Addressing phenoconversion: the Achilles' heel of personalized medicine

Phenoconversion is a phenomenon that converts genotypic extensive metabolizers (EMs) into phenotypic poor metabolizers (PMs) of drugs, thereby modifying their clinical response to that of genotypic PMs. Phenoconversion, usually resulting from nongenetic extrinsic factors, has a significant impact on the analysis and interpretation of genotype-focused clinical outcome association studies and personalizing therapy in routine clinical practice. The high phenotypic variability or genotype–phenotype mismatch, frequently observed due to phenoconversion within the genotypic EM population, means that the real number of phenotypic PM subjects may be greater than predicted from their genotype alone, because many genotypic EMs would be phenotypically PMs. If the phenoconverted population with genotype–phenotype mismatch, most extensively studied for CYP2D6, is as large as the evidence suggests, there is a real risk that genotype-focused association studies, typically correlating only the genotype with clinical outcomes, may miss clinically strong pharmacogenetic associations, thus compromising any potential for advancing the prospects of personalized medicine. This review focuses primarily on co-medication-induced phenoconversion and discusses potential approaches to rectify some of the current shortcomings. It advocates routine phenotyping of subjects in genotype-focused association studies and proposes a new nomenclature to categorize study populations. Even with strong and reliable data associating patients'' genotypes with clinical outcome(s), there are problems clinically in applying this knowledge into routine pharmacotherapy because of potential genotype–phenotype mismatch. Drug-induced phenoconversion during routine clinical practice remains a major public health issue. Therefore, the principal challenges facing personalized medicine, which need to be addressed, include identification of the following factors: (i) drugs that are susceptible to phenoconversion; (ii) co-medications that can cause phenoconversion; and (iii) dosage amendments that need to be applied during and following phenoconversion.     

Molecular mechanisms of platinum resistance: still searching for the Achilles' heel.

The platinum compounds cisplatin and carboplatin are commonly used in cancer chemotherapy. However, tumors frequently develop resistance to these compounds, significantly decreasing their usefulness in the clinic. In the past few years, basic research has unraveled novel and unexpected mechanisms for the development of platinum resistance. For example, it has been reported that MUC1 expression and particularly the localization of its C-terminal subunit to the mitochondria may affect cisplatin resistance. Another recent finding suggests that cisplatin damage may activate DNA-dependent protein kinase (DNA-PK) to initiate a death signal that can be transmitted to neighboring cells through gap junctions, adding to a growing belief that the interactions of cancer cells with their surroundings may be important to the outcome of chemotherapy. While most clinical efforts have focused on identifying alternative regimens for drug-resistant cancer, it might be possible to exploit our knowledge of the mechanism of platinum resistance to specifically reverse resistance and increase platinum efficacy. The strategy of drug resistance reversal therapy (DRRT) may have significant impact on our approaches to the treatment and management of drug-resistant tumors.     

Experimental therapeutics: targeting the redox Achilles heel of cancer

Reactive oxygen species (ROS) have recently emerged as promising targets for anticancer drug discovery. Constitutively elevated levels of cellular oxidative stress and dependence on mitogenic and anti-apoptotic ROS signaling represent a specific vulnerability of malignant cells that can be selectively targeted by novel pro- and antioxidant redox chemotherapeutics. This review discusses small-molecule anticancer redox drugs currently in various phases of preclinical and clinical development that are characterized by their unique mechanism of action, including small-molecule superoxide dismutase and catalase mimetics, bioreductively activated pro-oxidant redox catalysts, metal-based pro-oxidants, hypoxia-selective free radical precursors, and specific antagonists of the cancer cell antioxidant glutathione or thioredoxin redox systems. Based on ongoing redox biomarker discovery and validation, future redox phenotyping and genotyping may guide the selection of novel redox chemotherapeutics that efficiently target the redox Achilles heel of the individual tumor.     

Restenosis,the Achilles' Heel of Coronary Angioplasty

Coronary angioplasty is widely performed for the management of symptomatic coronary artery disease. With improvements in technique, operator experience, and tools, more complex lesions are being treated. Unfortunately, luminal renarrowing continues to limit the long-term success of the procedure, resulting in the need for repeat revascularization in approximately 30% of patients within 6 months. As the pathophysiologic process of restenosis is better defined, various pharmacologic and mechanical interventions have been tried to attenuate the process. Some agents are antithrombotics, antiplatelets, angiotensin-converting enzyme inhibitors, lipid-lowering drugs, and calcium channel blockers. Improvement has been noted with the newer glycoprotein IIb- and IIIa-blocking agents, mechanical stents, and radioactive materials. Whether these new compounds will withstand the test of time is unknown.     

Characterization and inhibition of SARS-coronavirus main protease

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus (CoV). During the 2003 epidemic, the disease rapidly spread from its origin in southern China to other countries and affected almost 8000 patients, which resulted in about 800 fatalities. A chymotrypsin-like cysteine protease named 3C-like protease (3CLpro) is essential for the life cycle of the SARS-CoV. This main protease is responsible for maturation of functional proteins and represents a key anti-viral target. HPLC and fluorescence-based assays have been used to characterize the protease and to determine the potency of the inhibitors. The fluorogenic method monitoring the increase of fluorescence from the cleavage of a peptide substrate containing an Edans-Dabcyl fluorescence quenching pair at two ends has enabled the use of high throughput screening to speed up the drug discovery process. Several groups of inhibitors have been identified through high throughput screening and rational drug design approaches. Thus, alpha,beta-unsaturated peptidomimetics, anilides, metal-conjugated compounds, boronic acids, quinolinecarboxylate derivatives, thiophenecarboxylates, phthalhydrazide-substituted ketoglutamine analogues, isatin and natural products have been identified as potent inhibitors of the SARS-CoV main protease. The different classes of inhibitors reported in these studies are summarized in this review. Some of these inhibitors could be developed into potential drug candidates, which may provide a solution to combat possible reoccurrence of the SARS and other life-threatening viruses with 3CL proteases.     

Drug resistance in Mycobacterium tuberculosis and targeting the l,d-transpeptidase enzyme

Tuberculosis (TB) is a disease that has afflicted mankind for thousands of years, but in the last seven decades, much progress has been made in anti-TB therapy. Early drugs, such as para-aminosalicylic acid, streptomycin, isoniazid, and rifamycins were very effective in combatting the disease, giving rise to the hope that TB would be eradicated from the face of the earth by 2010. Despite that optimism, TB continues to kill more than a million people annually worldwide. A major reason for our inability to contain TB is the emergence drug resistance in Mycobacterium tuberculosis. This commentary is based on our recent publication on the structure of l ,d -transpeptidase enzyme, relevant to drug resistance. As a background, we briefly outline the history and development of anti-TB therapy. Based on the crystal structure, we suggest a potential direction for designing more potent drugs against TB.     

Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease

SARS coronavirus main protease (SARS-CoV M^pro) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV M^pro. Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-CoV M^pro requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We determined four crystal structures of SARS-CoV M^pro in complex with pentapeptide aldehydes (Ac-ESTLQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhibit inhibitory activity towards SARS-CoV M^pro, with K_i values in the μM range. Surprisingly, the X-ray structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2 position was 160-times lower than that for the original substrate (P2 = Leu); furthermore, the substrate with aspartic acid in the P2 position was not cleaved at all. We also determined a crystal structure of SARS-CoV M^pro in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against SARS-CoV M^pro, with K_i = 2.24 ± 0.58 μM. These results show that the stringent substrate specificity of the SARS-CoV M^pro with respect to the P1 and P2 positions can be overruled by the highly electrophilic character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhibitors need to correspond to the observed cleavage specificity of the target protease.     

Structure-based drug design and structural biology study of novel nonpeptide inhibitors of severe acute respiratory syndrome coronavirus main protease

Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)), a protein required for the maturation of SARS-CoV, is vital for its life cycle, making it an attractive target for structure-based drug design of anti-SARS drugs. The structure-based virtual screening of a chemical database containing 58,855 compounds followed by the testing of potential compounds for SARS-CoV M(pro) inhibition leads to two hit compounds. The core structures of these two hits, defined by the docking study, are used for further analogue search. Twenty-one analogues derived from these two hits exhibited IC50 values below 50 microM, with the most potent one showing 0.3 microM. Furthermore, the complex structures of two potent inhibitors with SARS-CoV M(pro) were solved by X-ray crystallography. They bind to the protein in a distinct manner compared to all published SARS-CoV M(pro) complex structures. They inhibit SARS-CoV M(pro) activity via intensive H-bond network and hydrophobic interactions, without the formation of a covalent bond. Interestingly, the most potent inhibitor induces protein conformational changes, and the inhibition mechanisms, particularly the disruption of catalytic dyad (His41 and Cys145), are elaborated.     

Drug targeting to the colon with lectins and neoglycoconjugates

Targeting of drugs to specific sites of action provides several advantages over non-targeted drugs. These include the prevention of side effects of drugs on healthy tissues and enhancement of drug uptake by targeted cells. This review will cover traditional approaches of colon drug targeting as well as the use of lectins and neoglycoconjugates for the targeted delivery. Direct and reverse targeting strategies, potential molecular targets and targeting moieties for colon drug delivery, targeted drug delivery systems (DDS) for colon delivery, anticancer DDS targeted to colon cancer are examined. Directions of future development are discussed.     

Drug targeting to the kidney: Advances in the active targeting of therapeutics to proximal tubular cells

Activated signaling cascades in the proximal tubular cells of the kidneys play a crucial role in the development of tubulointerstitial fibrosis. Inhibition of these signaling cascades with locally delivered therapeutics is an attractive approach to minimize the risk of unwanted side effects and to enhance their efficacy within the renal tissue.This review describes the potential avenues to actively target drugs to proximal tubular cells by recognition of internalizing receptors and how drug carriers can reach this cell type from either the apical or basolateral side. Important characteristics of drug carrier systems such as size and charge are discussed, as well as linking technologies that have been used for the coupling of drugs to the presented carrier systems. Lastly, we discuss the cellular handling of drugs by proximal tubular cells after their delivery to the kidneys.     

Drug targeting to lungs by way of microspheres

In many conventional drug delivery systems in vogue, failure to deliver efficient drug delivery at the target site/organs; is evident as a result, less efficacious pharmacological response is elicited. Microspheres can be derived a remedial measure which can improve site-specific drug delivery to a considerable extent. As an application, Lung-targeting Ofloxacin-loaded gelatin microspheres (GLOME) were prepared by water in oil emulsion method. The Central Composite Design (CCD) was used to optimize the process of preparation, the appearance and size distribution were examined by scanning electron microscopy, the aspects such as in vitro release characteristics, stability, drug loading, loading efficiency, pharmacokinetics and tissue distribution in albino mice were studied. The experimental results showed that the microspheres in the range of 0.32-22 microm. The drug loading and loading efficiency were 61.05 and 91.55% respectively. The in vitro release profile of the microspheres matched the korsmeyer's peppas release pattern, and release at 1h was 42%, while for the original drug, ofloxacin under the same conditions 90.02% released in the first half an hour. After i.v. administration (15 min), the drug concentration of microspheres group in lung in albino mice was 1048 microg/g, while that of controlled group was 6.77 microg/g. GLOME found to release the drug to a maximum extent in the target tissue, lungs.     

Drug discovery by targeting the protein-protein interactions involved in autophagy

Autophagy is a cellular process in which proteins and organelles are engulfed in autophagosomal vesicles and transported to the lysosome/vacuole for degradation. Protein-protein interactions(PPIs) play a crucial role at many stages of autophagy, which present formidable but attainable targets for autophagy regulation. Moreover, selective regulation of PPIs tends to have a lower risk in causing undesired off-target effects in the context of a complicated biological network. Thus, small-molecule r...     

Structure-based design of non-peptide HIV protease inhibitors

A number of structurally novel P2-ligands have been designed and synthesized. Incorporation of these ligands in the (R)-(hydroxyethyl)sulfonamide isostere provided a series of potent non-peptidyl HIV protease inhibitors.