Tina Boville

The standard proteinogenic amino acids grant access to a myriad of chemistries that harmonize to create life. Outside of these twenty canonical protein building blocks are countless noncanonical amino acids (ncAAs), either found in nature or created by man. Interest in ncAAs has grown as research has unveiled their importance as precursors to natural products and pharmaceuticals, biological probes, and more. Despite their broad applications, synthesis of ncAAs remains a challenge, as poor… Show more

The standard proteinogenic amino acids grant access to a myriad of chemistries that harmonize to create life. Outside of these twenty canonical protein building blocks are countless noncanonical amino acids (ncAAs), either found in nature or created by man. Interest in ncAAs has grown as research has unveiled their importance as precursors to natural products and pharmaceuticals, biological probes, and more. Despite their broad applications, synthesis of ncAAs remains a challenge, as poor stereoselectivity and low functional-group compatibility stymie effective preparative routes. The use of enzymes has emerged as a versatile approach to prepare ncAAs, and nature's enzymes can be engineered to synthesize ncAAs more efficiently and expand the amino acid alphabet. In this tutorial review, we briefly outline different enzyme engineering strategies and then discuss examples where engineering has generated new ‘ncAA synthases’ for efficient, environmentally benign production of a wide and growing collection of valuable ncAAs. Show less

Non‐canonical amino acids (ncAAs) with dual stereocenters at the α and β positions are valuable precursors to natural products and therapeutics. Despite the potential applications of such bioactive β‐branched ncAAs, their availability is limited due to the inefficiency of the multi‐step methods used to prepare them. Here we report a stereoselective biocatalytic synthesis of β‐branched tryptophan analogs using an engineered variant of Pyrococcus furiosus tryptophan synthase (PfTrpB), PfTrpB7E6… Show more

Non‐canonical amino acids (ncAAs) with dual stereocenters at the α and β positions are valuable precursors to natural products and therapeutics. Despite the potential applications of such bioactive β‐branched ncAAs, their availability is limited due to the inefficiency of the multi‐step methods used to prepare them. Here we report a stereoselective biocatalytic synthesis of β‐branched tryptophan analogs using an engineered variant of Pyrococcus furiosus tryptophan synthase (PfTrpB), PfTrpB7E6. PfTrpB7E6 is the first biocatalyst to synthesize bulky β‐branched tryptophan analogs in a single step, with demonstrated access to 27 ncAAs. The molecular basis for the efficient catalysis and broad substrate tolerance of PfTrpB7E6 was explored through X‐ray crystallography and UV‐visible light spectroscopy, which revealed that a combination of active‐site and remote mutations increase the abundance and persistence of a key reactive intermediate. PfTrpB7E6 provides an operationally simple and environmentally benign platform for preparation of β‐branched tryptophan building blocks. Show less

The use of enzymes has become increasingly widespread in synthesis as chemists strive to reduce their reliance on organic solvents in favor of more environmentally benign aqueous media. With this in mind, we previously endeavored to engineer the tryptophan synthase β-subunit (TrpB) for production of noncanonical amino acids that had previously been synthesized through multistep routes involving water-sensitive reagents. This enzymatic platform proved effective for the synthesis of analogues of… Show more

The use of enzymes has become increasingly widespread in synthesis as chemists strive to reduce their reliance on organic solvents in favor of more environmentally benign aqueous media. With this in mind, we previously endeavored to engineer the tryptophan synthase β-subunit (TrpB) for production of noncanonical amino acids that had previously been synthesized through multistep routes involving water-sensitive reagents. This enzymatic platform proved effective for the synthesis of analogues of the amino acid tryptophan (Trp), which are frequently used in pharmaceutical synthesis as well as chemical biology. However, certain valuable compounds, such as the blue fluorescent amino acid 4-cyanotryptophan (4-CN-Trp), could only be made in low yield, even at elevated temperature (75 °C). Here, we describe the engineering of TrpB from Thermotoga maritima that improved synthesis of 4-CN-Trp from 24% to 78% yield. Remarkably, although the final enzyme maintains high thermostability (T50 = 93 °C), its temperature profile is shifted such that high reactivity is observed at ∼37 °C (76% yield), creating the possibility for in vivo 4-CN-Trp production. The improvements are not specific to 4-CN-Trp; a boost in activity at lower temperature is also demonstrated for other Trp analogues. Show less

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs in the world. While the role of NSAIDs as cyclooxygenase (COX) inhibitors is well established, other targets may contribute to anti-inflammation. Here we report caspases as a new pharmacological target for NSAID family drugs such as ibuprofen, naproxen, and ketorolac at physiologic concentrations both in vitro and in vivo. We characterize caspase activity in both in vitro and in cell culture, and combine… Show more

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs in the world. While the role of NSAIDs as cyclooxygenase (COX) inhibitors is well established, other targets may contribute to anti-inflammation. Here we report caspases as a new pharmacological target for NSAID family drugs such as ibuprofen, naproxen, and ketorolac at physiologic concentrations both in vitro and in vivo. We characterize caspase activity in both in vitro and in cell culture, and combine computational modeling and biophysical analysis to determine the mechanism of action. We observe that inhibition of caspase catalysis reduces cell death and the generation of pro-inflammatory cytokines. Further, NSAID inhibition of caspases is COX independent, representing a new anti-inflammatory mechanism. This finding expands upon existing NSAID anti-inflammatory behaviors, with implications for patient safety and next-generation drug design. Show less

Caspases are cellular executors, initiating cell death. In a recent study, Shi et al. report that caspases 4/5/11 are cytosolic LPS receptors, becoming activated through oligomerization upon interaction with LPS. These findings shed new light on the mechanisms underlying caspase-mediated pyroptosis, and have implications for the development of effective drugs to treat sepsis.

The last two decades have seen the rise of antimicrobial peptides (AMPs) to combat emerging antibiotic resistance. Herein we report the solid phase synthesis of short lipidated α/γ-AA hybrid peptides. This family of lipo-chimeric peptidomimetics displays potent and broad-spectrum antimicrobial activity against a range of multi-drug resistant Gram-positive bacteria and Gram-negative bacteria. These lipo-α/γ-AA hybrid peptides also demonstrate high biological specificity, with no hemolytic… Show more

The last two decades have seen the rise of antimicrobial peptides (AMPs) to combat emerging antibiotic resistance. Herein we report the solid phase synthesis of short lipidated α/γ-AA hybrid peptides. This family of lipo-chimeric peptidomimetics displays potent and broad-spectrum antimicrobial activity against a range of multi-drug resistant Gram-positive bacteria and Gram-negative bacteria. These lipo-α/γ-AA hybrid peptides also demonstrate high biological specificity, with no hemolytic activity towards red blood cells. Fluorescence microscopy suggests that these lipo-α/γ-AA chimeric peptides can mimic the mode of action of AMPs and kill bacterial pathogens via membrane disintegration. As the composition of these chimeric peptides is simple, therapeutic development may be economically feasible, and amenable for a variety of antimicrobial applications. Show less

A series of novel, saccharin-based antagonists have been identified for the interferon signaling pathway. Through in vitro high-throughput screening with the Colorado Center for Drug Discovery (C2D2) Pilot Library, we identified hit compound 1, which was the basis for extensive structure–activity relationship studies. Our efforts produced a lead anti-inflammatory compound, tert-butyl N-(furan-2-ylmethyl)-N-{4-[(1,1,3-trioxo-2,3-dihydro-1λ6,2-benzothiazol-2-yl)methyl]benzoyl}carbamate CU-CPD103… Show more

A series of novel, saccharin-based antagonists have been identified for the interferon signaling pathway. Through in vitro high-throughput screening with the Colorado Center for Drug Discovery (C2D2) Pilot Library, we identified hit compound 1, which was the basis for extensive structure–activity relationship studies. Our efforts produced a lead anti-inflammatory compound, tert-butyl N-(furan-2-ylmethyl)-N-{4-[(1,1,3-trioxo-2,3-dihydro-1λ6,2-benzothiazol-2-yl)methyl]benzoyl}carbamate CU-CPD103 (103), as a potent inhibitor using an established nitric oxide (NO) signaling assay. With further studies of its inhibitory mechanisms, we demonstrated that 103 carries out this inhibition through the JAK/STAT1 pathway, providing a drug-like small molecule inflammation suppressant for possible therapeutic uses. Show less

Herein we describe the development of a new class of antimicrobial and anti-infective peptidomimetics – cyclic lipo-α-AApeptides. They have potent and broad-spectrum antibacterial activity against a range of clinically relevant pathogens, including both multidrug-resistant Gram-positive and Gram-negative bacteria. Fluorescence microscopy suggests that cyclic lipo-α-AApeptides kill bacteria by disrupting bacterial membranes, possibly through a mechanism similar to that of cationic host defense… Show more

Herein we describe the development of a new class of antimicrobial and anti-infective peptidomimetics – cyclic lipo-α-AApeptides. They have potent and broad-spectrum antibacterial activity against a range of clinically relevant pathogens, including both multidrug-resistant Gram-positive and Gram-negative bacteria. Fluorescence microscopy suggests that cyclic lipo-α-AApeptides kill bacteria by disrupting bacterial membranes, possibly through a mechanism similar to that of cationic host defense peptides (HDPs). Furthermore, the cyclic lipo-α-AApeptide can mimic cationic host-defense peptides by antagonizing Toll-Like Receptor 4 (TLR4) signaling responses and suppressing pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α). Our results suggest that by mimicking host-defense peptides (HDPs), cyclic lipo-α-AApeptides may emerge to be a new class of antibiotic agents through direct bacteria killing, as well as novel anti-infective agents through immunomodulation. Show less

Antimicrobial peptides (AMPs) are host-defense agents capable of both bacterial membrane disruption and immunomodulation. However, the development of natural AMPs as potential therapeutics is hampered by their moderate activity and susceptibility to protease degradation. Herein we report lipidated cyclic γ-AApeptides that have potent antibacterial activity against clinically relevant Gram-positive and Gram-negative bacteria, many of which are resistant to conventional antibiotics. We show that… Show more

Antimicrobial peptides (AMPs) are host-defense agents capable of both bacterial membrane disruption and immunomodulation. However, the development of natural AMPs as potential therapeutics is hampered by their moderate activity and susceptibility to protease degradation. Herein we report lipidated cyclic γ-AApeptides that have potent antibacterial activity against clinically relevant Gram-positive and Gram-negative bacteria, many of which are resistant to conventional antibiotics. We show that lipidated cyclic γ-AApeptides mimic the bactericidal mechanism of AMPs by disrupting bacterial membranes. Interestingly, they also harness the immune response and inhibit lipopolysaccharide (LPS) activated Toll-Like Receptor 4 (TLR4) signaling, suggesting that lipidated cyclic γ-AApeptides have dual roles as novel antimicrobial and anti-inflammatory agents. Show less

Rifampin has been used for the treatment of bacterial infections for many years. Clinically, rifampin has been found to possess immunomodulatory effects. However, the molecular target responsible for the immunosuppressive effects of rifampin is not known. Herein, we show that rifampin binds to myeloid differentiation protein 2 (MD-2), the key coreceptor for innate immune TLR4. Rifampin blocked TLR4 signaling induced by LPS, including NF-κB activation and the overproduction of proinflammatory… Show more

Rifampin has been used for the treatment of bacterial infections for many years. Clinically, rifampin has been found to possess immunomodulatory effects. However, the molecular target responsible for the immunosuppressive effects of rifampin is not known. Herein, we show that rifampin binds to myeloid differentiation protein 2 (MD-2), the key coreceptor for innate immune TLR4. Rifampin blocked TLR4 signaling induced by LPS, including NF-κB activation and the overproduction of proinflammatory mediators nitric oxide, interleukin 1β, and tumor necrosis factor α in mouse microglia BV-2 cells and macrophage RAW 264.7 cells. Rifampin's inhibition of TLR4 signaling was also observed in immunocompetent rat primary macrophage, microglia, and astrocytes. Further, we show that rifampin (75 or 100 mg/kg b.i.d. for 3 d, intraperitoneal) suppressed allodynia induced by chronic constriction injury of the sciatic nerve and suppressed nerve injury-induced activation of microglia. Our findings indicate that MD-2 is a important target of rifampin in its inhibition of innate immune function and contributes to its clinically observed immune-suppressive effect. The results also suggest that rifampin may be repositioned as an agent for the treatment of neuropathic pain.—Wang, X., Grace, P. M., Pham, M. N., Cheng, K., Strand, K. A., Smith, C., Li, J., Watkins, L. R., Yin, H. Rifampin inhibits Toll-like receptor 4 signaling by targeting myeloid differentiation protein 2 and attenuates neuropathic pain. Show less

Toll-like receptors (TLRs) are type I transmembrane proteins that are key regulators of both innate and adaptive immune responses. To protect the host from viral and bacterial threats, TLRs trigger a pro-inflammatory immune response by detecting pathogen and danger associated molecular patterns. Considerable evidence has accumulated to show that the dysregulation of TLR signaling contributes to the development and progression of numerous diseases. Therefore, TLRs are emerging as important drug… Show more

Toll-like receptors (TLRs) are type I transmembrane proteins that are key regulators of both innate and adaptive immune responses. To protect the host from viral and bacterial threats, TLRs trigger a pro-inflammatory immune response by detecting pathogen and danger associated molecular patterns. Considerable evidence has accumulated to show that the dysregulation of TLR signaling contributes to the development and progression of numerous diseases. Therefore, TLRs are emerging as important drug discovery targets. Currently, there is great interest in the development of TLR small molecule modulators for interrogating TLR signaling and treating diseases caused by TLR signaling malfunctions. In this Tutorial Review, we will outline methods for the discovery of TLR small molecule modulators and the up-to-date progress in this field. Small molecules targeting TLRs not only provide an opportunity to identify promising drug candidates, but also unveil knowledge regarding TLR signaling pathways. Show less

The lateral transmembrane protein-protein interactions (PPI) have been regarded as “undruggable” despite their importance in many essential biological processes. The homo-trimerization of transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) is critical for the constitutive oncogenic activation of the Epstein-Barr virus (EBV). Herein we repurpose the antimicrobial agent pentamidine as a regulator of LMP-1 TMD-5 lateral interactions. The results of ToxR assay, tryptophan… Show more

The lateral transmembrane protein-protein interactions (PPI) have been regarded as “undruggable” despite their importance in many essential biological processes. The homo-trimerization of transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) is critical for the constitutive oncogenic activation of the Epstein-Barr virus (EBV). Herein we repurpose the antimicrobial agent pentamidine as a regulator of LMP-1 TMD-5 lateral interactions. The results of ToxR assay, tryptophan fluorescence assay, courmarin fluorescence dequenching assay, and Bis-Tris sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) consistently show pentamidine disrupts LMP-1 TMD-5 lateral interactions. Furthermore, pentamidine inhibits LMP-1 signaling, inducing cellular apoptosis and suppressing cell proliferation in the EBV infected B cells. In contrast, EBV negative cells are less susceptible to pentamidine. This study provides a novel non-peptide small molecule agent for regulating LMP-1 TMD-5 lateral interactions. Show less