Rachit Shah

A highly efficient catalyst for oxime ligation and hydrazone-oxime exchange suitable for bioconjugation.

Imine-based reactions are useful for a wide range of bioconjugation applications. Although aniline is known to catalyze the oxime ligation reaction under physiological conditions, it suffers from slow reaction kinetics, specifically when a ketone is being used or when hydrazone-oxime exchange is performed. Here, we report on the discovery of a new catalyst that is up to 15 times more efficient than aniline. That catalyst, m-phenylenediamine (mPDA), was initially used to analyze the kinetics of oxime ligation on aldehyde- and ketone-containing small molecules. While mPDA is only modestly more effective than aniline when used in equal concentrations (~2-fold), its much greater aqueous solubility relative to aniline allows it to be used at higher concentrations, resulting in significantly more efficient catalysis. In the context of protein labeling, it was first used to site-specifically label an aldehyde-functionalized protein through oxime ligation, and its kinetics were compared to reaction with aniline. Next, a protein was labeled with an aldehyde-containing substrate in crude cell lysate, captured with hydrazide-functionalized beads and then the kinetics of immobilized protein release via hydrazone-oxime exchange were analyzed. Our results show that mPDA can release and label 15 times more protein than aniline can in 3 h. Then, using the new catalyst, ciliary neurotrophic factor, a protein with therapeutic potential, was successfully labeled with a fluorophore in only 5 min. Finally, a protein containing the unnatural amino acid, p-acetyl phenylalanine, a ketone-containing residue, was prepared and PEGylated efficiently via oxime ligation using mPDA. This new catalyst should have a significant impact on the field of bioconjugation, where oxime ligation and hydrazone-oxime exchange are commonly employed.

Caught before Released: Structural Mapping of the Reaction Trajectory for the Sofosbuvir Activating Enzyme, Human Histidine Triad Nucleotide Binding Protein 1 (hHint1).

Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of nucleotide antiviral pronucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES1) and product (EP2) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during the formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis may be mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHint1 catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of the Hint family of enzymes.

In vivo Evaluation of Site-specifically PEGylated Chemically Self-assembled Protein Nanostructures

Chemically self-assembled nanorings (CSANs) are made of dihydrofolate reductase (DHFR) fusion proteins and have been successfully used in vitro for cellular cargo delivery and cell surface engineering applications. However, CSANs have yet to be evaluated for their in vivo stability, circulation, and tissue distribution. In an effort to evaluate CSANs in vivo, we engineered a site-specifically PEGylated epidermal growth factor receptor (EGFR) targeting DHFR molecules, characterized their self-assembly into CSANs with bivalent methotrexates (bis-MTX), visualized their in vivo tissue localization by microPET/CT imaging, and determined their ex vivo organ biodistribution by tissue-based gamma counting. A dimeric DHFR (DHFR(2)) molecule fused with a C-terminal EGFR targeting peptide (LARLLT) was engineered to incorporate a site-specific ketone functionality using unnatural amino acid mutagenesis. Aminooxy-PEG, of differing chain lengths, was successfully conjugated to the protein using oxime chemistry. These proteins were self-assembled into CSANs with bis-MTX DHFR dimerizers and characterized by size exclusion chromatography and dynamic light scattering. In vitro binding studies were performed with fluorescent CSANs assembled using bis-MTX-FITC, while in vivo microPET/CT imaging was performed with radiolabeled CSANs assembled using bis-MTX-DOTA[(64)Cu]. PEGylation reduced the uptake of anti-EGFR CSANs by mouse macrophages (RAW 264.7) up to 40% without altering the CSANs binding affinity toward U-87 MG glioblastoma cells in vitro. A significant time dependent tumor accumulation of (64)Cu labeled anti-EGFR-CSANs was observed by microPET/CT imaging and biodistribution studies in mice bearing U-87 MG xenografts. PEGylated CSANs demonstrated a reduced uptake by the liver, kidneys, and spleen resulting in high contrast tumor imaging within an hour of intravenous injection (9.6% ID/g), and continued to increase up to 24 h (11.7% ID/g) while the background signal diminished. CSANs displayed an in vivo profile between those of rapidly clearing small molecules and slow clearing antibodies. Thus, CSANs offer a modular, programmable, and stable protein based platform that can be used for in vivo drug delivery and imaging applications.

Design, Synthesis, and Characterization of Sulfamide and Sulfamate Nucleotidomimetic Inhibitors of hHint1.

Hint1 has recently emerged to be an important target of interest due to its involvement in the regulation of a broad range of CNS functions including opioid signaling, tolerance, neuropathic pain, and nicotine dependence. A series of inhibitors were rationally designed, synthesized, and tested for their inhibitory activity against hHint1 using isothermal titration calorimetry (ITC). The studies resulted in the development of the first small-molecule inhibitors of hHint1 with submicromolar binding affinities. A combination of thermodynamic and high-resolution X-ray crystallographic studies provides an insight into the biomolecular recognition of ligands by hHint1. These novel inhibitors have potential utility as molecular probes to better understand the role and function of hHint1 in the CNS.

A Crystal Structure Based Guide to the Design of Human Histidine Triad Nucleotide Binding Protein 1 (hHint1) Activated ProTides.

Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by these results may be exploited to tune the rate of substrate hydrolysis to strategically alter the intracellular release of the product nucleoside monophosphate from the ProTide.

Inhibition by divalent metal ions of human histidine triad nucleotide binding protein1 (hHint1), a regulator of opioid analgesia and neuropathic pain

Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidase and adenylate hydrolase that has emerged as a potential therapeutic target for the management of pain. However, the molecular mechanism of Hint1 in the signaling pathway has remained less clear. The role of metal ions in regulating postsynaptic transmission is well known, and the active site of hHint1 contains multiple histidines. Here we have investigated the effect of divalent metal ions (Cd2+, Cu2+, Mg2+, Mn2+, Ni2+, and Zn2+) on the structural integrity and catalytic activity of hHint1. With the exception of Mg2+, all the divalent ions inhibited hHint1, the rank of order was found to be Cu2+ >Zn2+ >Cd2+ ≥Ni2+ >Mn2+ based on their IC50 and kin/KI values. A crystal structure of hHint1 with bound Cu2+ is described to explain the competitive reversible inactivation of hHint1 by divalent cations. All the metal ions exhibited time- and concentration- dependent inhibition, with the rate of inactivation highly dependent on alterations of the C-terminus. With the exception of Cu2+; restoration of inhibition was observed for all the metal ions after treatment with EDTA. Our studies reveal a loss in secondary structure and aggregation of hHint1 upon incubation with 10-fold excess of copper. Thus, hHint1 appears to be structurally sensitive to irreversible inactivation by copper, which may be of neurotoxicological and pharmacological significance.

A structure-based guide to building hHint-1 activated nucleotide prodrugs

The success of sofosbuvir in the treatment of chronic Hepatitis C viral infections has invigorated interest in human Histidine Triad Nucleotide Binding Protein 1 (hHint1). This enzyme hydrolyzes the sofosbuvir phosphoramidate to expose the nucleotide monophosphate, which is then further modified to the active triphosphate species. Before the hHint1-catalyzed activation step, sofosbuvir phosphoramidate is taken up orally and transported to the liver; this circumvents problems with absorption and metabolic halflife that reduce the therapeutic utility of antiviral and anticancer nucleotides. To establish a structural basis for the understanding the diversity of substrates that might be activated through this pathway, we have undertaken a systematic study of nucleotide phosphoramidate hydrolysis by hHint1. Our work includes the structural characterization of binding by natural and unnatural purine and pyrimidine nucleobases, altered ribose analogs and different amine leaving groups. Observations are correlated with enzyme catalytic efficiency and the thermodynamics of binding from ITC. A guide to the design of good and not-so-good substrates has emerged that should inform the design of future phosphoramidate-based nucleotide prodrugs.