A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

Diverse Catalytic Reactions for the Stereoselective Synthesis of Cyclic Dinucleotide MK-1454.

As practitioners of organic chemistry strive to deliver efficient syntheses of the most complex natural products and drug candidates, further innovations in synthetic strategies are required to facilitate their efficient construction. These aspirational breakthroughs often go hand-in-hand with considerable reductions in cost and environmental impact. Enzyme-catalyzed reactions have become an impressive and necessary tool that offers benefits such as increased selectivity and waste limitation. These benefits are amplified when enzymatic processes are conducted in a cascade in combination with novel bond-forming strategies. In this article, we report a highly diastereoselective synthesis of MK-1454, a potent agonist of the stimulator of interferon gene (STING) signaling pathway. The synthesis begins with the asymmetric construction of two fluoride-bearing deoxynucleotides. The routes were designed for maximum convergency and selectivity, relying on the same benign electrophilic fluorinating reagent. From these complex subunits, four enzymes are used to construct the two bridging thiophosphates in a highly selective, high yielding cascade process. Critical to the success of this reaction was a thorough understanding of the role transition metals play in bond formation.

Building blocks and blueprints for bacterial autolysins.

Bacteria utilize a wide variety of endogenous cell wall hydrolases, or autolysins, to remodel their cell walls during processes including cell division, biofilm formation, and programmed death. We here systematically investigate the composition of these enzymes in order to gain insights into their associated biological processes, potential ways to disrupt them via chemotherapeutics, and strategies by which they might be leveraged as recombinant antibacterial biotherapies. To do so, we developed LEDGOs (lytic enzyme domains grouped by organism), a pipeline to create and analyze databases of autolytic enzyme sequences, constituent domain annotations, and architectural patterns of multi-domain enzymes that integrate peptidoglycan binding and degrading functions. We applied LEDGOs to eight pathogenic bacteria, gram negatives Acinetobacter baumannii, Klebsiella pneumoniae, Neisseria gonorrhoeae, and Pseudomonas aeruginosa; and gram positives Clostridioides difficile, Enterococcus faecium, Staphylococcus aureus, and Streptococcus pneumoniae. Our analysis of the autolytic enzyme repertoires of these pathogens reveals commonalities and differences in their key domain building blocks and architectures, including correlations and preferred orders among domains in multi-domain enzymes, repetitions of homologous binding domains with potentially complementarity recognition modalities, and sequence similarity patterns indicative of potential divergence of functional specificity among related domains. We have further identified a variety of unannotated sequence regions within the lytic enzymes that may themselves contain new domains with important functions.

Deep Dive into Machine Learning Models for Protein Engineering.

Protein redesign and engineering has become an important task in pharmaceutical research and development. Recent advances in technology have enabled efficient protein redesign by mimicking natural evolutionary mutation, selection, and amplification steps in the laboratory environment. For any given protein, the number of possible mutations is astronomical. It is impractical to synthesize all sequences or even to investigate all functionally interesting variants. Recently, there has been an increased interest in using machine learning to assist protein redesign, since prediction models can be used to virtually screen a large number of novel sequences. However, many state-of-the-art machine learning models, especially deep learning models, have not been extensively explored. Moreover, only a small selection of protein sequence descriptors has been considered. In this work, the performance of prediction models built using an array of machine learning methods and protein descriptor types, including two novel, single amino acid descriptors and one structure-based three-dimensional descriptor, is benchmarked. The predictions were evaluated on a diverse collection of public and proprietary data sets, using a variety of evaluation metrics. The results of this comparison suggest that Convolution Neural Network models built with amino acid property descriptors are the most widely applicable to the types of protein redesign problems faced in the pharmaceutical industry.

Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 ß-lactamase (P99ßL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 109 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99ßL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.

Variations within class-A ß-lactamase physiochemical properties reflect evolutionary and environmental patterns, but not antibiotic specificity.

The bacterial enzyme ß-lactamase hydrolyzes the ß-lactam ring of penicillin and chemically related antibiotics, rendering them ineffective. Due to rampant antibiotic overuse, the enzyme is evolving new resistance activities at an alarming rate. Related, the enzyme's global physiochemical properties exhibit various amounts of conservation and variability across the family. To that end, we characterize the extent of property conservation within twelve different class-A ß-lactamases, and conclusively establish that the systematic variations therein parallel their evolutionary history. Large and systematic differences within electrostatic potential maps and pairwise residue-to-residue couplings are observed across the protein, which robustly reflect phylogenetic outgroups. Other properties are more conserved (such as residue pKa values, electrostatic networks, and backbone flexibility), yet they also have systematic variations that parallel the phylogeny in a statistically significant way. Similarly, the above properties also parallel the environmental condition of the bacteria they are from in a statistically significant way. However, it is interesting and surprising that the only one of the global properties (protein charge) parallels the functional specificity patterns; meaning antibiotic resistance activities are not significantly constraining the global physiochemical properties. Rather, extended spectrum activities can emerge from the background of nearly any set of electrostatic and dynamic properties.

Changes in Lysozyme Flexibility upon Mutation Are Frequent, Large and Long-Ranged.

We investigate changes in human c-type lysozyme flexibility upon mutation via a Distance Constraint Model, which gives a statistical mechanical treatment of network rigidity. Specifically, two dynamical metrics are tracked. Changes in flexibility index quantify differences within backbone flexibility, whereas changes in the cooperativity correlation quantify differences within pairwise mechanical couplings. Regardless of metric, the same general conclusions are drawn. That is, small structural perturbations introduced by single point mutations have a frequent and pronounced affect on lysozyme flexibility that can extend over long distances. Specifically, an appreciable change occurs in backbone flexibility for 48% of the residues, and a change in cooperativity occurs in 42% of residue pairs. The average distance from mutation to a site with a change in flexibility is 17-20 Å. Interestingly, the frequency and scale of the changes within single point mutant structures are generally larger than those observed in the hen egg white lysozyme (HEWL) ortholog, which shares 61% sequence identity with human lysozyme. For example, point mutations often lead to substantial flexibility increases within the ß-subdomain, which is consistent with experimental results indicating that it is the nucleation site for amyloid formation. However, ß-subdomain flexibility within the human and HEWL orthologs is more similar despite the lowered sequence identity. These results suggest compensating mutations in HEWL reestablish desired properties.

Using enzymes to tame nitrogen-centred radicals for enantioselective hydroamination.

The formation of C-N bonds-of great importance to the pharmaceutical industry-can be facilitated enzymatically using nucleophilic and nitrene transfer mechanisms. However, neither natural nor engineered enzymes are known to generate and control nitrogen-centred radicals, which serve as valuable species for C-N bond formation. Here we use flavin-dependent 'ene'-reductases with an exogenous photoredox catalyst to selectively generate amidyl radicals within the protein active site. These enzymes are engineered through directed evolution to catalyse 5-exo, 6-endo, 7-endo, 8-endo, and intermolecular hydroamination reactions with high levels of enantioselectivity. Mechanistic studies suggest that radical initiation occurs via an enzyme-gated mechanism, where the protein thermodynamically activates the substrate for reduction by the photocatalyst. Molecular dynamics studies indicate that the enzymes bind substrates using non-canonical binding interactions, which may serve as a handle to further manipulate reactivity. This approach demonstrates the versatility of these enzymes for controlling the reactivity of high-energy radical intermediates and highlights the opportunity for synergistic catalyst strategies to unlock previously inaccessible enzymatic functions.

Functional Deimmunization of Botulinum Neurotoxin Protease Domain via Computationally Driven Library Design and Ultrahigh-Throughput Screening.

Botulinum neurotoxin serotype A (BoNT/A) is a widely used cosmetic agent that also has diverse therapeutic applications; however, adverse antidrug immune responses and associated loss of efficacy have been reported in clinical uses. Here, we describe computational design and ultrahigh-throughput screening of a massive BoNT/A light-chain (BoNT/A-LC) library optimized for reduced T cell epitope content and thereby dampened immunogenicity. We developed a functional assay based on bacterial co-expression of BoNT/A-LC library members with a Förster resonance energy transfer (FRET) sensor for BoNT/A-LC enzymatic activity, and we employed high-speed fluorescence-activated cell sorting (FACS) to identify numerous computationally designed variants having wild-type-like enzyme kinetics. Many of these variants exhibited decreased immunogenicity in humanized HLA transgenic mice and manifested in vivo paralytic activity when incorporated into full-length toxin. One variant achieved near-wild-type paralytic potency and a 300% reduction in antidrug antibody response in vivo. Thus, we have achieved a striking level of BoNT/A-LC functional deimmunization by combining computational library design and ultrahigh-throughput screening. This strategy holds promise for deimmunizing other biologics with complex superstructures and mechanisms of action.

Similarity based enzymatic retrosynthesis.

Enzymes synthesize complex natural products effortlessly by catalyzing chemo-, regio-, and enantio-selective transformations. Further, biocatalytic processes are increasingly replacing conventional organic synthesis steps because they use mild solvents, avoid the use of metals, and reduce overall non-biodegradable waste. Here, we present a single-step retrosynthesis search algorithm to facilitate enzymatic synthesis of natural product analogs. First, we develop a tool, RDEnzyme, capable of extracting and applying stereochemically consistent enzymatic reaction templates, i.e., subgraph patterns that describe the changes in connectivity between a product molecule and its corresponding reactant(s). Using RDEnzyme, we demonstrate that molecular similarity is an effective metric to propose retrosynthetic disconnections based on analogy to precedent enzymatic reactions in UniProt/RHEA. Using ∼5500 reactions from RHEA as a knowledge base, the recorded reactants to the product are among the top 10 proposed suggestions in 71% of ∼700 test reactions. Second, we trained a statistical model capable of discriminating between reaction pairs belonging to homologous enzymes and evolutionarily distant enzymes using ∼30 000 reaction pairs from SwissProt as a knowledge base. This model is capable of understanding patterns in enzyme promiscuity to evaluate the likelihood of experimental evolution success. By recursively applying the similarity-based single-step retrosynthesis and evolution prediction workflow, we successfully plan the enzymatic synthesis routes for both active pharmaceutical ingredients (e.g. Islatravir, Molnupiravir) and commodity chemicals (e.g. 1,4-butanediol, branched-chain higher alcohols/biofuels), in a retrospective fashion. Through the development and demonstration of the single-step enzymatic retrosynthesis strategy using natural transformations, our approach provides a first step towards solving the challenging problem of incorporating both enzyme- and organic-chemistry based transformations into a computer aided synthesis planning workflow.

Successive remodeling of IgG glycans using a solid-phase enzymatic platform.

The success of glycoprotein-based drugs in various disease treatments has become widespread. Frequently, therapeutic glycoproteins exhibit a heterogeneous array of glycans that are intended to mimic human glycopatterns. While immunogenic responses to biologic drugs are uncommon, enabling exquisite control of glycosylation with minimized microheterogeneity would improve their safety, efficacy and bioavailability. Therefore, close attention has been drawn to the development of glycoengineering strategies to control the glycan structures. With the accumulation of knowledge about the glycan biosynthesis enzymes, enzymatic glycan remodeling provides a potential strategy to construct highly ordered glycans with improved efficiency and biocompatibility. In this study, we quantitatively evaluate more than 30 enzymes for glycoengineering immobilized immunoglobulin G, an impactful glycoprotein class in the pharmaceutical field. We demonstrate successive glycan remodeling in a solid-phase platform, which enabled IgG glycan harmonization into a series of complex-type N-glycoforms with high yield and efficiency while retaining native IgG binding affinity.

Engineered Ribosyl-1-Kinase Enables Concise Synthesis of Molnupiravir, an Antiviral for COVID-19.

Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compound, it was critical to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manufacture and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approximately 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general.

Enantioselective Enzymatic Reduction of Acrylic Acids.

An ene-reductase (ERED 36) with broad substrate specificity was identified, and optimization studies led to the development of an enzymatic protocol for the reduction of α,ß-unsaturated acids under mild, aqueous conditions. The substrate scope includes aromatic- and aliphatic-substituted acrylic acids, as well as cyclic α,ß-substituted acrylic acids, yielding chiral α-substituted acids with exquisite levels of enantioselectivity (>99% ee).

Globally deimmunized lysostaphin evades human immune surveillance and enables highly efficacious repeat dosing.

There is a critical need for novel therapies to treat methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant pathogens, and lysins are among the vanguard of innovative antibiotics under development. Unfortunately, lysins' own microbial origins can elicit detrimental antidrug antibodies (ADAs) that undermine efficacy and threaten patient safety. To create an enhanced anti-MRSA lysin, a novel variant of lysostaphin was engineered by T cell epitope deletion. This "deimmunized" lysostaphin dampened human T cell activation, mitigated ADA responses in human HLA transgenic mice, and enabled safe and efficacious repeated dosing during a 6-week longitudinal infection study. Furthermore, the deimmunized lysostaphin evaded established anti-wild-type immunity, thereby providing significant anti-MRSA protection for animals that were immune experienced to the wild-type enzyme. Last, the enzyme synergized with daptomycin to clear a stringent model of MRSA endocarditis. By mitigating T cell-driven antidrug immunity, deimmunized lysostaphin may enable safe, repeated dosing to treat refractory MRSA infections.

Pareto Optimization of Combinatorial Mutagenesis Libraries.

In order to increase the hit rate of discovering diverse, beneficial protein variants via high-throughput screening, we have developed a computational method to optimize combinatorial mutagenesis libraries for overall enrichment in two distinct properties of interest. Given scoring functions for evaluating individual variants, POCoM (Pareto Optimal Combinatorial Mutagenesis) scores entire libraries in terms of averages over their constituent members, and designs optimal libraries as sets of mutations whose combinations make the best trade-offs between average scores. This represents the first general-purpose method to directly design combinatorial libraries for multiple objectives characterizing their constituent members. Despite being rigorous in mapping out the Pareto frontier, it is also very fast even for very large libraries (e.g., designing 30 mutation, billion-member libraries in only hours). We here instantiate POCoM with scores based on a target's protein structure and its homologs' sequences, enabling the design of libraries containing variants balancing these two important yet quite different types of information. We demonstrate POCoM's generality and power in case study applications to green fluorescent protein, cytochrome P450, and ß-lactamase. Analysis of the POCoM library designs provides insights into the trade-offs between structure- and sequence-based scores, as well as the impacts of experimental constraints on library designs. POCoM libraries incorporate mutations that have previously been found favorable experimentally, while diversifying the contexts in which these mutations are situated and maintaining overall variant quality.

Characterization of potent RSV neutralizing antibodies isolated from human memory B cells and identification of diverse RSV/hMPV cross-neutralizing epitopes.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children and older adults. Currently, no licensed vaccine is available, and therapeutic options are limited. The primary target of neutralizing antibodies to RSV is the surface fusion (F) glycoprotein. Understanding the recognition of antibodies with high neutralization potencies to RSV F antigen will provide critical insights in developing efficacious RSV antibodies and vaccines. In this study, we isolated and characterized a panel of monoclonal antibodies (mAbs) with high binding affinity to RSV prefusion F trimer and neutralization potency to RSV viruses. The mAbs were mapped to previously defined antigenic sites, and some that mapped to the same antigenic sites showed remarkable diversity in specificity, binding, and neutralization potencies. We found that the isolated site III mAbs shared highly conserved germline V-gene usage, but had different cross-reactivities to human metapneumovirus (hMPV), possibly due to the distinct modes/angles of interaction with RSV and hMPV F proteins. Furthermore, we identified a subset of potent RSV/hMPV cross-neutralizing mAbs that target antigenic site IV and the recently defined antigenic site V, while the majority of the mAbs targeting these two sites only neutralize RSV. Additionally, the isolated mAbs targeting site Ø were mono-specific for RSV and showed a wide range of neutralizing potencies on different RSV subtypes. Our data exemplify the diversity of anti-RSV mAbs and provide new insights into the immune recognition of respiratory viruses in the Pneumoviridae family.

Design of an in vitro biocatalytic cascade for the manufacture of islatravir.

Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.

Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen.

Structure-based approaches to antigen design utilize insights from antibody (Ab):antigen interactions and a refined understanding of protective Ab responses to engineer novel antigens presenting epitopes with conformations relevant to eliciting or discovering protective humoral responses. For human immunodeficiency virus-1 (HIV-1), one model of protection is provided by broadly neutralizing Abs (bnAbs) against epitopes present in the closed prefusion trimeric conformation of HIV-1 envelope glycoprotein, such as the variable loops 1-2 (V1V2) apex. Here, computational design and directed evolution yielded a novel V1V2 sequence variant with potential utility for inclusion in an immunogen for eliciting bnAbs, or as an epitope probe for their detection. The computational design goal was to engineer a minimal single-chain antigen with three copies of the V1V2 loops to support maintenance of closed prefusion V1V2 trimeric conformation and presentation of bnAb epitopes. Via directed evolution of this computationally designed single-chain antigen, we isolated a V1V2 sequence variant that in monomeric form exhibited preferential recognition by quaternary-preferring and conformation-dependent mAbs. Structural context and transferability of this phenotype to V1V2 sequences from all strains of HIV-1 tested suggest a conformation-stabilizing effect. This example demonstrates the potential utility of computational design and directed evolution-based protein engineering strategies to develop minimal, conformation-stabilized epitope-specific antigens.

Docking mode of delvardine and its analogues into the p66 domain of HIV-1 reverse transcriptase: screening using molecular mechanics-generalized born/surface area and absorption, distribution, metabolism and excretion properties.

Delvardine and its structural derivatives are important non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs). In this work,15 delvardine analogues were studied. A free energy-of-binding (FEB)expression was developed in the form of an optimized linear combination of van der Waal (vdW), electrostatic, solvation and solvent-accessible surface area (SASA) energy terms. The solvation energy terms estimated by generalized born/surface area (GB/SA) play an important role in predicting the binding affinity of delvardine analogues. Out of 15 derivatives, substitution of CH3 with H at the Y and R positions, as well as substitution of SO2CH3 with only CH2 at the Z position in S2, S8 and S12 analogues, were found to be the most potent (glide score = -7.60, -8.06 and -7.44; pIC50 =7.28, 7.37 and 7.64) in comparison with the template delvardine (which is used currently as the drug candidate). All the three analogues also passed the absorption, distribution,metabolism and excretion (ADME) screening and Lipinski's rule of 5, and have the potential to be used for second-generation drug development. The work demonstrates that dock molecular mechanics-generalized born/surface area (MM-GB/SA-ADME) is a promising approach to predict the binding activity of ligands to the receptor and further screen for a successful candidate drug in a computer-aided rational drug design.

EpiSweep: Computationally Driven Reengineering of Therapeutic Proteins to Reduce Immunogenicity While Maintaining Function.

Therapeutic proteins are yielding ever more advanced and efficacious new drugs, but the biological origins of these highly effective therapeutics render them subject to immune surveillance within the patient's body. When recognized by the immune system as a foreign agent, protein drugs elicit a coordinated response that can manifest a range of clinical complications including rapid drug clearance, loss of functionality and efficacy, delayed infusion-like allergic reactions, more serious anaphylactic shock, and even induced auto-immunity. It is thus often necessary to deimmunize an exogenous protein in order to enable its clinical application; critically, the deimmunization process must also maintain the desired therapeutic activity.To meet the growing need for effective, efficient, and broadly applicable protein deimmunization technologies, we have developed the EpiSweep suite of protein design algorithms. EpiSweep seamlessly integrates computational prediction of immunogenic T cell epitopes with sequence- or structure-based assessment of the impacts of mutations on protein stability and function, in order to select combinations of mutations that make Pareto optimal trade-offs between the competing goals of low immunogenicity and high-level function. The methods are applicable both to the design of individual functionally deimmunized variants as well as the design of combinatorial libraries enriched in functionally deimmunized variants. After validating EpiSweep in a series of retrospective case studies providing comparisons to conventional approaches to T cell epitope deletion, we have experimentally demonstrated it to be highly effective in prospective application to deimmunization of a number of different therapeutic candidates. We conclude that our broadly applicable computational protein design algorithms guide the engineer towards the most promising deimmunized therapeutic candidates, and thereby have the potential to accelerate development of new protein drugs by shortening time frames and improving hit rates.