Small Molecule Enzyme Inhibitors Research Articles

Shaping enzyme inhibitors

Small-molecule enzyme-inhibitors often display insufficient affinity and selectivity for their targets causing unwanted side effects when used as drugs. Molecularly imprinted polymers prepared using the enzyme as a template could offer a solution.

Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State

The arduous task of rationally designing small-molecule enzyme inhibitors is complicated by the inherent flexibility of the protein scaffold. To gain insight into the changes in dynamics associated with small-molecule-based inhibition, we have characterized, using NMR spectroscopy, Escherichia coli dihydrofolate reductase in complex with two drugs: methotrexate and trimethoprim. The complexes allowed the intrinsic dynamic effects of drug binding to be revealed within the context of the "closed" structural ensemble. Binding of both drugs results in an identical decoupling of global motion on the micro- to millisecond timescale. Consistent with a change in overall dynamic character, the drugs' perturbations to pico- to nanosecond backbone and side-chain methyl dynamics are also highly similar. These data show that the inhibitors simultaneously modulate slow concerted switching and fast motions at distal regions of dihydrofolate reductase, providing a dynamic link between the substrate binding site and distal loop residues known to affect catalysis.

Mechanisms of resistance to tyrosine kinase inhibitors in chronic myeloid leukemia and recent therapeutic strategies to overcome resistance

Given its relative rarity, it may at first seem surprising that chronic myeloid leukemia (CML) has garnered so much attention over the last decade. Yet, the advances in molecular pathogenesis that have been derived from studying this leukemia have clearly benefited all of oncology. Moreover, the strides in drug design and development that have also ensued around CML have given rise to what others have called a molecular revolution in cancer therapy. While a majority of patients with chronic phase CML (CP-CML) have an excellent durable response to imatinib (Gleevec, Novartis, Basel, Switzerland), a clear minority will unfortunately have signs of primary or secondary resistance to therapy. Significant efforts geared toward understanding the molecular mechanisms of imatinib resistance have yielded valuable insights into the biology of drug trafficking into and out of cells, epigenetic control of cellular processes, alterations in enzymatic structures, and the rational structural-based design of small molecule enzyme inhibitors. This review will describe the efforts at understanding the pathogenesis of imatinib resistance and the molecular rationale for the development of second- and now third-generation therapies for patients with CML.

Ischemia‐reperfusion injury is attenuated in VAP‐1‐deficient mice and by VAP‐1 inhibitors

Neutrophils mediate the damage caused by ischemia-reperfusion both at the site of primary injury and in remote organs. Vascular adhesion protein-1 (VAP-1) is an ectoenzyme expressed on endothelial cells and it has been shown to regulate leukocyte extravasation. Here we show for the first time using VAP-1-deficient mice that VAP-1 plays a significant role in the intestinal damage and acute lung injury after ischemia-reperfusion. Separate inhibition of VAP-1 by small molecule enzyme inhibitors and a function-blocking monoclonal antibody in WT mice revealed that the catalytic activity of VAP-1 is responsible for its pro-inflammatory action. The use of transgenic humanized VAP-1 mice also showed that the enzyme inhibitors alleviate both the ischemia-reperfusion injury in the gut and neutrophil accumulation in the lungs. These data thus indicate that VAP-1 regulates the inflammatory response in ischemia-reperfusion injury and suggest that blockade of VAP-1 may have therapeutic value.

NOX enzymes as novel targets for drug development

The members of the NOX/DUOX family of NADPH oxidases mediate such physiologic functions as host defense, cell signaling, and thyroid hormone biosynthesis through the generation of reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide. Moreover, ROS are involved in a broad range of fundamental biochemical and cellular processes, and data accumulated in recent years indicate that the NOX enzymes comprise one of the most important biological sources of ROS. Given the high biochemical reactivity of ROS, it is not surprising that they have been implicated in a wide variety of pathologies and diseases. Prominent among the settings that feature ROS-mediated tissue injury are disorders associated with inflammation, aging, and progressive degenerative changes in cells and organ systems, and it appears that essentially no organ system is exempt. Among the disorders currently believed to be mediated at least in part by NOX-derived ROS are hypertension, aortic aneurysm, myocardial infarction (and other ischemia-reperfusion disorders), pulmonary fibrosis and hypertension, amyotropic lateral sclerosis, Alzheimer's disease, Parkinson's disease, ischemic stroke, diabetic nephropathy, and renal cell carcinoma. Several small-molecule and peptide inhibitors of the NOX enzymes have been useful in experimental studies, but issues of specificity, potency, and toxicity militate against any of the existing published compounds as candidates for drug development. Given the broad array of disease targets documented in recent work, the time is here for vigorous efforts to develop clinically useful inhibitors of the NOX enzymes. As most (though not all) NOX-related diseases appear to be mediated by a single member of the NOX family, agents with isoform specificity will be preferred, although broadly active NOX inhibitors may prove to be useful in some settings.

Structure‐based tailoring of compound libraries for high‐throughput screening: Discovery of novel EphB4 kinase inhibitors

High-throughput docking is a computational tool frequently used to discover small-molecule inhibitors of enzymes or receptors of known three-dimensional structure. Because of the large number of molecules in chemical libraries, automatic procedures to prune multimillion compound collections are useful for high-throughput docking and necessary for in vitro screening. Here, we propose an anchor-based library tailoring approach (termed ALTA) to focus a chemical library by docking and prioritizing molecular fragments according to their binding energy which includes continuum electrostatics solvation. In principle, ALTA does not require prior knowledge of known inhibitors, but receptor-based pharmacophore information (hydrogen bonds with the hinge region) is additionally used here to identify molecules with optimal anchor fragments for the ATP-binding site of the EphB4 receptor tyrosine kinase. The 21,418 molecules of the focused library (from an initial collection of about 730,000) are docked into EphB4 and ranked by force-field-based energy including electrostatic solvation. Among the 43 compounds tested in vitro, eight molecules originating from two different anchors show low-micromolar activity in a fluorescence-based enzymatic assay. Four of them are active in a cell-based assay and are potential anti-angiogenic compounds.

Chemical approaches to understanding O-GlcNAc glycosylation in the brain

O-GlcNAc glycosylation is a unique, dynamic form of glycosylation found on intracellular proteins of all multicellular organisms. Studies suggest that O-GlcNAc represents a key regulatory modification in the brain, contributing to transcriptional regulation, neuronal communication and neurodegenerative disease. Recently, several new chemical tools have been developed to detect and study the modification, including chemoenzymatic tagging methods, quantitative proteomics strategies and small-molecule inhibitors of O-GlcNAc enzymes. Here we highlight some of the emerging roles for O-GlcNAc in the nervous system and describe how chemical tools have significantly advanced our understanding of the scope, functional significance and cellular dynamics of this modification.

VAP-1 and CD73, Endothelial Cell Surface Enzymes in Leukocyte Extravasation

Leukocyte extravasation from the blood into tissues is crucial for normal immune surveillance and in inflammation. Traditionally molecules belonging to selectin, chemokine, integrin, and immunoglobulin super families are thought to mediate the multiple adhesive and activation events needed for a successful emigration cascade. Recently, emerging evidence suggests that enzymes expressed on the surface of endothelial cells and leukocytes also contribute to the leukocyte extravasation cascade. Here we briefly review the role of vascular adhesion protein-1 (VAP-1) and CD73, 2 cell surface enzymes, in leukocyte migration form the blood into the tissues. Importantly, specific enzyme inhibitors, gene-deficient mice, and recombinant enzymes have recently unambiguously shown that the catalytic activity of these enzymes regulates the leukocyte traffic. The concept of enzymatic regulation of leukocyte extravasation provides new insight into the multi-step adhesion cascade and opens new possibilities for inhibiting inappropriate inflammatory reaction through the use of small molecule enzyme inhibitors.

VAP-1-Deficient Mice Display Defects in Mucosal Immunity and Antimicrobial Responses: Implications for Antiadhesive Applications

VAP-1, an ecto-enzyme expressed on the surface of endothelial cells, is involved in leukocyte trafficking between the blood and tissues under physiological and pathological conditions. In this study, we used VAP-1-deficient mice to elucidate whether absence of VAP-1 alters the immune system under normal conditions and upon immunization and microbial challenge. We found that VAP-1-deficient mice display age-dependent paucity of lymphocytes, in the Peyer's patches of the gut. IgA concentration in serum was also found to be lower in VAP-1(-/-) animals than in wild-type mice. Although there were slightly less CD11a on B and T cells isolated from VAP-1-deficient mice than on those from wild-type mice, there were no differences in the expression of gut-homing-associated adhesion molecules or chemokine receptors. Because anti-VAP-1 therapies are being developed for clinical use to treat inflammation, we determined the effect of VAP-1 deletion on useful immune responses. Oral immunization with OVA showed defective T and B cell responses in VAP-1-deficient mice. Antimicrobial immune responses against Staphylococcus aureus and coxsackie B4 virus were also affected by the absence of VAP-1. Importantly, when the function of VAP-1 was acutely neutralized using small molecule enzyme inhibitors and anti-VAP-1 Abs rather than by gene deletion, no significant impairment in antimicrobial control was detected. In conclusion, VAP-1-deficient mice have mild deviations in the mucosal immune system and therapeutic targeting of VAP-1 does not appear to cause a generalized increase in the risk of infection.

Challenges and Optimism for Nanoengineering

Recent advances in nanotechnology and materials science have made it possible to design and synthesize structures with nanometer-scale features that have the potential to interphase in novel and productive ways with cells and tissues. Further progress in this field in the near future will probably result in more sensitive and complex biosensors, refined drug targeting, in vivo diagnostic applications and, eventually, novel avenues for the treatment of human diseases, many of which are presently refractory to the small-molecule drugs currently available. This editorial discusses some of the prospects and main obstacles for nanomedicine. I propose that the new field of nanoengineering should turn to the nanoscale features of biology to learn how to design nanodevices for therapeutic uses. In my view, the best reason for optimism is that life has already solved the obstacles we are grappling with and produced ‘nanodevices’ with capabilities that far exceed the ones nanomedicine is hoping for. Biology could well be defined as the science of nanostructures made of predominantly carbon, oxygen, nitrogen and hydrogen. Indeed, it is their subnanometer structural features that give biomolecules their properties and allow them to self-assemble into functional units with the autonomous and self-replicating properties we call ‘life’. In the last century we have come a long way in our quest to understand how biomolecules work and how their smallest errors can be associated with severe human disease. By contrast, we have not yet advanced far at all on the road to learning how to repair cellular machinery. Currently, the best avenue for therapy of human disease consists of small-molecule enzyme inhibitors – drugs, many of which are nonselective and have

Patented inhibitors (2002 – 2005) of the transcription factor NF-κB

Nuclear factor-κB (NF-κB) is a central transcriptional factor and a pleiotropic regulator of many genes involved in immunological responses. Dysregulation of NF-κB can lead to a wide variety of diseases, including those related to inflammation and cancer. Therefore, NF-κB and the signalling pathways that regulate its activity have become a focal point for intense drug discovery and development by a number of drug companies and academic groups. Small-molecule inhibitors of regulatory enzymes or of NF-κB itself, gene therapy, decoy oligonucleotides or various biologicals have been reported and patented as potential candidates for drug development. This article reviews patents claiming NF-κB inhibitors during the period 2002 – 2005. Some of these inhibitors may be developed to therapeutics and used for the treatment of inflammatory diseases and tumour chemoresistance.

Peptide inhibitor of pancreatic lipase selected by phage display using different elution strategies

Interference with fat hydrolysis results in the reduced use of ingested lipids. Inhibition of pancreatic lipase reduces the efficiency of fat absorption in the small intestine and thereby initiates modest long-term reduction in body weight. In an attempt to select peptides with affinity for the surface of pancreatic lipase and potential inhibitory activity, a random, cyclic heptapeptide phage-displayed library was used. Five independent selections, differing in elution step, were performed. In three selection protocols, a sequential elution strategy was applied in anticipation of improving the selection of high-affinity clones. Four heptapeptides with the highest affinity, seemingly for pancreatic lipase, were selected, synthesized, and characterized for their capacity to inhibit enzyme function. Although no clear consensus among the sequenced peptides was found, one of the selected peptides inhibited pancreatic lipase with an apparent inhibition constant of 16 muM.

New Strategies for the Solid‐Phase Synthesis of Highly Functionalized, Small Molecules: Sequential Nucleophilic Substitutions on Polymer‐Bound Polyelectrophiles

AbstractFor Abstract see ChemInform Abstract in Full Text.

Identification of Small Molecule Synthetic Inhibitors of DNA Polymerase β by NMR Chemical Shift Mapping

DNA polymerase beta (beta-pol) plays a central role in repair of damaged DNA bases by base excision repair (BER) pathways. A predominant phenotype of beta-pol null mouse fibroblasts is hypersensitivity to the DNA-methylating agent methyl methanesulfonate. Residues in the 8-kDa domain of beta-pol that seem to interact with a known natural product beta-pol inhibitor, koetjapic acid, were identified by NMR chemical shift mapping. The data implicate the binding pocket as the hydrophobic cleft between helix-2 and helix-4, which provides the DNA binding and deoxyribose phosphate lyase activities of the enzyme. Nine structurally related synthetic compounds, containing aromatic or other hydrophobic groups in combination with two carboxylate groups, were then tested. They were found to bind to the same or a very similar region on the surface of the enzyme. The ability of these compounds to potentiate methyl methanesulfonate cytotoxicity, an indicator of cellular BER capacity, in wild-type and beta-pol null mouse fibroblasts, was next ascertained. The most active and beta-pol-specific of these agents, pamoic acid, was further characterized and found to be an inhibitor of the deoxyribose phosphate lyase and DNA polymerase activities of purified beta-pol on a BER substrate. Our results illustrate that NMR-based mapping techniques can be used in the design of small molecule enzyme inhibitors including those with potential use in a clinical setting.

Therapeutic potential of antisense nucleic acid molecules.

Elucidation of many disease-related signal transduction and gene expression pathways has provided unparalleled opportunities for the development of targeted therapeutics. The types of molecules in development are increasingly varied and include small-molecule enzyme inhibitors, humanized antibodies to cell surface receptors, and antisense nucleic acids for silencing the expression of specific genes. This Perspective reviews the basis for various antisense strategies for modulating gene expression, including RNA interference, and discusses the prospects for their clinical use.

New Strategies for the Solid-Phase Synthesis of Highly Functionalized, Small Molecules: Sequential Nucleophilic Substitutions on Polymer-Bound Polyelectrophiles

Publisher Summary The screening of large and diverse arrays of compounds prepared on insoluble supports is one of the most efficient approaches for fast identification of lead compounds for novel, small molecule enzyme inhibitors, or other ligands for proteins. Parallel solid-phase synthesis is particularly well suited for the preparation of such arrays of diverse compounds, since multistep synthetic sequences on insoluble supports can be conducted on fully automated synthesizers. The advantages of using nucleophilic reagents for library production can be exploited by performing sequential nucleophilic substitutions on a polymer-bound polyelectrophile. A suitable polyelectrophile would be a polymer-bound compound with two or more leaving groups that could be orthogonally displaced by different nucleophiles. The sequential nucleophilic substitutions on support-bound polyelectrophiles gives quick access to highly functionalized, pharmacophore-rich small molecules. Because no protective groups are required, the scope of these syntheses is broad and a multitude of different pharmacophoric patterns can be generated by using exclusively simple, commercially available reagents. The use of 2,3-dichloropropionic acid and 4,5-difluoro-2-nitrobenzoic acid is discussed in the chapter to illustrate the scope and limitations of this strategy for the preparation of lead-like compound arrays.

Assays for the identification and evaluation of histone acetyltransferase inhibitors

There is presently enormous interest in the function and regulatory roles of histone acetyltransferase enzymes. Along with deacetylases it is now evident that these enzymes play a key role in many cellular processes including chromatin remodeling and gene transcription. As such, effective small molecule enzyme inhibitors would be useful tools for molecular pharmacology and may also be suitable for further development into agents for the treatment of diseases such as cancer. A high-throughput assay based on the use of scintillating microplates (FlashPlates) suitable for screening libraries of compounds for inhibitors of acetylase activity is described here. Confirmation of activity of selected compounds is achieved with a conventional filter assay, the details of which are also described. In addition, an assay suitable for confirming that cellular protein acetylation has been altered by inhibition of acetylases or deacetylases is also presented. On the same plate, cells are grown, exposed to compound, fixed, and permeabilized, and protein acetylation is determined using standard ELISA methodology and a europium-labeled second antibody. This latter method provides a medium-throughput alternative to the use of immunoblotting for mechanistic studies.

Untangling the ErbB signalling network.

When epidermal growth factor and its relatives bind the ErbB family of receptors, they trigger a rich network of signalling pathways, culminating in responses ranging from cell division to death, motility to adhesion. The network is often dysregulated in cancer and lends credence to the mantra that molecular understanding yields clinical benefit: over 25,000 women with breast cancer have now been treated with trastuzumab (Herceptin), a recombinant antibody designed to block the receptor ErbB2. Likewise, small-molecule enzyme inhibitors and monoclonal antibodies to ErbB1 are in advanced phases of clinical testing. What can this pathway teach us about translating basic science into clinical use?

Anticancer drug targets: growth factors and growth factor signaling.

Signaling mechanisms that drive cell proliferation are closely associated with tumor malignancy. Components of these pathways, encoded by some of the very first oncogenes identified, include the PDGF-like ligand Sis, the tyrosine kinases Src and HER-2/c-Neu (HER-2), and the GTP-binding switch Ras. The study of communication by these oncoproteins has identified a complex array of intracellular circuits. In some cancers, mutations in key components lead to constitutive activation of these pathways; this activation is associated with the proliferative properties of the tumor cells. In this Perspective, I provide a broad overview of a growth factor signal transduction system, with a focus on those points that have been translated to drugs or clinical candidates. Due to editorial restrictions limiting the number of reference citations, much of the clinical data gleaned from abstracts is not listed in the references. Instead, the reader is directed to the 1999 Proceedings of the American Society of Clinical Oncology and the 1999 Proceedings of the AACR-NCI-EORTC International Conference. Signaling pathways are initiated with the binding of a ligand, such as PDGF, EGF, EGF-like ligands (e.g., TGF-α and amphiregulin), or IGF, to its cognate transmembrane receptor (1). Ligand binding induces the dimerization of receptor subunits, promoting autophosphorylation of the receptor and recruiting a variety of intracellular docking proteins (such as Grb2, Shc, and Nck) to the plasma membrane. These docking proteins create a molecular scaffold from which subsequent signals emanate. For example, the guanine nucleotide exchange factor Sos binds to Grb2, which in turn interacts with the Ras protein. Ras serves as a molecular switch in the plasma membrane that alternates between an inactive GDP-bound state and an active GTP-bound state. Normally, Ras is bound to GDP because of the abundance of GTPase-activating protein and neurofibromin, which both suppress Ras function. However, upon recruitment of Sos to the membrane, Sos binds Ras-GDP and facilitates release of GDP. In cells, the nucleotide GTP is about 10-fold more abundant than GDP; GTP binds to Ras by mass action. Ras-GTP adopts a conformation that permits interaction with downstream targets called effector molecules. These effectors include the protein kinase Raf, which activates the MAP kinase cascade; GTPase-activating protein, which links Ras to the Rho/Rac pathway; and phosphoinositide (PI) 3′-kinase and Ral–guanine nucleotide dissociation stimulator (Ral-GDS), which activate lipid pathways (2). The dysregulation of these signals in tumor cells leads to multiple cellular changes, including alterations in DNA synthesis, lipid metabolism, cellular morphology, cell adhesion properties, and gene expression. In the broadest sense, the study of signaling mechanisms has already yielded therapeutic agents in the treatment of cancer, as evidenced by antiestrogens, antiandrogens, agonists of gonadotropin-releasing hormone, and stem cell growth factors, for example. However, research into oncoproteins that function within the signal transduction system is only beginning to be applied in the clinic. Therapeutic approaches of interest include tools such as mAbs against the extracellular domain of receptors, oligonucleotides that are antisense to key target proteins, and small molecule inhibitors of enzymes (Table ​(Table11). Table 1 Examples of inhibitors of growth factor signaling for cancer treatment

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries

Background: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. Results: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. Conclusions: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.