Krishna Kodukula

SRI International, Menlo Park, California, United States

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Publications (18)56.34 Total impact

  • D C Anderson, Krishna Kodukula
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    ABSTRACT: Biomarkers, quantitatively measurable indicators of biological or pathogenic processes, once validated play a critical role in disease diagnostics, the prediction of disease progression, and/or monitoring of the response to treatment. They may also represent drug targets. A number of different methods can be used for biomarker discovery and validation, including proteomics methods, metabolomics, imaging, and genome wide association studies (GWASs) and can be analysed using receiver operating characteristic (ROC) plots. The relative utility of single biomarkers with biomarker panels is discussed, along with paradigms for biomarker development, the latter in the context of three large-scale biomarker consortia, the Critical Path Predictive Safety Testing Consortium (PSTC), the NCI Early Detection Research Network (EDRN) and the Alzheimer's Disease Neuroimaging Initiative (ADNI). The importance of systematic optimization of many parameters in biomarker analysis, including validation, reproducibility, study design, statistical analysis and avoidance of bias are critical features used by these consortia. Problems including introduction of bias into study designs, data reporting or data analysis are also reviewed.
    Biochemical pharmacology 08/2013; · 4.25 Impact Factor
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    ABSTRACT: Improved diagnostics are needed to detect invasive pulmonary aspergillosis, a life-threatening infection caused by the pathogenic fungus Aspergillus fumigatus. We are investigating secreted fungal proteases as novel biomarkers for the diagnosis of this disease. Although the A. fumigatus genome encodes a multitude of secreted proteases, few have been experimentally characterized. Here, we employed an unbiased combinatorial library of internally quenched fluorogenic probes to detect infection-associated proteolysis in the lungs of guinea pigs experimentally infected with A. fumigatus. Comparative protease activity profiling revealed a prolyl endopeptidase activity that is reproducibly induced during infection but is not observed in healthy animals. This proteolytic activity was found in four independent animal experiments involving two A. fumigatus isolates. We synthesized a small, focused fluorogenic probe library to define the substrate specificity of the prolyl endopeptidase substrate motif and to identify optimal Probe sequences. These efforts resulted in the identification of a panel of six individual substrate-based fluorescent probes capable of detecting infection in guinea pigs with high statistical significance (P<0.005 in most cases). Receiver operating characteristic analyses demonstrated that this fluorogenic assay could detect A. fumigatus infection-associated proteolysis with comparable sensitivity and specificity as existing diagnostic procedures, suggesting that further optimization of the methodology may lead to improved diagnostics options for invasive pulmonary aspergillosis.
    Medical mycology: official publication of the International Society for Human and Animal Mycology 01/2013; · 2.13 Impact Factor
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    ABSTRACT: Proteases play an important role in many cellular functions and represent promising biomarkers in addition to being targets for many diseases ranging from cardiovascular and neurological disorders to respiratory problems and cancer. Sensitive and robust methods are necessary to quantify proteolytic activities in complex biological samples, e.g., serum and plasma to compliment existing techniques. We have previously utilized a combinatorial library of internally quenched fluorogenic probes to map the proteolytic profiles of guinea pig serum and bronchoalveolar lavage fluid (BALF). The technique was extended to identify disease-specific proteolytic activity in guinea pig BALF infected with invasive aspergillosis. Here, the library was utilized to map the global proteolytic specificities of plasma samples obtained from patients with the nephrotic syndrome, known as focal segmental glomerulosclerosis (FSGS). FSGS compromises both native and transplanted kidneys and has limited treatment options due to poorly understood pathophysiology. The circulating factor hypothesis suggests that some factor (or lack thereof) within the blood causes FSGS and recent research points to proteases as one of these. Comparison of the global proteolytic profiles of FSGS and healthy plasma samples identified many peptide motifs that were cleaved only in FSGS or in healthy plasma samples. The observed results reveal not only the protease specificities of plasma samples but also potential targets that can be pursued further to understand the role of proteases in FSGS pathology.
    Drug Development Research 01/2013; · 0.87 Impact Factor
  • Joseph Perrone, Krishna Kodukula, Walter H Moos
    Drug Development Research 01/2013; · 0.87 Impact Factor
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    ABSTRACT: Proteases have garnered interest as candidate biomarkers and therapeutic targets for many human diseases. A key challenge is the identification and characterization of disease-relevant proteases in the complex milieu of biological fluids such as serum, plasma, and bronchoalveolar lavage, in which a multitude of hydrolases act in concert. This unit describes a protocol to map the global proteolytic substrate specificities of complex biological samples using a concise combinatorial library of internally quenched fluorogenic peptide probes (IQFPs). This substrate profiling approach provides a global and quantitative comparison of protease specificities between different biological samples. Such a comparative analysis can lead to the identification of disease-specific 'fingerprints' of proteolytic activities with potential utility in diagnosis and therapy. Curr. Protoc. Protein Sci. 70:21.22.1-21.22.14. © 2012 by John Wiley & Sons, Inc.
    Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 11/2012; Chapter 21:Unit21.22.
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    ABSTRACT: Post-proline cleaving peptidases are promising therapeutic targets for neurodegenerative diseases, psychiatric conditions, metabolic disorders, and many cancers. Prolyl oligopeptidase (POP; E.C. 3.4.21.26) and fibroblast activation protein α (FAP; E.C. 3.4.24.B28) are two post-proline cleaving endopeptidases with very similar substrate specificities. Both enzymes are implicated in numerous human diseases, but their study is impeded by the lack of specific substrate probes. We interrogated a combinatorial library of proteolytic substrates and identified novel and selective substrates of POP and FAP. These new sequences will be useful as probes for fundamental biochemical study, scaffolds for inhibitor design, and triggers for controlled drug delivery.
    FEBS letters 06/2012; 586(16):2507-12. · 3.54 Impact Factor
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    ABSTRACT: The nonapeptide hemopressin, which is derived from the α chain of hemoglobin, has been reported to exhibit inverse agonist activity against the CB1 receptor. Administration of this peptide in animal models led to decreased food intake and elicited hypotensive and antinociceptive effects. On the basis of hemopressin's potential in therapeutic applications and the lack of a structure-activity relationship study in literature, we aimed to determine the conformational features of hemopressin under physiological conditions. We conducted transmission electron microscopy experiments of hemopressin, revealing that it self-assembles into fibrils under aqueous conditions at pH 7.4. Circular dichroism and nuclear magnetic resonance experiments indicate that the peptide adopts a mostly extended β-like structure, which may contribute to its self-assembly and fibril formation.
    Biomacromolecules 02/2012; 13(3):579-83. · 5.37 Impact Factor
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    ABSTRACT: The ST Pinch is a 12-membered hydrogen-bonded motif (Ser/Thr-Xaa-Ser/Thr) involving the side chain oxygen atoms of two Ser/Thr residues. We identified the ST Pinch in 104 proteins in a database containing high-resolution crystal structures. Conformational analysis of the ST Pinch in these proteins points to specific preferences for the Xaa residue and a high propensity of this residue to adopt positive φ angles. Our results suggest that this motif serves as a linker of secondary structural elements within proteins and is a new addition to the existing list of short hydrogen bond-stabilized motifs in proteins.
    Proteins Structure Function and Bioinformatics 02/2012; 80(5):1259-63. · 3.34 Impact Factor
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    ABSTRACT: Serine-Proline (SP) dipeptide motifs have been shown to form unique hydrogen-bonding patterns in protein crystal structures. Peptides were designed to mimic these patterns by forming the 6 + 10 and the 9 + 10 hydrogen-bonded rings. Factors that contribute to the formation of SP turns include controlling backbone flexibility and amino acid chirality along with creating a hydrophobic environment around the intramolecular hydrogen bonds.
    Organic Letters 02/2012; 14(3):732-5. · 6.14 Impact Factor
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    ABSTRACT: Aiming to design short linear peptides featuring strong intramolecular hydrogen bonds in water, a series of tetrapeptides based on the sequence Ac-Ala-Pro-Ala-Ala-NH(2) containing all possible combinations of L- and D-amino acids was synthesized. A regiospecific combination of heterochiral residues (DDLL or its mirror image LLDD) can be used to increase turn formation and stability within short peptides in water.
    Organic Letters 11/2011; 13(21):5878-81. · 6.14 Impact Factor
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    ABSTRACT: Proteases are candidate biomarkers and therapeutic targets for many diseases. Sensitive and robust techniques are needed to quantify proteolytic activities within the complex biological milieu. We hypothesized that a combinatorial protease substrate library could be used effectively to identify similarities and differences between serum and bronchoalveolar lavage fluid (BALF), two body fluids that are clinically important for developing targeted therapies and diagnostics. We used a concise library of fluorogenic probes to map the protease substrate specificities of serum and BALF from guinea pigs. Differences in the proteolytic fingerprints of the two fluids were striking: serum proteases cleaved substrates containing cationic residues and proline, whereas BALF proteases cleaved substrates containing aliphatic and aromatic residues. Notably, cleavage of proline-containing substrates dominated all other protease activities in both human and guinea pig serum. This substrate profiling approach provides a foundation for quantitative comparisons of protease specificities between complex biological samples.
    BioTechniques 08/2011; 51(2):95-104. · 2.40 Impact Factor
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    ABSTRACT: The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers. As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures. This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.
    PLoS ONE 01/2011; 6(6):e21001. · 3.53 Impact Factor
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    Formal Modeling: Actors, Open Systems, Biological Systems - Essays Dedicated to Carolyn Talcott on the Occasion of Her 70th Birthday; 01/2011
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    ABSTRACT: Non-thiazide loop diuretics such as bumetanide and furosemide are well-established medicaments, with varying substituents that alter their chemical properties in ways that affect biological parameters, including biodistribution, efficacy, and safety. However, literature data supporting the assignment of pKa values and ionization sequence of bumetanide are limited. The present study summarizes available literature data and then characterizes nuclear magnetic resonance (NMR) and ultraviolet (UV) spectral changes over a range of pH values to delineate the apparent sequence of deprotonations. Three macroscopic pKa values, 1.44, 3.74, and 10.37, were determined in water via pH titration experiments monitored by 1H NMR. Both 1H and 13C analyses support assignment of the lowest pKa value to the anilinium ion, as described for another diuretic, piretanide. However, UV data suggest that initial deprotonation of the benzoic acid competes to some extent with deprotonation of the anilinium ion, such that both pathways may operate simultaneously in an aqueous environment. Thus, the observed pKa values likely represent the macroscopic averages of the two competing deprotonation sequences. Calculated pKa values for bumetanide, piretanide, and model compounds are contrary to the deprotonation sequence we and others have determined experimentally, which is not surprising given the complicated factors driving the acid–base properties of these molecules. Drug Dev Res 72: 416–426, 2011. © 2011 Wiley-Liss, Inc.
    Drug Development Research 01/2011; 72(5):416-426. · 0.87 Impact Factor
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    ABSTRACT: The rising occurrence of drug-resistant pathogens accentuates the need to identify novel antibiotics. We wanted to identify new scaffolds for drug discovery by repurposing FDA-approved drugs against Acinetobacter baumannii, an emerging Gram-negative nosocomial drug-resistant pathogen. In this study, we screened 1040 FDA-approved drugs against drug-susceptible A. baumannii ATCC 17978 and drug-resistant A. baumannii BAA-1605. Twenty compounds exhibited significant antimicrobial activity (MIC ≤8 mg/L) against ATCC 17978 while only five compounds showed such activity against BAA-1605. Among the most notable results, tyrothricin, a bactericidal antibiotic typically active only against Gram-positive bacteria, exhibited equipotent activity against both strains. The paucity of identified compounds active against drug-resistant A. baumannii exemplifies its ability to resist antimicrobials as well as the resilience of drug-resistant Gram-negative pathogens. Repurposing of approved drugs is a viable alternative to de novo drug discovery and development.
    Journal of Antimicrobial Chemotherapy 12/2010; 65(12):2598-601. · 5.34 Impact Factor
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    ABSTRACT: Using a combination of an aromatic amino acid, a homoserine side chain, and a d-amino acid, a series of linear tetrapeptides were designed that adopt an "Hse turn" in water. The conformation was stabilized by intramolecular hydrogen bonds even in the presence of surrounding water molecules. In particular, the peptide with sequence H-Abz-Homoser-Ser-d-Gln-NH(2) showed significant through-space interactions and its free energy of folding is estimated to be on the order of -4 kcal/mol. We report the design of the tetrapeptides using a novel mimicry approach and their characterization based on NMR spectroscopy and MD simulations.
    Journal of the American Chemical Society 03/2010; 132(13):4508-9. · 10.68 Impact Factor
  • Rajeev Vaidyanathan, Krishna Kodukula
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    ABSTRACT: The reductionist approach to biology has allowed us to understand the cell at a component level. But in recent years, with the deluge of genome-derived and empirically obtained data, we must interpret biological complexity holistically. Thus, systems biology research allows us to connect the seemingly independent elements in biological networks. Protozoan parasites in the genus Leishmania cause clinically distinct diseases in humans with visceral, cutaneous, and mucocutaneous pathologies. In this article, we take the example of Leishmania parasites and their interactions with the vertebrate host as a model to generate a global view of molecular and biochemical processes in both spatial and temporal compartments, based on genomic, transcriptomic, proteomic, and metabolomic data. Furthermore, we propose extending this approach to other pathogen-host relationships, thus enabling us to identify novel therapeutic targets, vaccine candidates, and to develop immune monitoring and diagnosis. Drug Dev Res 70:296–302, 2009 © 2009 Wiley-Liss, Inc.
    Drug Development Research 05/2009; 70(4):296 - 302. · 0.87 Impact Factor
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    ABSTRACT: Susceptibility to deadly diarrheal diseases is partly due to widespread pediatric vitamin A deficiency. To increase vitamin A coverage in malnourished children, we propose to engineer a probiotic bacterium that will produce β-carotene in the intestine, which will be metabolized to vitamin A. Such a therapy has the potential to broadly stimulate mucosal immunity and simultaneously reduce the incidence and duration of diarrheal disease. To that end, a β-carotene-producing variant of the probiotic Escherichia coli strain Nissle 1917 (EcN-BETA) was generated. Notably, the strain produces β-carotene under anaerobic conditions, reflective of the gut environment. EcN-BETA also retains β-carotene production capability after lyophilization, suggesting that it may be amenable to dry formulation. Moreover, EcN-BETA activates murine dendritic cells in vitro, suggesting that the presence of β-carotene may not diminish the immunostimulatory capacity of EcN. Finally, we present a framework through which further improvements may enable approaches such as the one described in this report to yield innovative life-saving therapies for the developing world.
    Probiotics and Antimicrobial Proteins 5(2).