong potential for drug discovery and design. The knowledge of the interface as well as the structural and chemical characteristics of the PPIs and their roles in the cellular pathways is necessary for a rational design of small molecules to modulate PPIs. In this study, we review the recent progress in the field and detail the physicochemical properties of PPIs including binding hot spots with a focus on structural methods. Then, we review recent approaches for structural prediction of PPIs. Finally, we revisit the concept of targeting PPIs from a systems biology perspective and we refer to approaches that are usually employed when the structural information is not present.

SPLINTS: small-molecule protein ligand interface stabilizers

p53 is a potent tumor suppressor with a crucial role in preventing uncontrolled cell proliferation and is therefore frequently deleted or mutated in cancer. For tumors with wild-type p53, its function can be overcome by overactive cellular antagonists, such as the ubiquitin ligase murine double minute clone 2 (MDM2). Restoring p53 activity by inhibiting MDM2 in such cancers can eradicate tumors. Consequently, the MDM2-p53 interaction has been extensively targeted for inhibition by small molecules. In recent years, MDM2-like protein (MDMX), another key downregulator of p53, has gained increasing importance as an additional target for drug development, in order to provide a complementary approach to MDM2 inhibition. In this review, we describe how detailed structural knowledge of the MDM2-p53 interface and, more recently, of the MDMX-p53 interaction have helped advance the development of inhibitors against the two targets. We present a summary of the functional biochemistry of MDM2, MDMX and p53 as well as their interactions and examine recent progress in the development of inhibitors of MDM2 and MDMX.

The development in crystallography is nothing short of phenomenal, showing an ever increasing trend towards applications, such as in molecular biology. ‘Traditional’ small molecule chemical crystallography tends to, in many aspects, be considered as trivial due to the exponential growth in computing power and the parallel expansion in software. However, many dynamic processes (still) occur at the molecular level. Thus, the fundamental understanding of subtle nuances in structural behaviour, and the associated influence on other (kinetic) properties, is often trivialized when conclusions are simply made based on thermodynamic observations alone. This review aims to emphasize the importance of crystallography in small molecule chemistry by presenting detailed evaluations of two extended ‘case studies’, i.e. the first on structure and dynamics of the middle transition metal cyanide-oxido complexes, and the second on rhodium model homogeneous catalyst precursors. Both underline the fact of understanding the overarching principles of dynamics, i.e. time-resolved behaviour of processes, but considered from a structural perspective. The reference point is to focus on an integrated and overarching mechanistic approach, utilizing structural data, but in particular reaction kinetics, to obtain a more complete picture of processes or systems being evaluated.

Maternal embryonic leucine zipper kinase (MELK), a member of the AMP-related serine-threonine kinase family, has been involved in regulating many cellular events, and aberrant MELK expression is associated with tumorigenesis and malignant progression in various cancers. More and more studies have found that MELK plays an essential regulatory role in tumor multidrug resistance or radio resistance. MELK inhibitors can also improve drug resistance caused by a gene mutation. These findings remind us that MELK could be a chemo- or radio-sensitizing target. However, it has also been found that most experiments on MELK rely on non-selective RNAi and small molecule reagents, which makes the results questionable, and thus the development of selective MELK inhibitors is still necessary. In this review, we summarized the identified regulatory pathways of MELK in tumor resistance and reclassified MELK inhibitors from a structural perspective. In addition, we discovered the glycosylation modification site of the MELK protein and discussed the possibility of continuing to develop small molecule inhibitors targeting the glycosylation modification site. These provide new strategies for developing selective MELK inhibitors and understanding the essential biological role of MELK in cancer.

ABC (ATP Binding Cassette) proteins form one of the largest protein superfamilies. Most members are active membrane transporters translocating their substrates across the lipid bilayer of the plasma membrane or intracellular organelles. Multidrug transporters exhibit broad substrate specificity, exporting molecules with diverse chemical structures to protect organisms from xenotoxic compounds, and also play an important role in influencing the efficacy of therapeutic agents. High resolution structural information is required to reveal the conformational changes associated with the transport cycle and the interaction with small molecules, with the ultimate aim to develop strategies to pharmacologically modulate function and predict substrates properties. In this chapter we review available ABC protein structures and discuss advances in using this structural information for computational approaches that are aimed at elucidating the mechanism of substrate recognition and cargo translocation in the context of the ATP catalytic cycle of human multidrug ABC transporters.

The use of nucleic acids as potential therapeutic tools, sensors, or biomaterials, among other applications, has dramatically increased. Among these, RNA aptamers are of interest due to an innate high specificity toward their cognate targets, which include small molecules, proteins, ions, or cells. In this work, we took advantage of the ability that 8-oxo-7,8-dihydroguanine (8-oxoG) has to participate in unique H-bonding interactions, and probed its use to increase/control the selectivity/affinity of aptamers of RNA and DNA. The chosen model is a 23-nt long RNA (Neo61/Neo1-5'-GGA CUG GGC GAG AAG UUU AGU CC) strand that folds into a pentaloop hairpin with a stretch of three G·U Wobble pairs within the stem, which is known to have affinity toward various aminoglycosides. 8-OxoG was incorporated at positions G6, G7, G10, G12, or G15, within aptamers composed of RNA, DNA, or 2'-OMe modified RNA. Their recognition was tested toward 9 small molecule targets with aminoglycoside (x8) or antibiotic (x1) backbones, and their affinities were measured via circular dichroism (CD). Isothermal titration calorimetry (ITC) was used to corroborate the use of CD as a reliable technique. It was determined that incorporation of 8-oxodG at position-12 within DNA (OG12-DNA) led to increased selectivity toward neomycin or ribostamycin (Kd ≈ 2.5 and 2.2 μM), displaying 1-2 orders of magnitude tighter binding compared to other targets. Furthermore, functionalization with 8-oxodG at position-6 (OG6-DNA) displayed increased selectivity toward neomycin or tobramycin, albeit with decreased affinities (Kd ≈ 46 and 53 μM). Interestingly, the canonical DNA aptamer also displayed 4-10 fold enhanced selectivity toward neomycin, ribostamycin, and gentamicin, compared to its RNA homologue. On the other hand, the corresponding RNA analogues containing 8-oxoG or other modifications, specifically 8-oxoinosine, inosine, 8-oxoadenosine, or uridine, resulted in a high level of promiscuity toward most aminoglycosides, with kanamycin and streptomycin generally exhibiting higher dissociation constants. The presence of 2'-OMe-modified ribose led to trends similar to those obtained with their corresponding canonical RNA constructs. From a structural perspective, all nucleobase modifications led to thermal destabilization of the aptamer (including the DNA analogues), while the presence of the 2'-OMe ribose modification resulted in increased thermal stability. Among the molecules tested, neomycin and ribostamycin induced significant structural changes (measured via CD) on aptamers of RNA or DNA. Changes in RNA included the formation of a new band with positive ellipticity (λmax ∼ 285 nm), associated with glycosyl bond rotation along the G·U wobble pairs that presumably facilitates recognition. On the other hand, binding by canonical and OG12 DNA aptamers resulted in a B-to-A form transition, where the smaller major groove may serve to facilitate DNA-target interaction. Further structural data were obtained by carrying out structural probing assays in the presence of RNase A, T1, or DNase I; which displayed varying degradation patterns and thus changes in secondary structure as a function of the position of 8-oxoG/8-oxodG and presence/absence of the small-molecule target. Overall, the results reported herein show that (1) the use of 8-oxodG within DNA increases aptamer selectivity toward neomycin and/or ribostamycin; (2) the presence of 8-oxoguanine can alter the function of RNA and DNA, which is of broad biological relevance; and (3) the introduction of 2'-OMe modifications does not affect the selectivity of the aptamers in this work. While it is early to predict how 8-oxoG will affect the selectivity of aptamers at large, this work provides a link between the structure and function of oxidized RNA.