Kannan Gunasekaran - Academia.edu (original) (raw)

Papers by Kannan Gunasekaran

Research paper thumbnail of Conformational Analysis of Human Calcitonin in Aqueous Solution and Comparison with a Highly Potent Lactam-Bridged Analogue

Springer eBooks, 2001

Human calcitonin (hCt) is a peptide hormone of 32 amino acid residues that plays a central role i... more Human calcitonin (hCt) is a peptide hormone of 32 amino acid residues that plays a central role in calcium metabolism [1,2]. The receptor-bound conformation of hCt has not yet been determined. In an effort to understand the bioactive conformation of hCt we have recently designed a lactam-bridged hCt analogue with the structure cyclo17,21-[Asp17,Orn21]hCt (1) and found it to be 400-times more potent than hCt and 4-times more potent than salmon Ct (sCt) in the in vivo hypocalcemic assay in mice, making this the most potent known hCt analogue [3]. By contrast, a linear control peptide [Asp17, Orn21]hCt (2) showed similar in vivo potency to hCt [3]. These results suggested that the conformation of 1 may correspond to a bioactive conformation of hCt [3]. Here, we present studies on the solution conformation of hCt and 1 based on NMR in combination with molecular modeling and CD spectroscopy. We also present data on the hCt receptor-binding affinity of 1 as compared to 2, hCt and sCt.

Research paper thumbnail of Conformational interconversions in peptide β-turns: Discrimination between enantiomeric conformations by chiral perturbation

Biopolymers, 1998

ABSTRACT

Research paper thumbnail of Improved brain uptake and efficacy of iduronate 2-sulfatase with the enzyme transport vehicle

Molecular Genetics and Metabolism, 2019

burden of disease for caregivers, among others. All patients affected by AM can participate into ... more burden of disease for caregivers, among others. All patients affected by AM can participate into the registry, regardless of their treatment. Based on the current protocol, the study will be opened for participation for an indefinite time and each patient will be followed up for 15 years. The protocol does not foresee a pre-specified sample size and aims at including most of the patients treated with VA in clinical practice. The SPARKLE study will start in Europe in the second half of 2019.

Research paper thumbnail of Bioinformation by Biomedical Informatics Publishing Group www.bioinformation.net open access Hypothesis

Structural features differentiate the mechanisms between 2S (2 state) and 3S (3 state) folding of... more Structural features differentiate the mechanisms between 2S (2 state) and 3S (3 state) folding of homodimers.

Research paper thumbnail of A brain penetrant progranulin biotherapeutic rescues lysosomal and inflammatory phenotypes in the brain of GRN knockout mice

Alzheimer's & Dementia, 2020

Background: Heterozygous loss of function (LOF) mutations in GRN cause frontotemporal dementia (F... more Background: Heterozygous loss of function (LOF) mutations in GRN cause frontotemporal dementia (FTD), a neurodegenerative disorder associated with lysosomal dysfunction, TDP-43 pathology and inflammation in the CNS. Additionally, homozygous LOF mutations cause neuronal ceroid lipofuscinosis, a lysosomal storage disorder. GRN encodes progranulin (PGRN), a soluble protein that is most abundantly expressed in microglia, where it localizes to lysosomes and can undergo secretion. Although the precise function of PGRN is unknown, growing evidence suggests that it regulates lysosomal function, inflammatory responses and promotes neuronal survival. Accordingly, PGRN LOF models are associated with lysosomal defects, hyperinflammatory responses, and decreased neuronal viability, both in vitro and in vivo. Because GRN-FTD patients exhibit reduced levels of PGRN in biofluids and tissues, including the brain, a protein replacement therapy analogous to enzyme replacement therapy and capable of crossing the blood brain barrier (BBB) more efficiently may represent a powerful approach to slow or prevent disease progression. Method: Here we describe a novel therapeutic for increasing brain penetrance of PGRN, referred to as Protein Transport Vehicle (PTV):PGRN. PTV:PGRN consists of recombinant human PGRN fused to a modified Fc domain engineered to bind to the human transferrin receptor (huTfR), thus facilitating receptor-mediated transcytosis across the BBB.

Research paper thumbnail of Molecular architecture determines brain delivery of a transferrin-receptor targeted lysosomal enzyme

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches ... more Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brai...

Research paper thumbnail of Best Practices in Developability Assessments of Therapeutic Protein Candidates in the Biopharmaceutical Industry

Computational Approaches, 2015

Research paper thumbnail of Sequence and structural analysis of cellular retinoic acid-binding proteins reveals a network of conserved hydrophobic interactions

Proteins: Structure, Function, and Bioinformatics, 2003

Proteins in the intracellular lipidbinding protein (iLBP) family show remarkably high structural ... more Proteins in the intracellular lipidbinding protein (iLBP) family show remarkably high structural conservation despite their lowsequence identity. A multiple-sequence alignment using 52 sequences of iLBP family members revealed 15 fully conserved positions, with a disproportionately high number of these (n ‫؍‬ 7) located in the relatively small helical region. The conserved positions displayed high structural conservation based on comparisons of known iLBP crystal structures. It is striking that the ␤-sheet domain had few conserved positions, despite its high structural conservation. This observation prompted us to analyze pair-wise interactions within the ␤-sheet region to ask whether structural information was encoded in interacting amino acid pairs. We conducted this analysis on the iLBP family member, cellular retinoic acid-binding protein I (CRABP I), whose folding mechanism is under study in our laboratory. Indeed, an analysis based on a simple classification of hydrophobic and polar amino acids revealed a network of conserved interactions in CRABP I that cluster spatially, suggesting a possible nucleation site for folding. Significantly, a small number of residues participated in multiple conserved interactions, suggesting a key role for these sites in the structure and folding of CRABP I. The results presented here correlate well with available experimental evidence on folding of CRABPs and their family members and suggest future experiments. The analysis also shows the usefulness of considering pairwise conservation based on a simple classification of amino acids, in analyzing sequences and structures to find common core regions among homologues.

Research paper thumbnail of β2-Microglobulin Amyloidosis: Insights from Conservation Analysis and Fibril Modelling by Protein Docking Techniques

Journal of Molecular Biology, 2003

Current data suggest that globular domains may form amyloids via different mechanisms. Neverthele... more Current data suggest that globular domains may form amyloids via different mechanisms. Nevertheless, there are indications that the initiation of the process takes place invariably in the less stable segments of a protein domain. We have studied the sequence and structural conservation of b 2-microglobulin that deposits into fibrils in dialysisrelated amyloidosis. The dataset includes 51 high-resolution nonredundant structures of the antibody constant domain-like proteins (C1) and 132 related sequences. We describe a set of 30 conserved residues. Among them, 23 are conserved structurally, 16 are conserved sequentially and nine are conserved both sequentially and structurally. Strands A (12-18), G (91-95) and D (45-55) are the less conserved and stable segments of the domain, while strands B (22-28), C (36-41), E (62-70) and F (78-83) are the conserved and stable segments. We find that the conserved residues form a cluster with a network of interactions. The observed pattern of conservation is consistent with experimental data including H/D exchange, urea denaturation and limited proteolysis that suggest that strands A and G do not participate in the amyloid fibril. Additionally, the low conservation of strand D is consistent with the observation that this strand may acquire different conformations as seen in crystal structures of bound and isolated b 2-microglobulin. We used a docking technique to suggest a model for a fibril via stacking of b 2-microglobulin monomers. Our analysis suggests that the favored monomer building block for fibril elongation is the conformation of the isolated b 2-microglobulin, without the b-bulge on strand D and without strands A and G participating in the fibril b-sheet structure. This monomer retains all the conserved residues and their network of interactions, increasing the likelihood of its existence in solution. The inter-strand interaction between the two (monomer) building blocks forms a new continuous b-sheet such that addition of monomers results in a fibril model that has the characteristic cross-b structure.

Research paper thumbnail of Keeping it in the family: folding studies of related proteins

Current Opinion in Structural Biology, 2001

Abbreviations ACBP acyl coenzyme A-binding protein AcP acylphosphatase ADA2h activation domain of... more Abbreviations ACBP acyl coenzyme A-binding protein AcP acylphosphatase ADA2h activation domain of procarboxypeptidase A2 apoMb apomyoglobin CD2.d1 domain 1 of immunoglobulin CD2 CoC conservatism of conservatism CRABP cellular retinoic acid-binding protein Csp cold shock protein fn fibronectin-like domain GuHCl guanidinium hydrochloride IFABP intestinal fatty acid-binding protein Ig immunoglobulin iLBP intracellular lipid-binding protein ILBP ileal lipid-binding protein Lb leghemoglobin PDB Protein Data Bank SH Src homology TN tenascin Recent work by Ptitsyn and Shakhnovich has exploited the availability of large numbers of sequences to extract folding information from evolutionarily related proteins. Ptitsyn and Ting [7 • ] looked for conserved residues among globins. This approach led to the identification of two conserved clusters, one clearly involved in interactions with the heme and the other, a group of large nonpolar residues on helices A, G and H. The authors suggested that interactions among these residues could be critical to initiation of folding in the globin family, pointing out that indeed the A, G and H helices form early in apomyoglobin (apoMb) folding [8]. Interestingly, this prediction has been called into question in a recent experimental study comparing the folding of leghemoglobin (Lb) with that of myoglobin [9 • ], as discussed below. In order to identify amino acids conserved for folding and eliminate those conserved for function, Mirny and Shakhnovich [10 • ] developed the 'conservatism of conservatism' (CoC) approach, in which they compare members of superfamilies, that is, proteins that share folds, but are highly divergent in sequence and function. Not surprisingly, sites

Research paper thumbnail of Triggering Loops and Enzyme Function: Identification of Loops that Trigger and Modulate Movements

Journal of Molecular Biology, 2003

Enzyme function often involves a conformational change. There is a general agreement that loops p... more Enzyme function often involves a conformational change. There is a general agreement that loops play a vital role in correctly positioning the catalytically important residues. Nevertheless, predicting the functional loops and most importantly their role in enzyme function remains a difficult task. A major reason for this difficulty is that loops that undergo conformational change are frequently not well conserved in their primary sequence. b1,4-Galactosyltransferase is one such enzyme. There, the amino acid sequence of a long loop that undergoes a large conformational change upon substrate binding is not well conserved. Our molecular dynamics simulations show that the large conformational change in the long loop is brought about by a second, interacting loop. Interestingly, while the structural change of the second loop is much smaller than that of the long loop, its sequence (particularly glycine residues) is highly conserved. We further examine the generality of the proposition that there are loops that trigger movements but nevertheless show little or no structural changes in crystals. We focus on two other enzymes, enolase and lipase. We chose these enzymes, since they too undergo conformational change upon ligand binding, however, they have different folds and different functions. Through multiple sets of simulations we show that the conformational change of the functional loop(s) is brought about through communication of flexibility by triggering loops that have several glycine residues. We further propose that similar to the conservation of common favorable fold types and structural motifs, evolution has also conserved common "skillful" mechanisms. Mechanisms may be conserved across different folds, sequences and functions, with adaptation to specific enzymatic roles.

Research paper thumbnail of Side chain interactions determine the amyloid organization: a single layer  -sheet molecular structure of the calcitonin peptide segment 15–19

Physical Biology, 2004

In this paper we present a detailed atomic model for a protofilament, the most basic organization... more In this paper we present a detailed atomic model for a protofilament, the most basic organization level, of the amyloid fibre formed by the peptide DFNKF. This pentapeptide is a segment derived from the human calcitonin, a natural amyloidogenic protein. Our model, which represents the outcome of extensive explicit solvent molecular dynamics (MD) simulations of different strand/sheet organizations, is a single β-sheet filament largely without a hydrophobic core. Nevertheless, this structure is capable of reproducing the main features of the characteristic amyloid fibril organization and provides clues to the molecular basis of its experimental aggregation behaviour. Our results show that the side chains' chemical diversity induces the formation of a complex network of interactions that finally determine the microscopic arrangement of the strands at the protofilament level. This network of interactions, consisting of both side chain-side chain and backbone-side chain interactions, confers on the final single β-sheet arrangement an unexpected stability, both by enhancing the association of related chemical groups and, at the same time, by shielding the hydrophobic segments from the polar solvent. The chemical physical characterization of this protofilament provides hints to the possible thermodynamical basis of the supra molecular organization that allows the formation of the filaments by lateral association of the preformed protofibrils. Its regular, highly polarized structure shows how other protofilaments can assemble. In terms of structural biology, our results clearly indicate that an amyloid organization implies a degree of complexity far beyond a simple nonspecific association of peptide strands via amide hydrogen bonds.

Research paper thumbnail of Structural and thermodynamic effects of ANS binding to human interleukin-1 receptor antagonist

Protein Science, 2008

Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies... more Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies, the structural and thermodynamic effects of its binding to proteins are not well understood. Using highresolution two-dimensional NMR and human interleukin-1 receptor antagonist (IL-1ra) as a model protein, we obtained detailed information on ANS-protein interactions in the absence and presence of urea. The effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed from experiments performed at 25°C and 37°C. Overall, the affinity of ANS was lower at 37°C compared to 25°C, but no significant change in the site specificity of binding was observed from the chemical shift perturbation data. The same site-specific binding was evident in the presence of 5.2 M urea, well within the unfolding transition region, and resulted in selective stabilization of the folded state. Based on the two-state denaturation mechanism, ANS-dependent changes in the protein stability were estimated from relative intensities of two amide resonances specific to the folded and unfolded states of IL-1ra. No evidence was found for any ANS-induced partially denatured or aggregated forms of IL-1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The NMR results support earlier observations on the tendency of ANS to interact with solventexposed positively charged sites on proteins. Under denaturing conditions, ANS binding appears to be selective to structured states rather than unfolded conformations. Interestingly, the binding occurs within a previously identified aggregation-critical region in IL-1ra, thus providing an insight into ligand-dependent protein aggregation.

Research paper thumbnail of Brain delivery and efficacy of an intravenously-administered lysosomal enzyme using a blood-brain barrier transport vehicle

Molecular Genetics and Metabolism

Research paper thumbnail of Local and Long-Range Sequence Contributions to the Folding of a Predominantly β-Sheet Protein

Peptides: The Wave of the Future, 2001

Understanding how a linear sequence of amino acids guides acquisition of the native state of a pr... more Understanding how a linear sequence of amino acids guides acquisition of the native state of a protein — the protein folding problem — is key to interpreting information present in the human genome and to developing therapeutic approaches to diseases caused by protein misfolding. While considerable progress has been made in elucidating the interplay of local and long-range forces in the folding of α-helical proteins, such understanding has lagged for primarily β-sheet proteins [1]. We have undertaken studies aimed at determining the roles of local and global sequence information in the folding of the predominantly β-sheet protein, cellular retinoic acid binding protein I (CRABP I). CRABP I is a 136 residue β-clamshell protein (Figure 1) whose physiological role is the sequestration and transport of the hydrophobic ligand retinoic acid. CRABP I is a member of the large family of intracellular lipid binding proteins (iLBPs) [2], which contains 52 members with sequence identity greater than 30% with respect to CRABP I. Past work in our laboratory using stopped-flow (SF) mixing to follow the fluorescence of CRABP I has allowed us to define a series of kinetic phases from the urea-denatured conformational ensemble to the native fold [3]. Within the ≈10 ms dead time of the SF instrument, the unfolded ensemble forms a hydro-phobically collapsed state with considerable secondary structure. The presence of significant secondary structure along with hydrophobic collapse suggests that both global and local forces are acting in the earliest folding events.

Research paper thumbnail of Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic

Research paper thumbnail of Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice

Science Translational Medicine

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairmen... more Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS i...

Research paper thumbnail of High-resolution mass spectrometry confirms the presence of a hydroxyproline (Hyp) post-translational modification in the GGGGP linker of an Fc-fusion protein

mAbs, Jul 1, 2017

Flexible and protease resistant (G4S)n linkers are used extensively in protein engineering to con... more Flexible and protease resistant (G4S)n linkers are used extensively in protein engineering to connect various protein domains. Recently, several groups have observed xylose-based O-glycosylation at linker Ser residues that yield unwanted heterogeneity and may affect product quality. Because of this, an engineering effort was implemented to explore different linker sequence constructs. Here, we demonstrate the presence of an unexpected hydroxylation of a prolyl residue in the linker, made possible through the use of high-resolution mass spectrometry (HR-MS) and MSn. The discovery started with the detection of a poorly resolved ∼+17 Da mass addition at the reduced protein chain level of an Fc-fusion construct by liquid chromatography-MS. Upon further investigation at the peptide level using HR-MS, the mass increase was determined to be +15.99 Da and was localized to the linker peptide SLSLSPGGGGGPAR [210-223]. This peptide corresponds to the C-terminus of Fc [210-216], the G4P linker ...

Research paper thumbnail of Engineering Antibody Reactivity for Efficient Derivatization to Generate NaV1.7 Inhibitory GpTx-1 Peptide-Antibody Conjugates

ACS chemical biology, Sep 11, 2017

The voltage-gated sodium channel NaV1.7 is a genetically-validated pain target under investigatio... more The voltage-gated sodium channel NaV1.7 is a genetically-validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain, however functional NaV1.7 targeting antibodies are not known. In this report we describe NaV1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the NaV1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of thirteen potential cysteine conjugation site...

Research paper thumbnail of Biological characterization of a Stable Effector Functionless (SEFL) monoclonal antibody scaffold in vitro

The Journal of biological chemistry, Feb 19, 2016

The Stable Effector FunctionLess (SEFL) antibody was designed as an IgG1 antibody with a constant... more The Stable Effector FunctionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article titled "Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability" by Jacobsen et al. The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared to the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro against Daudi cells with cynomolgus monkey PBMC, and had minimal complement depende...

Research paper thumbnail of Conformational Analysis of Human Calcitonin in Aqueous Solution and Comparison with a Highly Potent Lactam-Bridged Analogue

Springer eBooks, 2001

Human calcitonin (hCt) is a peptide hormone of 32 amino acid residues that plays a central role i... more Human calcitonin (hCt) is a peptide hormone of 32 amino acid residues that plays a central role in calcium metabolism [1,2]. The receptor-bound conformation of hCt has not yet been determined. In an effort to understand the bioactive conformation of hCt we have recently designed a lactam-bridged hCt analogue with the structure cyclo17,21-[Asp17,Orn21]hCt (1) and found it to be 400-times more potent than hCt and 4-times more potent than salmon Ct (sCt) in the in vivo hypocalcemic assay in mice, making this the most potent known hCt analogue [3]. By contrast, a linear control peptide [Asp17, Orn21]hCt (2) showed similar in vivo potency to hCt [3]. These results suggested that the conformation of 1 may correspond to a bioactive conformation of hCt [3]. Here, we present studies on the solution conformation of hCt and 1 based on NMR in combination with molecular modeling and CD spectroscopy. We also present data on the hCt receptor-binding affinity of 1 as compared to 2, hCt and sCt.

Research paper thumbnail of Conformational interconversions in peptide β-turns: Discrimination between enantiomeric conformations by chiral perturbation

Biopolymers, 1998

ABSTRACT

Research paper thumbnail of Improved brain uptake and efficacy of iduronate 2-sulfatase with the enzyme transport vehicle

Molecular Genetics and Metabolism, 2019

burden of disease for caregivers, among others. All patients affected by AM can participate into ... more burden of disease for caregivers, among others. All patients affected by AM can participate into the registry, regardless of their treatment. Based on the current protocol, the study will be opened for participation for an indefinite time and each patient will be followed up for 15 years. The protocol does not foresee a pre-specified sample size and aims at including most of the patients treated with VA in clinical practice. The SPARKLE study will start in Europe in the second half of 2019.

Research paper thumbnail of Bioinformation by Biomedical Informatics Publishing Group www.bioinformation.net open access Hypothesis

Structural features differentiate the mechanisms between 2S (2 state) and 3S (3 state) folding of... more Structural features differentiate the mechanisms between 2S (2 state) and 3S (3 state) folding of homodimers.

Research paper thumbnail of A brain penetrant progranulin biotherapeutic rescues lysosomal and inflammatory phenotypes in the brain of GRN knockout mice

Alzheimer's & Dementia, 2020

Background: Heterozygous loss of function (LOF) mutations in GRN cause frontotemporal dementia (F... more Background: Heterozygous loss of function (LOF) mutations in GRN cause frontotemporal dementia (FTD), a neurodegenerative disorder associated with lysosomal dysfunction, TDP-43 pathology and inflammation in the CNS. Additionally, homozygous LOF mutations cause neuronal ceroid lipofuscinosis, a lysosomal storage disorder. GRN encodes progranulin (PGRN), a soluble protein that is most abundantly expressed in microglia, where it localizes to lysosomes and can undergo secretion. Although the precise function of PGRN is unknown, growing evidence suggests that it regulates lysosomal function, inflammatory responses and promotes neuronal survival. Accordingly, PGRN LOF models are associated with lysosomal defects, hyperinflammatory responses, and decreased neuronal viability, both in vitro and in vivo. Because GRN-FTD patients exhibit reduced levels of PGRN in biofluids and tissues, including the brain, a protein replacement therapy analogous to enzyme replacement therapy and capable of crossing the blood brain barrier (BBB) more efficiently may represent a powerful approach to slow or prevent disease progression. Method: Here we describe a novel therapeutic for increasing brain penetrance of PGRN, referred to as Protein Transport Vehicle (PTV):PGRN. PTV:PGRN consists of recombinant human PGRN fused to a modified Fc domain engineered to bind to the human transferrin receptor (huTfR), thus facilitating receptor-mediated transcytosis across the BBB.

Research paper thumbnail of Molecular architecture determines brain delivery of a transferrin-receptor targeted lysosomal enzyme

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches ... more Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brai...

Research paper thumbnail of Best Practices in Developability Assessments of Therapeutic Protein Candidates in the Biopharmaceutical Industry

Computational Approaches, 2015

Research paper thumbnail of Sequence and structural analysis of cellular retinoic acid-binding proteins reveals a network of conserved hydrophobic interactions

Proteins: Structure, Function, and Bioinformatics, 2003

Proteins in the intracellular lipidbinding protein (iLBP) family show remarkably high structural ... more Proteins in the intracellular lipidbinding protein (iLBP) family show remarkably high structural conservation despite their lowsequence identity. A multiple-sequence alignment using 52 sequences of iLBP family members revealed 15 fully conserved positions, with a disproportionately high number of these (n ‫؍‬ 7) located in the relatively small helical region. The conserved positions displayed high structural conservation based on comparisons of known iLBP crystal structures. It is striking that the ␤-sheet domain had few conserved positions, despite its high structural conservation. This observation prompted us to analyze pair-wise interactions within the ␤-sheet region to ask whether structural information was encoded in interacting amino acid pairs. We conducted this analysis on the iLBP family member, cellular retinoic acid-binding protein I (CRABP I), whose folding mechanism is under study in our laboratory. Indeed, an analysis based on a simple classification of hydrophobic and polar amino acids revealed a network of conserved interactions in CRABP I that cluster spatially, suggesting a possible nucleation site for folding. Significantly, a small number of residues participated in multiple conserved interactions, suggesting a key role for these sites in the structure and folding of CRABP I. The results presented here correlate well with available experimental evidence on folding of CRABPs and their family members and suggest future experiments. The analysis also shows the usefulness of considering pairwise conservation based on a simple classification of amino acids, in analyzing sequences and structures to find common core regions among homologues.

Research paper thumbnail of β2-Microglobulin Amyloidosis: Insights from Conservation Analysis and Fibril Modelling by Protein Docking Techniques

Journal of Molecular Biology, 2003

Current data suggest that globular domains may form amyloids via different mechanisms. Neverthele... more Current data suggest that globular domains may form amyloids via different mechanisms. Nevertheless, there are indications that the initiation of the process takes place invariably in the less stable segments of a protein domain. We have studied the sequence and structural conservation of b 2-microglobulin that deposits into fibrils in dialysisrelated amyloidosis. The dataset includes 51 high-resolution nonredundant structures of the antibody constant domain-like proteins (C1) and 132 related sequences. We describe a set of 30 conserved residues. Among them, 23 are conserved structurally, 16 are conserved sequentially and nine are conserved both sequentially and structurally. Strands A (12-18), G (91-95) and D (45-55) are the less conserved and stable segments of the domain, while strands B (22-28), C (36-41), E (62-70) and F (78-83) are the conserved and stable segments. We find that the conserved residues form a cluster with a network of interactions. The observed pattern of conservation is consistent with experimental data including H/D exchange, urea denaturation and limited proteolysis that suggest that strands A and G do not participate in the amyloid fibril. Additionally, the low conservation of strand D is consistent with the observation that this strand may acquire different conformations as seen in crystal structures of bound and isolated b 2-microglobulin. We used a docking technique to suggest a model for a fibril via stacking of b 2-microglobulin monomers. Our analysis suggests that the favored monomer building block for fibril elongation is the conformation of the isolated b 2-microglobulin, without the b-bulge on strand D and without strands A and G participating in the fibril b-sheet structure. This monomer retains all the conserved residues and their network of interactions, increasing the likelihood of its existence in solution. The inter-strand interaction between the two (monomer) building blocks forms a new continuous b-sheet such that addition of monomers results in a fibril model that has the characteristic cross-b structure.

Research paper thumbnail of Keeping it in the family: folding studies of related proteins

Current Opinion in Structural Biology, 2001

Abbreviations ACBP acyl coenzyme A-binding protein AcP acylphosphatase ADA2h activation domain of... more Abbreviations ACBP acyl coenzyme A-binding protein AcP acylphosphatase ADA2h activation domain of procarboxypeptidase A2 apoMb apomyoglobin CD2.d1 domain 1 of immunoglobulin CD2 CoC conservatism of conservatism CRABP cellular retinoic acid-binding protein Csp cold shock protein fn fibronectin-like domain GuHCl guanidinium hydrochloride IFABP intestinal fatty acid-binding protein Ig immunoglobulin iLBP intracellular lipid-binding protein ILBP ileal lipid-binding protein Lb leghemoglobin PDB Protein Data Bank SH Src homology TN tenascin Recent work by Ptitsyn and Shakhnovich has exploited the availability of large numbers of sequences to extract folding information from evolutionarily related proteins. Ptitsyn and Ting [7 • ] looked for conserved residues among globins. This approach led to the identification of two conserved clusters, one clearly involved in interactions with the heme and the other, a group of large nonpolar residues on helices A, G and H. The authors suggested that interactions among these residues could be critical to initiation of folding in the globin family, pointing out that indeed the A, G and H helices form early in apomyoglobin (apoMb) folding [8]. Interestingly, this prediction has been called into question in a recent experimental study comparing the folding of leghemoglobin (Lb) with that of myoglobin [9 • ], as discussed below. In order to identify amino acids conserved for folding and eliminate those conserved for function, Mirny and Shakhnovich [10 • ] developed the 'conservatism of conservatism' (CoC) approach, in which they compare members of superfamilies, that is, proteins that share folds, but are highly divergent in sequence and function. Not surprisingly, sites

Research paper thumbnail of Triggering Loops and Enzyme Function: Identification of Loops that Trigger and Modulate Movements

Journal of Molecular Biology, 2003

Enzyme function often involves a conformational change. There is a general agreement that loops p... more Enzyme function often involves a conformational change. There is a general agreement that loops play a vital role in correctly positioning the catalytically important residues. Nevertheless, predicting the functional loops and most importantly their role in enzyme function remains a difficult task. A major reason for this difficulty is that loops that undergo conformational change are frequently not well conserved in their primary sequence. b1,4-Galactosyltransferase is one such enzyme. There, the amino acid sequence of a long loop that undergoes a large conformational change upon substrate binding is not well conserved. Our molecular dynamics simulations show that the large conformational change in the long loop is brought about by a second, interacting loop. Interestingly, while the structural change of the second loop is much smaller than that of the long loop, its sequence (particularly glycine residues) is highly conserved. We further examine the generality of the proposition that there are loops that trigger movements but nevertheless show little or no structural changes in crystals. We focus on two other enzymes, enolase and lipase. We chose these enzymes, since they too undergo conformational change upon ligand binding, however, they have different folds and different functions. Through multiple sets of simulations we show that the conformational change of the functional loop(s) is brought about through communication of flexibility by triggering loops that have several glycine residues. We further propose that similar to the conservation of common favorable fold types and structural motifs, evolution has also conserved common "skillful" mechanisms. Mechanisms may be conserved across different folds, sequences and functions, with adaptation to specific enzymatic roles.

Research paper thumbnail of Side chain interactions determine the amyloid organization: a single layer  -sheet molecular structure of the calcitonin peptide segment 15–19

Physical Biology, 2004

In this paper we present a detailed atomic model for a protofilament, the most basic organization... more In this paper we present a detailed atomic model for a protofilament, the most basic organization level, of the amyloid fibre formed by the peptide DFNKF. This pentapeptide is a segment derived from the human calcitonin, a natural amyloidogenic protein. Our model, which represents the outcome of extensive explicit solvent molecular dynamics (MD) simulations of different strand/sheet organizations, is a single β-sheet filament largely without a hydrophobic core. Nevertheless, this structure is capable of reproducing the main features of the characteristic amyloid fibril organization and provides clues to the molecular basis of its experimental aggregation behaviour. Our results show that the side chains' chemical diversity induces the formation of a complex network of interactions that finally determine the microscopic arrangement of the strands at the protofilament level. This network of interactions, consisting of both side chain-side chain and backbone-side chain interactions, confers on the final single β-sheet arrangement an unexpected stability, both by enhancing the association of related chemical groups and, at the same time, by shielding the hydrophobic segments from the polar solvent. The chemical physical characterization of this protofilament provides hints to the possible thermodynamical basis of the supra molecular organization that allows the formation of the filaments by lateral association of the preformed protofibrils. Its regular, highly polarized structure shows how other protofilaments can assemble. In terms of structural biology, our results clearly indicate that an amyloid organization implies a degree of complexity far beyond a simple nonspecific association of peptide strands via amide hydrogen bonds.

Research paper thumbnail of Structural and thermodynamic effects of ANS binding to human interleukin-1 receptor antagonist

Protein Science, 2008

Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies... more Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies, the structural and thermodynamic effects of its binding to proteins are not well understood. Using highresolution two-dimensional NMR and human interleukin-1 receptor antagonist (IL-1ra) as a model protein, we obtained detailed information on ANS-protein interactions in the absence and presence of urea. The effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed from experiments performed at 25°C and 37°C. Overall, the affinity of ANS was lower at 37°C compared to 25°C, but no significant change in the site specificity of binding was observed from the chemical shift perturbation data. The same site-specific binding was evident in the presence of 5.2 M urea, well within the unfolding transition region, and resulted in selective stabilization of the folded state. Based on the two-state denaturation mechanism, ANS-dependent changes in the protein stability were estimated from relative intensities of two amide resonances specific to the folded and unfolded states of IL-1ra. No evidence was found for any ANS-induced partially denatured or aggregated forms of IL-1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The NMR results support earlier observations on the tendency of ANS to interact with solventexposed positively charged sites on proteins. Under denaturing conditions, ANS binding appears to be selective to structured states rather than unfolded conformations. Interestingly, the binding occurs within a previously identified aggregation-critical region in IL-1ra, thus providing an insight into ligand-dependent protein aggregation.

Research paper thumbnail of Brain delivery and efficacy of an intravenously-administered lysosomal enzyme using a blood-brain barrier transport vehicle

Molecular Genetics and Metabolism

Research paper thumbnail of Local and Long-Range Sequence Contributions to the Folding of a Predominantly β-Sheet Protein

Peptides: The Wave of the Future, 2001

Understanding how a linear sequence of amino acids guides acquisition of the native state of a pr... more Understanding how a linear sequence of amino acids guides acquisition of the native state of a protein — the protein folding problem — is key to interpreting information present in the human genome and to developing therapeutic approaches to diseases caused by protein misfolding. While considerable progress has been made in elucidating the interplay of local and long-range forces in the folding of α-helical proteins, such understanding has lagged for primarily β-sheet proteins [1]. We have undertaken studies aimed at determining the roles of local and global sequence information in the folding of the predominantly β-sheet protein, cellular retinoic acid binding protein I (CRABP I). CRABP I is a 136 residue β-clamshell protein (Figure 1) whose physiological role is the sequestration and transport of the hydrophobic ligand retinoic acid. CRABP I is a member of the large family of intracellular lipid binding proteins (iLBPs) [2], which contains 52 members with sequence identity greater than 30% with respect to CRABP I. Past work in our laboratory using stopped-flow (SF) mixing to follow the fluorescence of CRABP I has allowed us to define a series of kinetic phases from the urea-denatured conformational ensemble to the native fold [3]. Within the ≈10 ms dead time of the SF instrument, the unfolded ensemble forms a hydro-phobically collapsed state with considerable secondary structure. The presence of significant secondary structure along with hydrophobic collapse suggests that both global and local forces are acting in the earliest folding events.

Research paper thumbnail of Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic

Research paper thumbnail of Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice

Science Translational Medicine

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairmen... more Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS i...

Research paper thumbnail of High-resolution mass spectrometry confirms the presence of a hydroxyproline (Hyp) post-translational modification in the GGGGP linker of an Fc-fusion protein

mAbs, Jul 1, 2017

Flexible and protease resistant (G4S)n linkers are used extensively in protein engineering to con... more Flexible and protease resistant (G4S)n linkers are used extensively in protein engineering to connect various protein domains. Recently, several groups have observed xylose-based O-glycosylation at linker Ser residues that yield unwanted heterogeneity and may affect product quality. Because of this, an engineering effort was implemented to explore different linker sequence constructs. Here, we demonstrate the presence of an unexpected hydroxylation of a prolyl residue in the linker, made possible through the use of high-resolution mass spectrometry (HR-MS) and MSn. The discovery started with the detection of a poorly resolved ∼+17 Da mass addition at the reduced protein chain level of an Fc-fusion construct by liquid chromatography-MS. Upon further investigation at the peptide level using HR-MS, the mass increase was determined to be +15.99 Da and was localized to the linker peptide SLSLSPGGGGGPAR [210-223]. This peptide corresponds to the C-terminus of Fc [210-216], the G4P linker ...

Research paper thumbnail of Engineering Antibody Reactivity for Efficient Derivatization to Generate NaV1.7 Inhibitory GpTx-1 Peptide-Antibody Conjugates

ACS chemical biology, Sep 11, 2017

The voltage-gated sodium channel NaV1.7 is a genetically-validated pain target under investigatio... more The voltage-gated sodium channel NaV1.7 is a genetically-validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain, however functional NaV1.7 targeting antibodies are not known. In this report we describe NaV1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the NaV1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of thirteen potential cysteine conjugation site...

Research paper thumbnail of Biological characterization of a Stable Effector Functionless (SEFL) monoclonal antibody scaffold in vitro

The Journal of biological chemistry, Feb 19, 2016

The Stable Effector FunctionLess (SEFL) antibody was designed as an IgG1 antibody with a constant... more The Stable Effector FunctionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article titled "Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability" by Jacobsen et al. The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared to the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro against Daudi cells with cynomolgus monkey PBMC, and had minimal complement depende...