Current understanding of the link between angiotensin-converting enzyme and pain perception.

The renin-angiotensin system (RAS) is known to affect diverse physiological processes that affect the functioning of many key organs. Angiotensin-converting enzyme (ACE) modulates a variety of bioactive peptides associated with pain. ACE inhibitors (ACEis) have found applications in the treatment of cardiovascular, kidney, neurological and metabolic disorders. However, ACEis also tend to display undesirable effects, resulting in increased pain sensitization and mechanical allodynia. In this review, we provide comprehensive discussion of preclinical and clinical studies involving the evaluation of various clinically approved ACEis. With the emerging knowledge of additional factors involved in RAS signaling and the indistinct pharmacological role of ACE substrates in pain, extensive studies are still required to elucidate the mechanistic role of ACE in pain perception.

Modulation of endogenous opioid signaling by inhibitors of puromycin-sensitive aminopeptidase.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin-sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

Deletion of Glyoxalase 1 Exacerbates Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) overdose triggers a cascade of intracellular oxidative stress events, culminating in acute liver injury. The clinically used antidote, N-acetylcysteine (NAC), has a narrow therapeutic window, and early treatment is essential for a satisfactory therapeutic outcome. For more versatile therapies that can be effective even at late presentation, the intricacies of APAP-induced hepatotoxicity must be better understood. Accumulation of advanced glycation end products (AGEs) and the consequent activation of the receptor for AGEs (RAGE) are considered one of the key mechanistic features of APAP toxicity. Glyoxalase 1 (Glo-1) regulates AGE formation by limiting the levels of methylglyoxal (MEG). In this study, we studied the relevance of Glo-1 in the APAP-mediated activation of RAGE and downstream cell death cascades. Constitutive Glo-1-knockout mice (GKO) and a cofactor of Glo-1, ψ-GSH, were used as tools. Our findings showed elevated oxidative stress resulting from the activation of RAGE and hepatocyte necrosis through steatosis in GKO mice treated with high-dose APAP compared to wild-type controls. A unique feature of the hepatic necrosis in GKO mice was the appearance of microvesicular steatosis as a result of centrilobular necrosis, rather than the inflammation seen in the wild type. The GSH surrogate and general antioxidant ψ-GSH alleviated APAP toxicity irrespective of the Glo-1 status, suggesting that oxidative stress is the primary driver of APAP toxicity. Overall, the exacerbation of APAP hepatotoxicity in GKO mice suggests the importance of this enzyme system in antioxidant defense against the initial stages of APAP overdose.

Modulation of endogenous opioid signaling by inhibitors of puromycin sensitive aminopeptidase.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

Recombinant human endostatin as a potential anti-angiogenic agent: therapeutic perspective and current status.

Angiogenesis is the physiological process that results in the formation of new blood vessels develop from pre-existing vasculature and plays a significant role in several physiological and pathological processes. Inhibiting angiogenesis, a crucial mechanism in the growth and metastasis of cancer, has been proposed as a potential anticancer therapy. Different studies showed the beneficial effects of angiogenesis inhibitors either in patients suffering from different cancers, alone or in combination with conventional therapies. Even though there are currently a number of efficient anti-angiogenic drugs, including monoclonal antibodies and kinase inhibitors, the associated toxicity profile and their affordability constraints are prompting researchers to search for a safe and affordable angiostatic agent for cancer treatment. Endostatin is one of the endogenous anti-angiogenic candidates that have been extensively pursued for the treatment of cancer, but even over three decades after its discovery, we have not made much advancement in employing it as an anticancer therapeutic despite of its remarkable anti-angiogenic effect with low toxicity profile. A recombinant human endostatin (rh-Es) variant for non-small cell lung cancer was approved by China in 2006 and has since been used effectively. Several other successful clinical trials related to endostatin for various malignancies are either ongoing or have already been completed with promising results. Thus, in this review, we have provided an overview of existing anti-angiogenic drugs developed for cancer therapy, with a summary of tumour angiogenesis in the context of Endostatin, and clinical status of rh-Es in cancer treatment. Furthermore, we briefly discuss the various strategies to improve endostatin features (poor pharmacokinetic properties) for developing rh-Es as a safe and effective agent for cancer treatment.

Role of 3-Mercaptopyruvate Sulfurtransferase (3-MST) in Physiology and Disease.

3-mercaptopyruvate sulfurtransferase (3-MST) plays the important role of producing hydrogen sulfide. Conserved from bacteria to Mammalia, this enzyme is localized in mitochondria as well as the cytoplasm. 3-MST mediates the reaction of 3-mercaptopyruvate with dihydrolipoic acid and thioredoxin to produce hydrogen sulfide. Hydrogen sulfide is also produced through cystathionine beta-synthase and cystathionine gamma-lyase, along with 3-MST, and is known to alleviate a variety of illnesses such as cancer, heart disease, and neurological conditions. The importance of cystathionine beta-synthase and cystathionine gamma-lyase in hydrogen sulfide biogenesis is well-described, but documentation of the 3-MST pathway is limited. This account compiles the current state of knowledge about the role of 3-MST in physiology and pathology. Attempts at targeting the 3-MST pathway for therapeutic benefit are discussed, highlighting the potential of 3-MST as a therapeutic target.

Fused human paraoxonase 1 as a prophylactic agent against organophosphate poisoning.

Organophosphates (OPs) are highly neurotoxic compounds and certain OP-compounds are also exploited as a weapon of mass destruction and chemical warfare in terrorist attacks. Available prophylactic and post-exposure treatments are less effective and also have serious side-effects. Thus, there is a dire need to develop effective and safe prophylactic agent(s) against OP-poisoning. Human Paraoxonase 1 (hPON1) can hydrolyze a wide range of OP molecules and can be developed as an effective and safe prophylactic agent. Thus, there is a dire need in the art to develop variant(s) of rhPON1 that not only possess 'good' OP-hydrolyzing activity but also have improved pharmacokinetic properties. In this report, we describe the characterization of the fused hPON1 (FHP) variant that not only exhibit enhanced in vivo pharmacokinetic properties but also delay / prevent the symptoms of OP-poisoning and prevents OP-induced mortality in rats.

Deletion of Glyoxalase 1 exacerbates acetaminophen-induced hepatotoxicity in mice.

Acetaminophen (APAP) overdose triggers a cascade of intracellular oxidative stress events culminating in acute liver injury. The clinically used antidote, N-acetylcysteine (NAC) has a narrow therapeutic window and early treatment is essential for satisfactory therapeutic outcome. For more versatile therapies that can be effective even at late-presentation, the intricacies of APAP-induced hepatotoxicity must be better understood. Accumulation of advanced glycation end-products (AGEs) and consequent activation of the receptor for AGEs (RAGE) are considered one of the key mechanistic features of APAP toxicity. Glyoxalase-1 (Glo-1) regulates AGE formation by limiting the levels of methylglyoxal (MEG). In this study, we studied the relevance of Glo-1 in APAP mediated activation of RAGE and downstream cell-death cascades. Constitutive Glo-1 knockout mice (GKO) and a cofactor of Glo-1, ψ-GSH, were employed as tools. Our findings show elevated oxidative stress, activation of RAGE and hepatocyte necrosis through steatosis in GKO mice treated with high-dose APAP compared to wild type controls. A unique feature of the hepatic necrosis in GKO mice is the appearance of microvesicular steatosis as a result of centrilobular necrosis, rather than inflammation seen in wild type. The GSH surrogate and general antioxidant, ψ-GSH alleviated APAP toxicity irrespective of Glo-1 status, suggesting that oxidative stress being the primary driver of APAP toxicity. Overall, exacerbation of APAP hepatotoxicity in GKO mice suggests the importance of this enzyme system in antioxidant defense against initial stages of APAP overdose.

Optimization of medium composition to increase the expression of recombinant human interferon-ß using the Plackett-Burman and central composite design in E. coli SE1.

Recombinant human interferon-ß (rhIFN-ß) is therapeutically important and new commercially viable approaches are needed for its increased production. In this study, a codon-optimized gene encoding for rhIFN-ß(C17S) protein was designed and expressed in E. coli SE1. As a first step of medium optimization, growth of E. coli as a function of different media components was studied. Subsequently, to optimize the media composition, a response surface methodology (RSM) was used. Our results show that optimized medium (15.0 g/L tryptone, 12.3 g/L meat extract, 1.0 g/L MgSO4 and 0.5 g/L thiamine along with minimal medium) obtained in this study provide better growth of recombinant cells and the expression level of recombinant protein was ~ 1.7-fold more than Luria-Bertani medium. The optimized medium may be utilized for the large-scale production of rhIFN-ß.