Mahmoud Ghanem - Academia.edu (original) (raw)

Papers by Mahmoud Ghanem

mAbs, 2019

We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) a... more We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.

Experimental Neurology, 2015

Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a t... more Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for dopaminergic neurons. Direct parenchymal administration of GDNF is robustly neuroprotective and neurorestorative in multiple neurotoxin-based animal models (rat and non-human primate (NHP)) of Parkinson's Disease (PD), suggesting its potential as a therapeutic agent. Although small, open-label clinical trials of intra-putamenal administration of bacteria-derived, full length, wild type GDNF (GDNFwt) were efficacious in improving standardized behavioral scores, a double-blinded, randomized controlled trial failed to do so. We hypothesize that the lack of clinical efficacy of GDNFwt in the larger randomized trial was due to poor bio-distribution in the putamen and/or poor chemical stability while in the delivery device for prolonged time periods at 37°C. The development of neutralizing antibodies in some patients may also have been a contributing factor. GDNFv is an engineered form of GDNFwt, expressed and purified from mammalian cells, designed to overcome these limitations, including removal of the N-terminal heparin-binding domain to improve its diffusivity in brain parenchyma by reducing its binding to extracellular matrix (ECM), and key amino acid substitutions to improve chemical stability. Intra-striatal administration of a single injection of GDNFv in the rat produced significantly greater brain distribution than GDNFwt, consistent with reduced binding to ECM. Using liquid chromatography/mass spectrometry (LS/MS) methods GDNFv was shown to have improved chemical stability compared to GDNFwt when stored at 37°C for 4 weeks. In addition, GDNFv resulted in lower predicted clinical immunogenicity compared to GDNFwt, as demonstrated by reduced CD4 + T cell proliferation and reduced IL-2-induced secretion in peripheral blood mononucleated cells collected from volunteers representing the world's major histocompatibility complex (MHC) haplotypes. GDNFv was demonstrated to be pharmacologically equivalent to GDNFwt in the key parameters in vitro of GFRα1 receptor binding, c-Ret phosphorylation, neurite outgrowth, and in vivo in its ability to increase dopamine turnover (DA). GDNFv protected dopamine nerve terminals and neurons in a 6-hydroxy-dopamine (6-OHDA) rat model. In summary, we empirically demonstrate the superior properties of GDNFv compared to GDNFwt through enhanced

Innovation management has recently received much attention from research, industry and state supp... more Innovation management has recently received much attention from research, industry and state support. However, much of the research has focused on large enterprises with much available financial wealth and required planning infrastructure, in order to be effective at managing innovation. This paper aims to understand the factors that affect the innovation management process within Turkish SMEs. A number of interviews are conducted with 25 SMEs using three different innovation auditing models to capture and validate best practice techniques. The goals of the analysis are (a) to understand the Turkish SMEs current capabilities and innovation management practices in each organization and (b) to identify a set of recommendations that may facilitate and enhance innovation management in all participated SMEs. Results showed that the majority of the SMEs have apparent lacking of innovation strategic plan, culture, formal process and assessment approach to measure impact of innovation project. These factors impact to the overall success of the organization.

SME Congress 2012, 2012

The topic of innovation management has received much attention from research, industry and state ... more The topic of innovation management has received much attention from research, industry and state support in the past number of years. However, much of the research has focused on the large enterprises with much available financial wealth and planning ...

Chemistry & Biology, 2009

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to... more Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and aD -ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state of human PNP is protected from solvent by hydrophobic residues that sequester the catalytic site. The catalytic site was enlarged by replacing individual catalytic site amino acids with glycine. Reactivity of the ribocation transition state was tested for capture by water and other nucleophiles. In the absence of phosphate, inosine is hydrolyzed by native, Y88G, F159G, H257G, and F200G enzymes. Phosphorolysis but not hydrolysis is detected when phosphate is bound. An unprecedented N9-to-N3 isomerization of inosine is catalyzed by H257G and F200G in the presence of phosphate and by all PNPs in the absence of phosphate. These results establish a ribocation lifetime too short to permit capture by water. An enlarged catalytic site permits ribocation formation with relaxed geometric constraints, permitting nucleophilic rebound and N3inosine isomerization.

Biophysical Journal, 2008

It has been found that with mutation of two surface residues (Lys 22 / Glu and His 104 / Arg) in ... more It has been found that with mutation of two surface residues (Lys 22 / Glu and His 104 / Arg) in human purine nucleoside phosphorylase (hPNP), there is an enhancement of catalytic activity in the chemical step. This is true although the mutations are quite remote from the active site, and there are no significant changes in crystallographic structure between the wild-type and mutant active sites. We propose that dynamic coupling from the remote residues to the catalytic site may play a role in catalysis, and it is this alteration in dynamics that causes an increase in the chemical step rate. Computational results indicate that the mutant exhibits stronger coupling between promotion of vibrations and the reaction coordinate than that found in native hPNP. Power spectra comparing native and mutant proteins show a correlation between the vibrations of Immucillin-G (ImmG):O59Á Á ÁImmG:N49 and H257:NdÁ Á ÁImmG:O59 consistent with a coupling of these motions. These modes are linked to the protein promoting vibrations. Stronger coupling of motions to the reaction coordinate increases the probability of reaching the transition state and thus lowers the activation free energy. This motion has been shown to contribute to catalysis. Coincident with the approach to the transition state, the sum of the distances of ImmG:O49Á Á ÁImmG:O59Á Á ÁH257:Nd became smaller, stabilizing the oxacarbenium ion formed at the transition state. Combined results from crystallography, mutational analysis, chemical kinetics, and computational analysis are consistent with dynamic compression playing a significant role in forming the transition state. Stronger coupling of these pairs is observed in the catalytically enhanced mutant enzyme. That motion and catalysis are enhanced by mutations remote from the catalytic site implicates dynamic coupling through the protein architecture as a component of catalysis in hPNP.

Biochemistry, 2009

Human PNP is a homotrimer containing three tryptophan residues at positions 16, 94, and 178, all ... more Human PNP is a homotrimer containing three tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The catalytic sites of PNP are located near the subunit-subunit interfaces where F159 is a catalytic site residue donated from an adjacent subunit. F159 covers the top () surface of the ribosyl group at the catalytic site. QM/MM calculations of human PNP have shown that F159 is the center of the most mobile region of the protein providing access to the substrate in the active site. F159 is also the key residue in a cluster of hydrophobic residues that shield catalytic site ligands from bulk solvent. Trp-free human PNP (Leuko-PNP) was previously engineered by replacing the three Trp residues of native PNP with Tyr. From this active construct, a single Trp residue was placed in the catalytic site loop (F159W-Leuko-PNP) as a reporter group for the ribosyl region of the catalytic site. The F159W-Leuko-PNP fluorescence is red shifted compared to native PNP, suggesting a solventexposed Trp residue. Upon ligand binding (hypoxanthine), the 3-fold fluorescence quench confirms conformational packing of the catalytic site pocket hydrophobic cluster. F159W-Leuko-PNP has an onenzyme thermodynamic equilibrium constant (K eq) near unity in the temperature range between 20 and 30°C and nonzero enthalpic components, making it suitable for laser-induced T-jump analyses. T-jump relaxation kinetics of F159W-Leuko-PNP in equilibrium with substrates and/or products indicate the conformational equilibria of at least two ternary complex intermediates in the nano-to millisecond time scale (1000-10000 s-1) that equilibrate prior to the slower chemical step (∼200 s-1). F159W-Leuko-PNP provides a novel protein platform to investigate the protein conformational dynamics occurring prior to transition state formation.

Biochemistry, 2008

Human (HsPNP) and bovine (BtPNP) purine nucleoside phosphorylases are homotrimers with the cataly... more Human (HsPNP) and bovine (BtPNP) purine nucleoside phosphorylases are homotrimers with the catalytic sites located near the subunit-subunit interfaces. Despite the high amino acid sequence similarity (87% identical) and the fully conserved catalytic site contacts between BtPNP and HsPNP, crystal structures reveal that the subunits interact differently and isotope effect studies indicate distinct transition-state structures. The subunit interfaces and crystallographic packing properties of BtPNP differ from HsPNP. Hypothetically, mutating HsPNP toward BtPNP might alter the dynamic, catalytic and subunit packing properties of HsPNP to become more similar to BtPNP. Amino acids Lys22 and His104 in HsPNP were target candidates based on crystal packing contacts and were replaced with their BtPNP counterparts to give Lys22Glu:His104Arg (E:R-PNP). The kinetic properties (steady and pre-steady state), inhibition constants, and thermodynamic properties of E:R-PNP were compared to HsPNP and BtPNP. The E:R-PNP is similar to HsPNP in steady-state kinetic properties. However HsPNP and E:R-PNP show remarkable ratios for (K m guanosine)/(K i * DADMe-ImmG) of 2.8 × 10 7 and 4.7 × 10 7 respectively, suggesting that DADMe-ImmG is an excellent mimic of the transition states for both HsPNP and E:R-PNP with a preference for E:R-PNP. Thermodynamic parameters obtained from the temperature dependence studies of the chemical step establish E:R-PNP to be catalytically more efficient than the parent enzyme and reveal differences in the entropic component of catalysis. The two companion manuscripts (

Biochemistry, 2008

Human purine nucleoside phosphorylase (PNP) is a homotrimer, containing three nonconserved trypto... more Human purine nucleoside phosphorylase (PNP) is a homotrimer, containing three nonconserved tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The Trp residues were replaced with Tyr to produce Trp-free PNP (Leuko-PNP). Leuko-PNP showed near-normal kinetic properties. It was used (1) to determine the tautomeric form of guanine that produces strong fluorescence when bound to PNP, (2) for thermodynamic binding analysis of binary and ternary complexes with substrates, (3) in temperature-jump perturbation of complexes for evidence of multiple conformational complexes, and (4) to establish the ionization state of a catalytic site tyrosine involved in phosphate nucleophile activation. The 13 C NMR spectrum of guanine bound to Leuko-PNP, its fluorescent properties, and molecular orbital electronic transition analysis establish that its fluorescence originates from the lowest singlet excited state of the N1H, 6-keto, N7H guanine tautomer. Binding of guanine and phosphate to PNP and Leuko-PNP are random, with decreased affinity for formation of ternary complexes. Pre-steadystate kinetics and temperature-jump studies indicate that the ternary complex (enzyme-substrate-phosphate) forms in single binding steps without kinetically significant protein conformational changes as monitored by guanine fluorescence. Spectral changes of Leuko-PNP upon phosphate binding establish that the hydroxyl of Tyr88 is not ionized to the phenolate anion when phosphate is bound. A loop region (residues 243-266) near the purine base becomes highly ordered upon substrate/inhibitor binding. A single Trp residue was introduced into the catalytic loop of Leuko-PNP (Y249W-Leuko-PNP) to determine effects on catalysis and to introduce a fluorescence catalytic site probe. Although Y249W-Leuko-PNP is highly fluorescent and catalytically active, substrate binding did not perturb the fluorescence. Thermodynamic boxes, constructed to characterize the binding of phosphate, guanine, and hypoxanthine to native, Leuko-, and Y249W-Leuko-PNPs, establish that Leuko-PNP provides a versatile protein scaffold for introduction of specific Trp catalytic site probes.

Biochemistry, 2007

Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvag... more Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min and a K m /K d ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min and a K m /K d ratio of 203,300. Tight binding of DADMe-ImmA supports a late S N 1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP•ImmA•PO 4 and TvPNP•DADMe-ImmA•PO 4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 Å ionic interaction between a PO 4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP•ImmA•PO 4 structure at 3.5 Å. However, the TvPNP•ImmA•PO 4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP•DADMe-ImmA•PO 4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18 O]ImmH to establish that O6 is the keto tautomer in TvPNP•ImmH•PO 4 , causing an unfavorable leaving-group interaction.

Biochemistry, 2011

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of 6-oxypurine (2′-... more Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of 6-oxypurine (2′-deoxy)ribonucleosides, generating (2-deoxy)ribose 1-phosphate and the purine base. Transition-state models for inosine cleavage have been proposed with bovine, human, and malarial PNPs using arsenate as the nucleophile, since kinetic isotope effects (KIEs) are obscured on phosphorolysis due to high commitment factors. The Phe200Gly mutant of human PNP has a low forward and reverse commitment factors in the phosphorolytic reaction, permitting the measurement of competitive intrinsic KIEs on both arsenolysis and phosphorolysis of inosine. The intrinsic 1′-14 C, 1′-3 H, 2′-2 H, 9-15 N, and 5′-3 H 2 KIEs for inosine were measured for arsenolysis and phosphorolysis. Except for the remote 5′-3 H 2 , and some slight difference between the 2′-2 H KIEs, all isotope effects originating in the reaction coordinate are the same within experimental error. Hence, arsenolysis and phosphorolysis proceed through closely related transition states. Although electrostatically similar, the volume of arsenate is greater than phosphate and supports a steric influence to explain the differences in the 5′-3 H 2 KIEs. Density functional theory calculations provide quantitative models of the transition states for Phe200Gly human PNPcatalyzed arsenolysis and phosphorolysis, selected upon matching calculated and experimental KIEs. The models confirm the striking resemblance between the transition states for the two reactions. Purine nucleoside phosphorylase (PNP) 1 (EC 2.4.2.1) catalyzes the reversible phosphorolysis of 6-oxopurine (2′-deoxy)ribonucleosides to generate ribose 1-phosphate (R-1-P) and the corresponding purine base, a key step of the purine salvage pathway and an essential step in formation of uric acid in humans (1). The reaction causes inversion of configuration from β-nucleosides to α-D-(2-deoxy)ribose 1-phosphate (2) (Scheme 1A). The genetic deficiency of PNP activity in humans impairs T-cell-based immunological response, while other tissues are unaltered (3). This led to the recognition of PNP as a target for † This work was supported by NIH grant GM41964 to V. L. S.

Biochemistry, 2008

Transition-state structures of human and bovine of purine nucleoside phosphorylases differ, despi... more Transition-state structures of human and bovine of purine nucleoside phosphorylases differ, despite 87% homologous amino acid sequences. Human PNP (HsPNP) has a fully dissociated transition state, while that for bovine PNP (BtPNP) has early S N 1 character. Crystal structures and sequence alignment indicate that the active sites of these enzymes are the same within crystallographic analysis, but residues in the second-sphere from the active sites differ significantly. Residues in BtPNP have been mutated toward HsPNP, resulting in double (Asn123Lys; Arg210Gln) and triple mutant PNPs (Val39Thr; Asn123Lys; Arg210Gln). Steady-state kinetic studies indicated unchanged catalytic activity, while presteady-state studies indicate that the chemical step is slower in the triple mutant. The mutant enzymes have higher affinity for inhibitors that are mimics of a late dissociative transition state. Kinetic isotope effects (KIEs) and computational chemistry were used to identify the transition-state structure of the triple mutant. Intrinsic KIEs from [1′-3 H], [1′-14 C], [2′-3 H], [5′-3 H], and [9-15 N] inosines were 1.221, 1.035, 1.073, 1.062 and 1.025, respectively. The primary intrinsic [1′-14 C] and [9-15 N] KIEs indicate a highly dissociative S N 1 transition state with low bond order to the leaving group, a transition state different from the native enzyme. The [1′-14 C] KIE suggests significant nucleophilic participation at the transition state. The transition-state structure of triple mutant PNP is altered as a consequence of the amino acids in the second sphere from the catalytic site. These residues are implicated in linking the dynamic motion of the protein to formation of the transition state.

Biochemistry, 2007

The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been iden... more The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has k cat values of 54 and 41 s-1 for 2′-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5′-Deaza-1′-aza-2′-deoxy-1′-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2′-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable K m /K i * of 5.4 × 10 7 , and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Å to reveal the differences in substrate and inhibitor specificity. The distance from the N1′ cation to the phosphate O4 anion is shorter in the AgPNP‚DADMe-ImmH‚PO 4 complex than in HsPNP‚DADMe-ImmH‚SO 4 , offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.

The International Journal of Knowledge, Culture, and Change Management: Annual Review, 2009

ABSTRACT An important aspect of knowledge management is the implementation of methods to share th... more ABSTRACT An important aspect of knowledge management is the implementation of methods to share the unstructured knowledge of expert practitioners within an organization. The existence of unstructured and dynamic knowledge represents a challenge to experts due to the dynamic and non-sequential nature of such knowledge. In order to make such knowledge sharable, it is necessary to have both an effective elicitation method and a useful representation toolkit. In this paper we describe a Dynamic Knowledge Toolkit (DKT) that is used in knowledge elicitation and representation based upon Knowledge maps. Knowledge Maps content is different from the more general information in typical reference material and that is organized quite differently than standard textbook knowledge or mainstream hypermedia learning systems. These knowledge models tend to be large and complex with interwoven themes and rich interconnections of the concepts based on the expert's highly articulated mental model of the domain. Knowledge Maps have been used in all facets of education, training and business. With the fundamental goal of fostering learning and knowledge sharing they have been shown to be an effective tool for displaying prior knowledge, summarizing, planning, scaffolding for understanding, consolidating experiences, improving affective conditions for critical thinking, decision making, supporting cooperation and collaboration, and organizing unstructured knowledge content. We describe the use of the toolkit in a case study on the capture and representation of local weather forecasting knowledge. We also show how Knowledge maps can be used to support activities such as the preservation of institutional memory, the "recovery" of expertise that might reside in less accessible forms such as archived documents, for performance support, and for other knowledge-intensive pursuits such as weather forecasting or crisis management.

mAbs, 2019

We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) a... more We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.

Experimental Neurology, 2015

Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a t... more Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for dopaminergic neurons. Direct parenchymal administration of GDNF is robustly neuroprotective and neurorestorative in multiple neurotoxin-based animal models (rat and non-human primate (NHP)) of Parkinson's Disease (PD), suggesting its potential as a therapeutic agent. Although small, open-label clinical trials of intra-putamenal administration of bacteria-derived, full length, wild type GDNF (GDNFwt) were efficacious in improving standardized behavioral scores, a double-blinded, randomized controlled trial failed to do so. We hypothesize that the lack of clinical efficacy of GDNFwt in the larger randomized trial was due to poor bio-distribution in the putamen and/or poor chemical stability while in the delivery device for prolonged time periods at 37°C. The development of neutralizing antibodies in some patients may also have been a contributing factor. GDNFv is an engineered form of GDNFwt, expressed and purified from mammalian cells, designed to overcome these limitations, including removal of the N-terminal heparin-binding domain to improve its diffusivity in brain parenchyma by reducing its binding to extracellular matrix (ECM), and key amino acid substitutions to improve chemical stability. Intra-striatal administration of a single injection of GDNFv in the rat produced significantly greater brain distribution than GDNFwt, consistent with reduced binding to ECM. Using liquid chromatography/mass spectrometry (LS/MS) methods GDNFv was shown to have improved chemical stability compared to GDNFwt when stored at 37°C for 4 weeks. In addition, GDNFv resulted in lower predicted clinical immunogenicity compared to GDNFwt, as demonstrated by reduced CD4 + T cell proliferation and reduced IL-2-induced secretion in peripheral blood mononucleated cells collected from volunteers representing the world's major histocompatibility complex (MHC) haplotypes. GDNFv was demonstrated to be pharmacologically equivalent to GDNFwt in the key parameters in vitro of GFRα1 receptor binding, c-Ret phosphorylation, neurite outgrowth, and in vivo in its ability to increase dopamine turnover (DA). GDNFv protected dopamine nerve terminals and neurons in a 6-hydroxy-dopamine (6-OHDA) rat model. In summary, we empirically demonstrate the superior properties of GDNFv compared to GDNFwt through enhanced

Innovation management has recently received much attention from research, industry and state supp... more Innovation management has recently received much attention from research, industry and state support. However, much of the research has focused on large enterprises with much available financial wealth and required planning infrastructure, in order to be effective at managing innovation. This paper aims to understand the factors that affect the innovation management process within Turkish SMEs. A number of interviews are conducted with 25 SMEs using three different innovation auditing models to capture and validate best practice techniques. The goals of the analysis are (a) to understand the Turkish SMEs current capabilities and innovation management practices in each organization and (b) to identify a set of recommendations that may facilitate and enhance innovation management in all participated SMEs. Results showed that the majority of the SMEs have apparent lacking of innovation strategic plan, culture, formal process and assessment approach to measure impact of innovation project. These factors impact to the overall success of the organization.

SME Congress 2012, 2012

The topic of innovation management has received much attention from research, industry and state ... more The topic of innovation management has received much attention from research, industry and state support in the past number of years. However, much of the research has focused on the large enterprises with much available financial wealth and planning ...

Chemistry & Biology, 2009

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to... more Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and aD -ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state of human PNP is protected from solvent by hydrophobic residues that sequester the catalytic site. The catalytic site was enlarged by replacing individual catalytic site amino acids with glycine. Reactivity of the ribocation transition state was tested for capture by water and other nucleophiles. In the absence of phosphate, inosine is hydrolyzed by native, Y88G, F159G, H257G, and F200G enzymes. Phosphorolysis but not hydrolysis is detected when phosphate is bound. An unprecedented N9-to-N3 isomerization of inosine is catalyzed by H257G and F200G in the presence of phosphate and by all PNPs in the absence of phosphate. These results establish a ribocation lifetime too short to permit capture by water. An enlarged catalytic site permits ribocation formation with relaxed geometric constraints, permitting nucleophilic rebound and N3inosine isomerization.

Biophysical Journal, 2008

It has been found that with mutation of two surface residues (Lys 22 / Glu and His 104 / Arg) in ... more It has been found that with mutation of two surface residues (Lys 22 / Glu and His 104 / Arg) in human purine nucleoside phosphorylase (hPNP), there is an enhancement of catalytic activity in the chemical step. This is true although the mutations are quite remote from the active site, and there are no significant changes in crystallographic structure between the wild-type and mutant active sites. We propose that dynamic coupling from the remote residues to the catalytic site may play a role in catalysis, and it is this alteration in dynamics that causes an increase in the chemical step rate. Computational results indicate that the mutant exhibits stronger coupling between promotion of vibrations and the reaction coordinate than that found in native hPNP. Power spectra comparing native and mutant proteins show a correlation between the vibrations of Immucillin-G (ImmG):O59Á Á ÁImmG:N49 and H257:NdÁ Á ÁImmG:O59 consistent with a coupling of these motions. These modes are linked to the protein promoting vibrations. Stronger coupling of motions to the reaction coordinate increases the probability of reaching the transition state and thus lowers the activation free energy. This motion has been shown to contribute to catalysis. Coincident with the approach to the transition state, the sum of the distances of ImmG:O49Á Á ÁImmG:O59Á Á ÁH257:Nd became smaller, stabilizing the oxacarbenium ion formed at the transition state. Combined results from crystallography, mutational analysis, chemical kinetics, and computational analysis are consistent with dynamic compression playing a significant role in forming the transition state. Stronger coupling of these pairs is observed in the catalytically enhanced mutant enzyme. That motion and catalysis are enhanced by mutations remote from the catalytic site implicates dynamic coupling through the protein architecture as a component of catalysis in hPNP.

Biochemistry, 2009

Human PNP is a homotrimer containing three tryptophan residues at positions 16, 94, and 178, all ... more Human PNP is a homotrimer containing three tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The catalytic sites of PNP are located near the subunit-subunit interfaces where F159 is a catalytic site residue donated from an adjacent subunit. F159 covers the top () surface of the ribosyl group at the catalytic site. QM/MM calculations of human PNP have shown that F159 is the center of the most mobile region of the protein providing access to the substrate in the active site. F159 is also the key residue in a cluster of hydrophobic residues that shield catalytic site ligands from bulk solvent. Trp-free human PNP (Leuko-PNP) was previously engineered by replacing the three Trp residues of native PNP with Tyr. From this active construct, a single Trp residue was placed in the catalytic site loop (F159W-Leuko-PNP) as a reporter group for the ribosyl region of the catalytic site. The F159W-Leuko-PNP fluorescence is red shifted compared to native PNP, suggesting a solventexposed Trp residue. Upon ligand binding (hypoxanthine), the 3-fold fluorescence quench confirms conformational packing of the catalytic site pocket hydrophobic cluster. F159W-Leuko-PNP has an onenzyme thermodynamic equilibrium constant (K eq) near unity in the temperature range between 20 and 30°C and nonzero enthalpic components, making it suitable for laser-induced T-jump analyses. T-jump relaxation kinetics of F159W-Leuko-PNP in equilibrium with substrates and/or products indicate the conformational equilibria of at least two ternary complex intermediates in the nano-to millisecond time scale (1000-10000 s-1) that equilibrate prior to the slower chemical step (∼200 s-1). F159W-Leuko-PNP provides a novel protein platform to investigate the protein conformational dynamics occurring prior to transition state formation.

Biochemistry, 2008

Human (HsPNP) and bovine (BtPNP) purine nucleoside phosphorylases are homotrimers with the cataly... more Human (HsPNP) and bovine (BtPNP) purine nucleoside phosphorylases are homotrimers with the catalytic sites located near the subunit-subunit interfaces. Despite the high amino acid sequence similarity (87% identical) and the fully conserved catalytic site contacts between BtPNP and HsPNP, crystal structures reveal that the subunits interact differently and isotope effect studies indicate distinct transition-state structures. The subunit interfaces and crystallographic packing properties of BtPNP differ from HsPNP. Hypothetically, mutating HsPNP toward BtPNP might alter the dynamic, catalytic and subunit packing properties of HsPNP to become more similar to BtPNP. Amino acids Lys22 and His104 in HsPNP were target candidates based on crystal packing contacts and were replaced with their BtPNP counterparts to give Lys22Glu:His104Arg (E:R-PNP). The kinetic properties (steady and pre-steady state), inhibition constants, and thermodynamic properties of E:R-PNP were compared to HsPNP and BtPNP. The E:R-PNP is similar to HsPNP in steady-state kinetic properties. However HsPNP and E:R-PNP show remarkable ratios for (K m guanosine)/(K i * DADMe-ImmG) of 2.8 × 10 7 and 4.7 × 10 7 respectively, suggesting that DADMe-ImmG is an excellent mimic of the transition states for both HsPNP and E:R-PNP with a preference for E:R-PNP. Thermodynamic parameters obtained from the temperature dependence studies of the chemical step establish E:R-PNP to be catalytically more efficient than the parent enzyme and reveal differences in the entropic component of catalysis. The two companion manuscripts (

Biochemistry, 2008

Human purine nucleoside phosphorylase (PNP) is a homotrimer, containing three nonconserved trypto... more Human purine nucleoside phosphorylase (PNP) is a homotrimer, containing three nonconserved tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The Trp residues were replaced with Tyr to produce Trp-free PNP (Leuko-PNP). Leuko-PNP showed near-normal kinetic properties. It was used (1) to determine the tautomeric form of guanine that produces strong fluorescence when bound to PNP, (2) for thermodynamic binding analysis of binary and ternary complexes with substrates, (3) in temperature-jump perturbation of complexes for evidence of multiple conformational complexes, and (4) to establish the ionization state of a catalytic site tyrosine involved in phosphate nucleophile activation. The 13 C NMR spectrum of guanine bound to Leuko-PNP, its fluorescent properties, and molecular orbital electronic transition analysis establish that its fluorescence originates from the lowest singlet excited state of the N1H, 6-keto, N7H guanine tautomer. Binding of guanine and phosphate to PNP and Leuko-PNP are random, with decreased affinity for formation of ternary complexes. Pre-steadystate kinetics and temperature-jump studies indicate that the ternary complex (enzyme-substrate-phosphate) forms in single binding steps without kinetically significant protein conformational changes as monitored by guanine fluorescence. Spectral changes of Leuko-PNP upon phosphate binding establish that the hydroxyl of Tyr88 is not ionized to the phenolate anion when phosphate is bound. A loop region (residues 243-266) near the purine base becomes highly ordered upon substrate/inhibitor binding. A single Trp residue was introduced into the catalytic loop of Leuko-PNP (Y249W-Leuko-PNP) to determine effects on catalysis and to introduce a fluorescence catalytic site probe. Although Y249W-Leuko-PNP is highly fluorescent and catalytically active, substrate binding did not perturb the fluorescence. Thermodynamic boxes, constructed to characterize the binding of phosphate, guanine, and hypoxanthine to native, Leuko-, and Y249W-Leuko-PNPs, establish that Leuko-PNP provides a versatile protein scaffold for introduction of specific Trp catalytic site probes.

Biochemistry, 2007

Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvag... more Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min and a K m /K d ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min and a K m /K d ratio of 203,300. Tight binding of DADMe-ImmA supports a late S N 1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP•ImmA•PO 4 and TvPNP•DADMe-ImmA•PO 4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 Å ionic interaction between a PO 4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP•ImmA•PO 4 structure at 3.5 Å. However, the TvPNP•ImmA•PO 4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP•DADMe-ImmA•PO 4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18 O]ImmH to establish that O6 is the keto tautomer in TvPNP•ImmH•PO 4 , causing an unfavorable leaving-group interaction.

Biochemistry, 2011

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of 6-oxypurine (2′-... more Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of 6-oxypurine (2′-deoxy)ribonucleosides, generating (2-deoxy)ribose 1-phosphate and the purine base. Transition-state models for inosine cleavage have been proposed with bovine, human, and malarial PNPs using arsenate as the nucleophile, since kinetic isotope effects (KIEs) are obscured on phosphorolysis due to high commitment factors. The Phe200Gly mutant of human PNP has a low forward and reverse commitment factors in the phosphorolytic reaction, permitting the measurement of competitive intrinsic KIEs on both arsenolysis and phosphorolysis of inosine. The intrinsic 1′-14 C, 1′-3 H, 2′-2 H, 9-15 N, and 5′-3 H 2 KIEs for inosine were measured for arsenolysis and phosphorolysis. Except for the remote 5′-3 H 2 , and some slight difference between the 2′-2 H KIEs, all isotope effects originating in the reaction coordinate are the same within experimental error. Hence, arsenolysis and phosphorolysis proceed through closely related transition states. Although electrostatically similar, the volume of arsenate is greater than phosphate and supports a steric influence to explain the differences in the 5′-3 H 2 KIEs. Density functional theory calculations provide quantitative models of the transition states for Phe200Gly human PNPcatalyzed arsenolysis and phosphorolysis, selected upon matching calculated and experimental KIEs. The models confirm the striking resemblance between the transition states for the two reactions. Purine nucleoside phosphorylase (PNP) 1 (EC 2.4.2.1) catalyzes the reversible phosphorolysis of 6-oxopurine (2′-deoxy)ribonucleosides to generate ribose 1-phosphate (R-1-P) and the corresponding purine base, a key step of the purine salvage pathway and an essential step in formation of uric acid in humans (1). The reaction causes inversion of configuration from β-nucleosides to α-D-(2-deoxy)ribose 1-phosphate (2) (Scheme 1A). The genetic deficiency of PNP activity in humans impairs T-cell-based immunological response, while other tissues are unaltered (3). This led to the recognition of PNP as a target for † This work was supported by NIH grant GM41964 to V. L. S.

Biochemistry, 2008

Transition-state structures of human and bovine of purine nucleoside phosphorylases differ, despi... more Transition-state structures of human and bovine of purine nucleoside phosphorylases differ, despite 87% homologous amino acid sequences. Human PNP (HsPNP) has a fully dissociated transition state, while that for bovine PNP (BtPNP) has early S N 1 character. Crystal structures and sequence alignment indicate that the active sites of these enzymes are the same within crystallographic analysis, but residues in the second-sphere from the active sites differ significantly. Residues in BtPNP have been mutated toward HsPNP, resulting in double (Asn123Lys; Arg210Gln) and triple mutant PNPs (Val39Thr; Asn123Lys; Arg210Gln). Steady-state kinetic studies indicated unchanged catalytic activity, while presteady-state studies indicate that the chemical step is slower in the triple mutant. The mutant enzymes have higher affinity for inhibitors that are mimics of a late dissociative transition state. Kinetic isotope effects (KIEs) and computational chemistry were used to identify the transition-state structure of the triple mutant. Intrinsic KIEs from [1′-3 H], [1′-14 C], [2′-3 H], [5′-3 H], and [9-15 N] inosines were 1.221, 1.035, 1.073, 1.062 and 1.025, respectively. The primary intrinsic [1′-14 C] and [9-15 N] KIEs indicate a highly dissociative S N 1 transition state with low bond order to the leaving group, a transition state different from the native enzyme. The [1′-14 C] KIE suggests significant nucleophilic participation at the transition state. The transition-state structure of triple mutant PNP is altered as a consequence of the amino acids in the second sphere from the catalytic site. These residues are implicated in linking the dynamic motion of the protein to formation of the transition state.

Biochemistry, 2007

The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been iden... more The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has k cat values of 54 and 41 s-1 for 2′-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5′-Deaza-1′-aza-2′-deoxy-1′-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2′-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable K m /K i * of 5.4 × 10 7 , and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Å to reveal the differences in substrate and inhibitor specificity. The distance from the N1′ cation to the phosphate O4 anion is shorter in the AgPNP‚DADMe-ImmH‚PO 4 complex than in HsPNP‚DADMe-ImmH‚SO 4 , offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.

The International Journal of Knowledge, Culture, and Change Management: Annual Review, 2009

ABSTRACT An important aspect of knowledge management is the implementation of methods to share th... more ABSTRACT An important aspect of knowledge management is the implementation of methods to share the unstructured knowledge of expert practitioners within an organization. The existence of unstructured and dynamic knowledge represents a challenge to experts due to the dynamic and non-sequential nature of such knowledge. In order to make such knowledge sharable, it is necessary to have both an effective elicitation method and a useful representation toolkit. In this paper we describe a Dynamic Knowledge Toolkit (DKT) that is used in knowledge elicitation and representation based upon Knowledge maps. Knowledge Maps content is different from the more general information in typical reference material and that is organized quite differently than standard textbook knowledge or mainstream hypermedia learning systems. These knowledge models tend to be large and complex with interwoven themes and rich interconnections of the concepts based on the expert's highly articulated mental model of the domain. Knowledge Maps have been used in all facets of education, training and business. With the fundamental goal of fostering learning and knowledge sharing they have been shown to be an effective tool for displaying prior knowledge, summarizing, planning, scaffolding for understanding, consolidating experiences, improving affective conditions for critical thinking, decision making, supporting cooperation and collaboration, and organizing unstructured knowledge content. We describe the use of the toolkit in a case study on the capture and representation of local weather forecasting knowledge. We also show how Knowledge maps can be used to support activities such as the preservation of institutional memory, the "recovery" of expertise that might reside in less accessible forms such as archived documents, for performance support, and for other knowledge-intensive pursuits such as weather forecasting or crisis management.