Enzymatic Routes for Chiral Amine Synthesis: Protein Engineering and Process Optimization.

Chiral amines are essential motifs in pharmaceuticals, agrochemicals, and specialty chemicals. While traditional chemical routes to chiral amines often lack stereoselectivity and require harsh conditions, biocatalytic methods using engineered enzymes can offer high efficiency and selectivity under sustainable conditions. This review discusses recent advances in protein engineering of transaminases, oxidases, and other enzymes to improve catalytic performance. Strategies such as directed evolution, immobilization, and computational redesign have expanded substrate scope and enhanced efficiency. Furthermore, process optimization guided by techno-economic assessments has been crucial for establishing viable biomanufacturing routes. Combining state-of-the-art enzyme engineering with multifaceted process development will enable scalable, economical enzymatic synthesis of diverse chiral amine targets.

De Novo Designed Heterochiral Blue Fluorescent Protein.

Diversification of chain stereochemistry offers a tremendous increase in protein design space. We have designed a minimal fluorescent protein, pregnant with ß-(1-azulenyl)-l-alanine in the hydrophobic core of a heterotactic protein scaffold, employing automated design tools such as automated repetitive simulated annealing molecular dynamics and IDeAS. The de novo designed heterochiral protein can be selectively excited at 342 nm, quite distant from the intrinsic fluorophore, and emits in the blue region. The structure and stability of the designed proteins were evaluated by established spectroscopic and calorimetric methods.

Structural and functional comparability study of anti-CD20 monoclonal antibody with reference product.

BACKGROUND: Cell surface protein, CD20, is extensively expressed on the surface of B cells. Antibodies targeting CD20 protein are being used to treat B-cell malignancies and B-cell mediated autoimmune diseases. Considering the cost of therapy with innovator monoclonal antibodies for these diseases, development of biosimilar products for the treatment of such diseases provides affordable solution to rising healthcare costs. MATERIALS AND METHODS: Reference products of rituximab (six batches) were procured and stored as per manufacturer's instructions. Cell lines used in bioassay were procured from American Type Culture Collection and all other reagents used for analysis were of analytical grade. Primary structure was studied by intact mass analysis, peptide fingerprinting, peptide mass fingerprinting and sequence coverage analysis. Higher order structure was studied by circular dichroism, ultraviolet-visible spectroscopy, fluorescence spectroscopy, and disulfide bridge analysis. Different isoforms of reference product and SB-02 were identified using capillary isoelectric focusing and capillary zone electrophoresis. Glycosylation was studied by N-glycan mapping using LC-ESI-MS, point of glycosylation, released glycan analysis using ultra performance liquid chromatography (UPLC). Product related impurities such as oligomer content analysis and oxidized impurities were studied using size exclusion chromatography and reverse phase high performance liquid chromatography, respectively. RESULTS AND CONCLUSION: Here, we report physicochemical and biological characterizations of Sun Pharma's proposed biosimilar (SB-02) to rituximab, a monoclonal anti-CD20 antibody approved for the treatment of non-Hodgkin's lymphoma and chronic lymphocytic leukemia. SB-02 and rituximab exhibited indistinguishable primary as well as higher-order structure upon analyzing with the array of analytical and extended characterization methods according to statistical methods. The molecule also displayed comparability to reference product in post-translational modifications and charge heterogeneity. In functional bioassays, SB-02 demonstrated comparable potency with respect to reference product. Our results indicate highly similar quality profile between SB-02 and rituximab.

Glycan mapping of recombinant human follicle stimulating hormone by mass spectrometry.

In humans, regulation of reproductive functions are carried out mainly by glycoprotein hormones namely follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH) and chorionic gonadotropin (CG). Since glycans play an important role in binding of gonadotropins with their respective receptors, it is important to identify associated glycans and their pharmacological properties not only for the disease manipulation but also for making more efficacious and safer recombinant versions. With the advancement of mass spectrometry, it is possible to identify minute quantity of associated glycans. Here, we studied the N-glycans of the FSH based on mass spectrometry and report one more complex glycan species in addition to twenty four previously reported glycans. The new glycan was a tetra antennary species that may have important role in binding of FSH with receptor with higher biological activity as well as lower clearance rate and higher half-life.

IDeAS: automated design tool for hetero-chiral protein folds.

Incorporating D amino acids in the protein design alphabet can in principle multiply the design space by many orders of magnitude. All native proteins are polymers composed of L chiral amino acids. Practically limitless in diversity over amino acid sequences, protein structure is limited in folds and thus shapes, principally due to the poly L stereochemistry of their backbone. To diversify shapes, we introduced both L- and D α-amino acids as design alphabets to explore the possibility of generating novel folds, varied in chemical as well as stereo-chemical sequence. Now, to have stereochemically-defined proteins tuned chemically, we present the Inverse Design and Automation Software, IDeAS. Retro-fitting side chains on a backbone with L and D stereochemistry, the software demonstrate functional fits over stereo-chemically diverse folds in a range of applications of interest in protein design.

Analytical characterization of recombinant hCG and comparative studies with reference product.

INTRODUCTION: Regulatory agencies recommend a stepwise approach for demonstrating biosimilarity between a proposed biosimilar and reference biological product emphasizing for functional and structural characterization to trace if there is any difference which may impact safety and efficacy. We studied the comparative structural and biological attributes of recombinant human chorionic gonadotropin (rhCG), SB005, with reference product, Ovidrel® and Ovitrelle®. Recombiant hCG was approved in 2000 by the US Food and Drug Administration for the induction of final follicular maturation, early luteinization in infertile women as part of assisted reproductive technology program. It is also indicated for the induction of ovulation and pregnancy in ovulatory infertile patients whose cause of infertility is not due to ovarian failure. MATERIALS AND METHODS: Primary structure was studied by intact mass analysis, peptide fingerprinting, peptide mass fingerprinting and sequence coverage analysis. Higher order structure was studied by circular dichroism, ultraviolet-visible spectroscopy, fluorescence spectroscopy, and disulfide bridge analysis. Different isoforms of reference product and SB005 were identified using capillary isoelectric focusing and capillary zone electrophoresis. Glycosylation was studied by N-glycan mapping using LC-ESI-MS, point of glycosylation, released glycan analysis using ultra performance liquid chromatography and sialic acid analysis. Product related impurities such as oligomer content analysis and oxidized impurities were studied using size exclusion chromatography and reverse phase high performance liquid chromatography, respectively. Biological activity in term of potency of reference product and SB005 was studied by in vivo analysis. RESULTS AND CONCLUSION: In this study we have compared analytical similarity of recombinant rhCG (SB005) produced at Sun Pharmaceuticals with the reference product with respect to its primary, higher order structure, isoforms, charge variants, glycosylation, sialyation pattern, pharmacodynamic and in vivo efficacy. Our studies show that the in house produced rhCG has a high degree of structural and functional similarity with the reference product available in the market.

Automated design evolution of stereochemically randomized protein foldamers.

Diversification of chain stereochemistry opens up the possibilities of an 'in principle' increase in the design space of proteins. This huge increase in the sequence and consequent structural variation is aimed at the generation of smart materials. To diversify protein structure stereochemically, we introduced L- and D-α-amino acids as the design alphabet. With a sequence design algorithm, we explored the usage of specific variables such as chirality and the sequence of this alphabet in independent steps. With molecular dynamics, we folded stereochemically diverse homopolypeptides and evaluated their 'fitness' for possible design as protein-like foldamers. We propose a fitness function to prune the most optimal fold among 1000 structures simulated with an automated repetitive simulated annealing molecular dynamics (AR-SAMD) approach. The highly scored poly-leucine fold with sequence lengths of 24 and 30 amino acids were later sequence-optimized using a Dead End Elimination cum Monte Carlo based optimization tool. This paper demonstrates a novel approach for the de novo design of protein-like foldamers.

Automated protein design: Landmarks and operational principles.

Protein design has an eventful history spanning over three decades, with handful of success stories reported, and numerous failures not reported. Design practices have benefited tremendously from improvements in computer hardware and advances in scientific algorithms. Though protein folding problem still remains unsolved, the possibility of having multiple sequence solutions for a single fold makes protein design a more tractable problem than protein folding. One of the most significant advancement in this area is the implementation of automated design algorithms on pre-defined templates or completely new folds, optimized through deterministic and heuristic search algorithms. This progress report provides a succinct presentation of important landmarks in automated design attempts, followed by brief account of operational principles in automated design methods.

Synthesis and pharmacological evaluation of [(4-arylpiperazin-1-yl)-alkyl]-carbamic acid ethyl ester derivatives as potential anxiolytic agents.

On the basis of our earlier studies, a series of N-{4-[4-(aryl) piperazin-1-yl]-phenyl}-amine derivatives containing terminal carbamoyl fragment with alkyl spacer of different lengths (15-20) were synthesized as ligands, for 5-hydroxytryptamine-1A (5-HT(1A)) receptor. Molecular modeling studies were undertaken to explain the influence of spacer length on ligands affinity towards 5-HT(1A) receptor. Compound 19 showed all the specific interactions responsible for recognition. The protonated amine of the ligand forms an ionic hydrogen bond with the negatively charged Asp116 of transmembrane3 helix (TM3), while the carbamoyl moiety interacts with Asn386 and Tyr390 of TM7. The aryl group is involved in forming a CH-π interaction with Phe362. The strong interaction of compound 19 with 5-HT(1A) receptor in docking studies was confirmed by radio ligand binding studies. Compound 19 showed high affinity for the receptor (Ki = 0.018 nM). In vivo pharmacological testing of compound 19 (3 mg/kg body weight) showed increased open arm entries, as well as time spent in Elevated plus Maze test. Toxicological analysis also revealed no significant biochemical or morphological alterations in the vital organs of experimental animals. Furthermore our results suggest that these compounds share some pharmacological effects with established anxiolytics and might prove to be effective compounds for the treatment of anxiety.

Homochiral stereochemistry: the missing link of structure to energetics in protein folding.

The notion is tested that homochiral stereochemistry being ubiquitous to protein structure could be critical to protein folding as well, causing it to become frustrated energetically providing the basis for its solvent- and sequence-mediated control. The proof in support of the notion is found in a consensus of experiment and computation according to which suitable oligopeptides are in their folding-unfolding equilibria, at both macrostate and microstate levels, susceptible to dielectric because of the conflict of peptide-chain electrostatics with interpeptide hydrogen bonds when the structure is poly-L but not when it is alternating-L,D. The argument is thus made that homochiral stereochemistry may in protein folding provide the unifying basis for its solvent- and sequence-mediated control based on screening of peptide-chain electrostatics under conflict with folding of the chain due to homochiral stereochemistry. Dielectric is brought into spotlight as the effect comparatively obscure but presumably critical to the folding in protein structure for its control.

The interplay of sequence and stereochemistry in defining conformation in proteins and polypeptides.

Sequential specification of conformation in proteins and polypeptides is a triangular interplay involving the system of linked peptides, the sequences in side chains, and water as solvent. Stereochemistry in side chain linkages is obviously important in the interaction between all of the players, but no specification of its explicit role, if any, in linking sequence with conformation has been made. Flory and coworkers made a puzzling observation in 1967 that, when mutated from poly-L to alternating-L,D stereochemical structure, polypeptides will suffer a reduction in overall dimension or characteristic ratio by an astonishing factor of 10 and to a value even lower than that predicted for free rotation (Miller, W. G.; Brant, D. A.; Flory, P. J. J Mol Biol 1967, 23, 67-80). Enquiring into this longstanding puzzle, Durani and coworkers found that the stereochemical modification will also abolish conformational sensitivity in polypeptide structure to solvent, because electrostatic interactions in the system of linked peptides are transformed from a condition of mutual conflict to one of harmony (Ramakrishnan, V.; Ranbhor, R.; Kumar, A.; Durani, S. J Phys Chem B 2006, 110, 9314-9323). Thus, poly-L stereochemistry could be the fulcrum linking sequences with phi,psis in protein and polypeptide structures, via dielectric arbitrations in a conflicting type of interpeptide electrostatics, in agreement with the electrostatic screening model of Avbelj and Moult (Avbelj, F.; Moult, J. Biochemistry 1995, 34, 755-764).

The link between sequence and conformation in protein structures appears to be stereochemically established.

In search of the link between sequence and conformation in protein structures, we perform molecular dynamics analysis of the effect of stereochemical mutation in end-protected octa-alanine Ac-Ala8-NHMe from poly-L to an alternating-L,D structure. The mutation has a dramatic effect, transforming the peptide from a condition of extreme sensitivity to one of extreme insensitivity to solvent. Examining the molecular folds of poly-L and alternating-L,D structure in atomistic detail, we find them to differ in the relationship between peptide dipolar interactions at the local and nonlocal levels, either conflicting or harmonious depending upon the chain stereochemistry. The stereochemical transformation of interpeptide electrostatics from a condition of conflict to one of harmony explains the long-standing puzzle of why poly-L and alternating-L,D peptides strongly differ in properties such as "stiffness" and solvent sensitivity. Furthermore, it is possible that poly-L stereochemistry is also the fulcrum of protein sensitivity to the effects of amino acid side-chain structures via dielectric arbitrations in interpeptide electrostatics. Indeed the evidence is accumulating that the amino acid side chains differing in alpha-helix and beta-sheet propensities also differ in their desolvating effects in the adjacent and nearest-neighbor peptides and thus possibly in the solvent screening of peptide dipolar interactions.

Simulated folding in polypeptides of diversified molecular tacticity: implications for protein folding and de novo design.

Stereochemistry could be a powerful variable for conformational tune up of polypeptides for de novo design. It may be also useful probe of possible role of interamide energetics in selection and stabilization of conformation. The homopolypeptides Ac-Xxx30-NHMe, with Xxx = Ala, Val, and Leu, of diversified stereochemical structure are generated by simulated racemization with a modified GROMOS-96 force field. The polypeptides, and other systematic stereochemical variants, are folded by simulated annealing with another modified GROMOS-96 force field under the dielectric constant values 1, 4, and 10. The resultant 15,000 molecular folds of isotactic (poly-L-chiral), syndiotactic (alternating L,D-chiral), and heterotactic (random-L,D-chiral) stereochemical structure, belonging to three polypeptide series, achieved under three different folding conditions, are assessed statistically for structure-to-energy-to-conformation relationship. The results suggest that interamide electrostatics could be a major factor in secondary-structure selection in polypeptides while main-chain stereochemistry could dictate molecular packing and therefore the relative magnitude of hydrogen-bond and Lennard-Jones (LJ) contributions in conformational energy. A method for computational design of heterotactic molecular folds in polypeptide structure has been developed, and the first road map for a chiral tune up of polypeptide structure based on stereochemical engineering has been laid down. Broad implications for protein structure, folding, and de novo design are briefly discussed.

Existence of specific "folds" in polyproline II ensembles of an "unfolded"alanine peptide detected by molecular dynamics.

Equilibrium ensembles of octaalanine (Ac-Ala8-NHMe) in water, prepared with MD, are analyzed for contributing microstates with an RMSD-based conformational clustering algorithm. The extracted ensemble-averaged properties are in excellent agreement with numerous spectroscopic measurements reported with small alanine model peptides in water. However, the dominantly polyproline II-like ensemble of the peptide is found to be populated with a handful of highly position-specific "folds", including beta-turns, beta-hairpins, and helix nuclei, which could be the "seeds" that initiate proteins along their folding pathways.