The unique properties of dipeptidyl-peptidase IV (DPP IV / CD26) and the therapeutic potential of DPP IV inhibitors

This review deals with the properties and functions of dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5). This membrane anchored ecto-protease has been identified as the leukocyte antigen CD26. The following aspects of DPP IV/CD26 will be discussed : the structure of DPP IV and the new family of serine proteases to which it belongs, the substrate specificity, the distribution in the human body, specific DPP IV inhibitors and the role of CD26 in the intestinal and renal handling of proline containing peptides, in cell adhesion, in peptide metabolism, in the immune system and in HIV infection. Especially the latest developments in the search for new inhibitors will be reported as well as the discovery of new natural substrates for DPP IV such as the glucagon-like peptides and the chemokines. Finally the therapeutical perspectives for DPP IV inhibitors will be discussed.     

Efficient conversion of tetrapeptide-based TSAO prodrugs to the parent drug by dipeptidyl-peptidase IV (DPPIV/CD26)

A novel prodrug approach has been evaluated using the anti-HIV-active TSAO molecule as the prototype drug to prove the kinetics with purified enzyme and the principles of conversion to the parent compound in sera and cell culture. When a variety of tetrapeptidyl amide prodrugs of NAP-TSAO were synthesized and exposed to purified dipeptidyl-peptidase IV (DPPIV/CD26) as well as human and bovine sera, they are converted to the parent NAP-TSAO drug in two successive steps by both purified CD26 and human and bovine serum. The efficiency of conversion strongly depends on the nature of the amino acid that has to be cleaved-off from the prodrug molecule. The tetrapeptidyl prodrug 20 showed a more than 10-fold improved water-solubility in comparison to that of the parent compound NAP-TSAO. The antiviral activity of the prototype NAP-TSAO could also be modulated by introducing different tetrapeptide moieties on the molecule resulting, in some cases, in a superior antiviral potential in cell culture than the parent drug.     

CD26/dipeptidyl peptidase IV as a novel therapeutic target for cancer and immune disorders

CD26 is a 110 kDa surface glycoprotein with intrinsic dipeptidyl peptidase IV (DPPIV) activity that is expressed on numerous cell types and has a multitude of biological functions. An important aspect of CD26 biology is its peptidase activity and its functional and physical association with molecules with key roles in various cellular pathways and biological programs. CD26 role in immune regulation has been extensively characterized, with recent findings elucidating its linkage with signaling pathways and structures involved in T-lymphocyte activation as well as antigen presenting cell-T-cell interaction. Recent work also suggests that CD26 has a significant role in tumor biology, being both a marker of disease behavior clinically as well as playing an important role in tumor pathogenesis and development. In this paper, we will review emerging data that suggest CD26 may be an appropriate therapeutic target for the treatment of selected neoplasms and immune disorders. Through the use of various experimental approaches and agents to influence CD26/DPPIV expression and activity, such as anti-CD26 antibodies, CD26/DPPIV chemical inhibitors, siRNAs to inhibit CD26 expression, overexpressing CD26 transfectants and soluble CD26 molecules, our group has shown that CD26 interacts with structures with essential cellular functions. Its association with such key molecules as topoisomerase IIalpha, p38 MAPK, and integrin beta1, has important clinical implications, including its potential ability to regulate tumor sensitivity to selected chemotherapies and to influence tumor migration/metastases and tumorigenesis. Importantly, our recent in vitro and in vivo data support the hypothesis that CD26 may indeed be an appropriate target for therapy for selected cancers and immune disorders.     

Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Aminopeptidase N (APN/ CD13)--a novel approach for the treatment of inflammatory bowel disease

The ectopeptidases Dipeptidylpeptidase IV and Alanyl-Aminopeptidase N, strongly expressed by both, activated and regulatory T cells were shown to co-operate in T cell regulation. Based on the findings that DPIV and APN inhibitors induce the TGF-beta1 and IL-10 production and a suppression of T helper cell proliferation in parallel, and that particularly APN inhibitors amplify the suppressing activity of regulatory T cells, both peptidases represent a promising target complex for treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD). The aim of the present study was to analyze the therapeutic potential of DPIV and APN inhibitors in vivo in a mouse model of colitis. Balb/c mice received 3% (w/v) dextran sulphate sodium with the drinking water for 7 days. After onset of colitis symptoms, inhibitor treatment started at day 3. Disease activity index (DAI) was assessed daily, supplemented by histological and immunological analysis. While the DPIV inhibitor Lys-[Z(NO])(2)]-pyrrolidide or the APN-inhibitor Actinonin alone had marked but no significant therapeutic effects, the simultaneous administration of both inhibitors reduced colitis activity in comparison to placebo treated mice, significantly (DAI 4.8 vs. 7.7, p<0.005). A newly developed compound IP12.C6 with inhibitory capacity toward both enzymes significantly attenuated the clinical manifestation of colitis (DAI 3.2 vs. 7.6, p<0.0001). TGF-beta mRNA was found to be up-regulated in colon tissue of inhibitor-treated animals. In summary our results strongly suggest that combined DPIV and APN inhibition by synthetic inhibitors represents a novel and efficient approach for the pharmacological therapy of IBD by triggering endogenous immunosuppressive mechanisms.     

Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Aminopeptidase N (APN/ CD13) — A novel approach for the treatment of inflammatory bowel disease

The ectopeptidases Dipeptidylpeptidase IV and Alanyl-Aminopeptidase N, strongly expressed by both, activated and regulatory T cells were shown to co-operate in T cell regulation. Based on the findings that DPIV and APN inhibitors induce the TGF-β1 and IL-10 production and a suppression of T helper cell proliferation in parallel, and that particularly APN inhibitors amplify the suppressing activity of regulatory T cells, both peptidases represent a promising target complex for treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD).The aim of the present study was to analyze the therapeutic potential of DPIV and APN inhibitors in vivo in a mouse model of colitis. Balb/c mice received 3% (w/v) dextran sulphate sodium with the drinking water for 7 days. After onset of colitis symptoms, inhibitor treatment started at day 3. Disease activity index (DAI) was assessed daily, supplemented by histological and immunological analysis. While the DPIV inhibitor Lys-[Z(NO])₂]-pyrrolidide or the APN-inhibitor Actinonin alone had marked but no significant therapeutic effects, the simultaneous administration of both inhibitors reduced colitis activity in comparison to placebo treated mice, significantly (DAI 4.8 vs. 7.7, p < 0.005). A newly developed compound IP12.C6 with inhibitory capacity toward both enzymes significantly attenuated the clinical manifestation of colitis (DAI 3.2 vs. 7.6, p < 0.0001). TGF-β mRNA was found to be up-regulated in colon tissue of inhibitor-treated animals.In summary our results strongly suggest that combined DPIV and APN inhibition by synthetic inhibitors represents a novel and efficient approach for the pharmacological therapy of IBD by triggering endogenous immunosuppressive mechanisms.     

Dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) as regulators of T cell function and targets of immunotherapy in CNS inflammation

The ectoenzymes dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) have been implicated in the regulation of T cell activation and function. Both DP IV and APN serve as targets of efficient enzymatic inhibitors which induce autocrine production of TGF-beta1 and subsequent suppression of T cell proliferation and cytokine release. Here, we tested the hypothesis that the simultaneous inhibition of DP IV and APN enzymatic activity on leukocytes potentiates the anti-inflammatory effect of single DP IV or APN inhibitors. Our data show that the combined application of DP IV and APN inhibitors increased suppression of DNA synthesis in human peripheral blood mononuclear cells and isolated T cells in vitro when compared to the use of a single ectopeptidase inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF-beta1 production associated with the observed immunosuppressive effects. In vivo, targeting DP IV and APN provided a potent therapeutic approach for the treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Taken together, our study suggests that combined DP IV and APN inhibition on pathogenic T cells represents a novel and efficient therapy for autoimmune disease of the central nervous system by a mechanism that involves an active TGF-beta1-mediated anti-inflammatory effect at the site of pathology.     

Molecular topology as a novel approach for drug discovery

INTRODUCTION: Molecular topology (MT) has emerged in recent years as a powerful approach for the in silico generation of new drugs. One key part of MT is that, in the process of drug design/discovery, there is no need for an explicit knowledge of a drug's mechanism of action unlike other drug discovery methods. AREAS COVERED: In this review, the authors introduce the topic by explaining briefly the most common methodology used today in drug design/discovery and address the most important concepts of MT and the methodology followed (QSAR equations, LDA, etc.). Furthermore, the significant results achieved, from this approach, are outlined and discussed. EXPERT OPINION: The results outlined herein can be explained by considering that MT represents a new paradigm in the field of drug design. This means that it is not only an alternative method to the conventional methods, but it is also independent, that is, it represents a pathway to connect directly molecular structure with the experimental properties of the compounds (particularly drugs). Moreover, the process can be realized also in the reverse pathway, that is, designing new molecules from their topological pattern, what opens almost limitless expectations in new drugs development, given that the virtual universe of molecules is much greater than that of the existing ones.     

Molecular topology as a novel approach for drug discovery

Introduction: Molecular topology (MT) has emerged in recent years as a powerful approach for the in silico generation of new drugs. One key part of MT is that, in the process of drug design/discovery, there is no need for an explicit knowledge of a drug's mechanism of action unlike other drug discovery methods.

Areas covered: In this review, the authors introduce the topic by explaining briefly the most common methodology used today in drug design/discovery and address the most important concepts of MT and the methodology followed (QSAR equations, LDA, etc.). Furthermore, the significant results achieved, from this approach, are outlined and discussed.

Expert opinion: The results outlined herein can be explained by considering that MT represents a new paradigm in the field of drug design. This means that it is not only an alternative method to the conventional methods, but it is also independent, that is, it represents a pathway to connect directly molecular structure with the experimental properties of the compounds (particularly drugs). Moreover, the process can be realized also in the reverse pathway, that is, designing new molecules from their topological pattern, what opens almost limitless expectations in new drugs development, given that the virtual universe of molecules is much greater than that of the existing ones.     

Virogenomics: a novel approach to antiviral drug discovery

Target discovery in virology has been limited to the few open-reading frames encoded by viral genomes. However, several recent examples show that inhibiting host-cell proteins can prevent viral infection. The human genome sequence should, therefore, contain many more genes that are essential for viral propagation than viral genomes. A systematic approach to find these potential cellular antiviral targets is global host gene expression analysis using DNA microarrays. Several recent studies reveal both unique and common strategies by which viruses change the gene expression profile of the host cell. Moreover, work in progress shows that some of the host pathways discovered by expression profiling are important for viral replication. Thus, human genomics tools have the potential to deliver novel antiviral drugs.     

Design, synthesis, and discovery of a novel CCR1 antagonist

The CC chemokines may play an important role in the pathogenesis of chronic inflammatory diseases including rheumatoid arthritis, and their effects are thought to be mediated through CCR1 receptors. Several nonpeptide CCR1 receptor antagonists that showed high affinity for human CCR1 receptors have been identified; however, their effectiveness in animal models of inflammatory diseases has been scarcely demonstrated, probably due to species selectivity of the antagonists. To elucidate the pathophysiological role of CCR1 receptors in murine models of disease, we looked for a potent antagonist for both murine and human CCR1 receptors. Screening of our chemical collection for inhibition of (125)I-MIP-1alpha binding to human CCR1 receptors transfected in CHO cells led to the identification of xanthene-9-carboxamide 1a as the lead compound. Derivatization of 1a by quaternarizing the piperidine nitrogen with various alkyl groups and by installing substituents into the xanthene moiety dramatically improved the inhibitory activity against both human and murine CCR1 receptors. As a result, 2q-1 showing IC(50) values of 0.9 and 5.8 nM for human and murine CCR1 receptors, respectively, was discovered. This compound is the first murine CCR1 receptor antagonist and may be a useful tool for clarifying the role of CCR1 receptors in murine models of disease.     

L-Dihydroxyphenylserine (L-DOPS): a norepinephrine prodrug

L-threo-3,4-dihydroxyphenylserine (L-DOPS, droxydopa) is a synthetic catecholamino acid. When taken orally, L-DOPS is converted to the sympathetic neurotransmitter, norepinephrine (NE), via decarboxylation catalyzed by L-aromatic-amino-acid decarboxylase (LAAAD). Plasma L-DOPS levels peak at about 3 h, followed by a monoexponential decline with a half-time of 2 to 3 h. Plasma levels of NE and of its main neuronal metabolite, dihydroxyphenylglycol (DHPG) peak approximately concurrently but at much lower concentrations. The relatively long half-time for disappearance of L-DOPS from plasma, compared to that of NE, explains their very different attained plasma concentrations. In patients with neurogenic orthostatic hypotension, L-DOPS increases blood pressure and ameliorates orthostatic intolerance. Inhibition of LAAAD, such as by treatment with carbidopa, which does not penetrate the blood-brain barrier, prevents the blood pressure effects of the drug, indicating that L-DOPS increases blood pressure by augmenting NE production outside the brain. Patients with pure autonomic failure (which usually entails loss of sympathetic noradrenergic nerves), and patients with multiple system atrophy (in which noradrenergic innervation remains intact) have similar plasma NE responses to L-DOPS. This suggests mainly non-neuronal production of NE from L-DOPS. L-DOPS is very effective in treatment of deficiency of dopamine-beta-hydroxylase (DBH), the enzyme required for conversion of dopamine to NE in sympathetic nerves. L-DOPS holds promise for treating other much more common conditions involving decreased DBH activity or NE deficiency, such as a variety of syndromes associated with neurogenic orthostatic hypotension.     

Radiolytic and cellular reduction of a novel hypoxia-activated cobalt(III) prodrug of a chloromethylbenzindoline DNA minor groove alkylator

Metabolic reduction can be used to activate prodrugs in hypoxic regions of tumours, but reduction by ionising radiation is also theoretically attractive. Previously, we showed that a cobalt(III) complex containing 8-hydroxyquinoline (8-HQ) and cyclen ligands releases 8-HQ efficiently on irradiation in hypoxic solutions [Ahn G-O, Ware DC, Denny WA, Wilson WR. Optimization of the auxiliary ligand shell of cobalt(III)(8-hydroxyquinoline) complexes as model hypoxia-selective radiation-activated prodrugs. Radiat Res 2004;162:315-25]. Here we investigate an analogous Co(III) complex containing the potent DNA minor groove alkylator azachloromethylbenzindoline (azaCBI, 1) to determine whether it releases 1 on radiolytic and/or enzymatic reduction under hypoxia. Monitoring by HPLC, the azaCBI ligand in the Co(III)(cyclen)(azaCBI) complex (2) slowly hydrolysed in aqueous solution, in contrast to the free ligand 1 which readily converted to its reactive cyclopropyl form. Irradiation of 2 (30-50 microM) in hypoxic solutions released 1 with yields of 0.57 micromol/J in formate buffer and 0.13 micromol/J in human plasma. Using bioassay methods, cytotoxic activation by irradiation of 2 at 1 microM in hypoxic plasma was readily detectable at clinically relevant doses (> or = 1 Gy), with a estimated yield of 1 of 0.075 micromol/J. Release of 1 from 2 was also observed in hypoxic HT29 cultures without radiation, with subsequent conversion of 1 to its O-glucuronide. Surprisingly, overexpression of human cytochrome P450 reductase in A549 cells did not increase the rate of metabolic reduction of 2, suggesting that other reductases and/or non-enzymatic reductants are responsible. Thus the cobalt(III) complex 2 is a promising prodrug capable of being activated to release a very potent cytotoxin when reduced by either ionising radiation or cells under hypoxic conditions.     

The physicochemical approach to drug design and discovery (QSAR)

The study of physicochemical parameters to correlate mathematically chemical structure with biological activity induced by sets of congeneric drugs is now generally referred to as QSAR (quantitative structure-activity relationships). The ways in which the QSAR paradigm are developing are becoming more varied and complex. Many kinds of parameters are under study in many different groups; various types of mathematical models have been proposed and are being evaluated. Drug researchers are turning more to enzyme modulation to control various biological processes. It is the study of enzyme-ligand reactions of enzymes whose x-ray crystallographic structure is known that affords us the means for developing a deeper understanding of QSAR and, at the same time, enhancing our ability to make drugs more selective for various forms of a given enzyme. The union of x-ray crystallography, moleculargraphics, and QSAR is one of the most exciting new areas of drug development. This report is an introduction to how QSAR is being used to gain insight into the interaction of drugs with macromolecules and macromolecular systems.     

CD26/dipeptidyl peptidase IV: a regulator of immune function and a potential molecular target for therapy

CD26 is a 110 kDa surface-bound ectopeptidase with intrinsic dipeptidyl peptidase IV (DPP IV) activity, which has multiple biological functions. In this review, we will focus specifically on work demonstrating that CD26 has a key role in immune function as a T cell activation molecule and a regulator of the functional effect of selected biological factors through its DPP IV enzyme activity. As further evidence of the important role played by this multifaceted molecule in immune regulation, we will also discuss experimental attempts from our laboratory and others to influence immune-mediated conditions through CD26 monoclonal antibodies and DPP IV activity with various agents, including anti-CD26 monoclonal antibodies and DPP IV chemical inhibitors. Of special significance from a clinical perspective is also CD26 effect on glucose metabolism through its DPP IV activity and its potential role as a therapeutic target in diabetes. In addition, we will review recent studies that describe the physical and functional interaction of CD26 with other essential cellular structures and the biological consequences of their association. In particular, we will present recent data from our laboratory that demonstrates the correlation between CD26, especially its DPP IV activity, and the key nuclear protein topoisomerase II alpha, an interaction that has important clinical implications. In summary, we will examine the biology of the multifaceted CD26/DPP IV molecule, focusing particularly on its function in immune regulation and its potential role as a molecular target for novel treatment modalities for a number of disease states, ranging from autoimmune diseases, diabetes to malignancies.     

Dose adjustment of metformin and dipeptidyl-peptidase IV inhibitors in diabetic patients with renal dysfunction

Objectives: This analysis of real-world data aimed to (a) determine the proportion of Type II diabetes (T2DM) patients treated with metformin or dipeptidyl peptidase-4 inhibitors (DPP-4i) that require dose adjustment or therapy discontinuation due to chronic kidney disease (CKD), and (b) to assess the time required to dose adjustment from the time of worsening of CKD.

Methods: In this retrospective study, two study populations were defined in a large healthcare organization. In the cross-sectional analysis, the distribution of CKD stages and the appropriate dosage of metformin and DPP-4i in 2013 was examined according to renal function among T2DM patients. In the longitudinal analysis, a cohort was defined to assess the time elapsed from first indication worsening of CKD to dose adjustment, among patients treated with those medications during years 2006–2013.

Results: Among patients treated with metformin or DPP-4i, one third of patients with CKD failed to adjust the dosage or to discontinue metformin or DPP-4i as indicated. Median time for dose adjustment or discontinuation was significantly longer for DPP-4i than for metformin (9.8 compared to 16.8 months for metformin and DPP-4i, respectively; p-value <.001).

Conclusions: This real-world data analysis showed that adjustment of dose or discontinuation of metformin or DPP-4i in patients with worsening CKD occurred less often in DPP-4i users than metformin users and took a longer time.     

新型CDK9小分子抑制剂的设计和发现

目的通过同源模建、计算机虚拟筛选和生物活性筛选,寻找新型的CDK9小分子抑制剂。方法采用同源模建方法得到CDK9的三维晶体构象,用DOCK(分子对接)对小分子三维数据库进行虚拟筛选。采用MTT法对挑选出的化合物进行生物活性测定,然后挑选高活性的化合物进行CDK9与小分子之间相互作用的研究,验证化合物对CDK9激酶活性的抑制作用。结果用DOCK程序对三维化合物库虚拟筛选后挑选出得分高的前1000个化合物,按结构差异化分类,最终选择并购买了27个代表性化合物进行进一步生物学活性测定。27个化合物中12个化合物明显抑制肿瘤细胞的增殖,其中1个化合物C-21的半数抑制浓度(IC50)在20μmol.L-1以下。选择C-21类化合物进一步进行研究,结果显示此化合物能够有效抑制各类肿瘤细胞系的增殖。酶学水平研究表明此类化合物能够有效抑制CDK9激酶活性,并在较低浓度范围内呈明显的剂量依赖关系。结论通过同源模建、计算机虚拟筛选、生物活性实验和分子水平研究,得到了一类靶向CDK9的全新的先导化合物。     

Targeting information-processing deficit in schizophrenia: a novel approach to psychotherapeutic drug discovery

Current observations indicate that dysfunction of neuronal circuitry dynamics contributes to the abnormal information processing in the brain in schizophrenia. It is presumed that disrupted auditory gating, abnormal P300-evoked potentials and deficits in mismatch negativity in schizophrenic patients indicate impaired processing of information. Recently, abnormalities in neuronal synchrony and oscillatory activity have been postulated as the mechanisms that underlie the distorted perception and cognitive dysfunction associated with schizophrenia. These novel observations might reveal the pathophysiology of the disorder, and indicate potential targets for antipsychotic drug therapy. Neuronal circuitry dynamics, such as network oscillations and sensory-gating processes, are conserved phylogenetically, which provides excellent opportunities for designing translational biomarkers. Whether preclinical, experimental compounds that impact on network oscillations and sensory processing (such as agonists and modulators of alpha7 nicotinic acetylcholine receptors) elicit the same neurophysiological events in schizophrenic patients and, subsequently, improve perception and cognitive functions will be determined when these drug candidates are available clinically.     

Constitutively activated G protein-coupled receptors: a novel approach to CNS drug discovery

G protein-coupled receptors (GPCRs) represent a major class of signal transduction proteins that modulate various biological functions. GPCRs are one of the most common targets for drug development-currently, 39 of the top 100 marketed drugs in use act directly or indirectly through activation or blockade of GPCR-mediated receptors. Nearly 160 GPCRs have been identified based on their gene sequence and their ability to interact with known endogenous ligands. However, an estimated 500-800 additional GPCRs have been classified as "orphan" receptors (oGPCRs) because their endogenous ligands have not yet been identified. Given that known GPCRs have proven to be such clinically useful drug targets, these oGPCRs represent a rich group of receptor targets for the development of novel and improved medicines. To develop ligands for these potential drug targets requires the ability to identify groups or pools of GPCRs that are likely to be involved in a specific disease process (obesity, schizophrenia, depression, etc.) and to dissect out the pharmacological and signal transduction differences between these GPCR subtypes. It also requires the development of assays to detect ligands of GPCRs even when the endogenous ligands are unidentified. This paper will review novel strategies to identify clinically interesting oGPCRs and to screen for small molecules that act as ligands without prior knowledge of endogenous ligands. This involves the use of constitutively activated GPCRs, a technology that provides a unique opportunity to identify several classes of pharmacological agents, including agonists, inverse agonists and allosteric modulators.