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COVID-19, the disease responsible for the recent pandemic, is caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main protease (Mpro) of SARS-CoV-2 is an essential proteolytic enzyme that plays a number of important roles in the replication of the virus in human host cells. Blocking the function of SARS-CoV-2 Mpro offers a promising and targeted, therapeutic option for the treatment of the COVID-19 infection. Such an inhibitory strategy is currently successful in treating COVID-19 under FDA’s emergency use authorization, although with limited benefit to the immunocompromised along with an unfortunate number of side effects and drug-drug interactions. Current COVID vaccines protect against severe disease and death but are mostly ineffective toward long COVID which has been seen in 5–36% of patients. SARS-CoV-2 is a rapidly mutating virus and is here to stay endemically. Hence, alternate therapeutics to treat SARS-CoV-2 infections are still needed. Moreover, because of the high degree of conservation of Mpro among different coronaviruses, any newly developed antiviral agents should better prepare us for potential future epidemics or pandemics. In this paper, we first describe the design and computational docking of a library of novel 188 first-generation peptidomimetic protease inhibitors using various electrophilic warheads with aza-peptide epoxides, α-ketoesters, and β-diketones identified as the most effective. Second-generation designs, 192 compounds in total, focused on aza-peptide epoxides with drug-like properties, incorporating dipeptidyl backbones and heterocyclic ring motifs such as proline, indole, and pyrrole groups, yielding 8 hit candidates. These novel and specific inhibitors for SARS-CoV-2 Mpro can ultimately serve as valuable alternate and broad-spectrum antivirals against COVID-19. Communicated by Ramaswamy H. Sarma

Novel bacterial topoisomerase inhibitors (NBTIs) are among the most promising new antibiotics in preclinical/clinical development. We previously reported dioxane-linked NBTIs with potent antistaphylococcal activity and reduced hERG inhibition, a key safety liability. Herein, polarity-focused optimization enabled the delineation of clear structure-property relationships for both microsomal metabolic stability and hERG inhibition, resulting in the identification of lead compound 79. This molecule demonstrates potent antibacterial activity against diverse Gram-positive pathogens, inhibition of both DNA gyrase and topoisomerase IV, a low frequency of resistance, a favorable in vitro cardiovascular safety profile, and in vivo efficacy in a murine model of methicillin-resistant Staphylococcus aureus infection.

Aim: To develop and evaluate detergent-free, triple-drug-loaded, hyaluronate-coated elastic nanovesicles (H-ENVs) for the topical treatment of cutaneous leishmaniasis. Materials & methods: H-ENVs were developed and evaluated for vesicle size, entrapment efficiency, skin permeation and antileishmanial potential. Results: A 15.7 and 28.6% decrease in the cytotoxicity of paromomycin and amphotericin B, respectively, was observed in detergent-free ENVs compared with conventional ENVs. H-ENVs improved the efficacy of paromomycin against promastigote and amastigote models of leishmaniasis by 4- and 7.5-fold, respectively. In vivo investigation of H-ENVs demonstrated efficient topical management of cutaneous leishmaniasis. Conclusion: The results indicate the potential of H-ENVs as a safe topical treatment choice for cutaneous leishmaniasis.

Stroke causes devastating sensory-motor deficits and long-term disability due to disruption of descending motor pathways. Restoration of these functions enables independent living and therefore represents a high priority for those afflicted by stroke. Here, we report that daily administration of gabapentin, a clinically approved drug already used to treat various neurological disorders, promotes structural and functional plasticity of the corticospinal pathway after photothrombotic cortical stroke in adult mice. We found that gabapentin administration had no effects on vascular occlusion, haemodynamic changes nor survival of corticospinal neurons within the ipsilateral sensory-motor cortex in the acute stages of stroke. Instead, using a combination of tract tracing, electrical stimulation and functional connectivity mapping, we demonstrated that corticospinal axons originating from the contralateral side of the brain in mice administered gabapentin extend numerous collaterals, form new synaptic contacts and better integrate within spinal circuits that control forelimb muscles. Not only does gabapentin daily administration promote neuroplasticity, but it also dampens maladaptive plasticity by reducing the excitability of spinal motor circuitry. In turn, mice administered gabapentin starting 1 h or 1 day after stroke recovered skilled upper extremity function. Functional recovery persists even after stopping the treatment at 6 weeks following a stroke. Finally, chemogenetic silencing of cortical projections originating from the contralateral side of the brain transiently abrogated recovery in mice administered gabapentin, further supporting the conclusion that gabapentin-dependent reorganization of spared cortical pathways drives functional recovery after stroke. These observations highlight the strong potential for repurposing gabapentinoids as a promising treatment strategy for stroke repair.

Summary Cerebral organoids (COs) are rapidly accelerating the rate of translational neuroscience based on their potential to model complex features of the developing human brain. Several studies have examined the electrophysiological and neural network features of COs; however, no study has comprehensively investigated the developmental trajectory of electrophysiological properties in whole-brain COs and correlated these properties with developmentally linked morphological and cellular features. Here, we profiled the neuroelectrical activities of COs over the span of 5 months with a multi-electrode array platform and observed the emergence and maturation of several electrophysiologic properties, including rapid firing rates and network bursting events. To complement these analyses, we characterized the complex molecular and cellular development that gives rise to these mature neuroelectrical properties with immunohistochemical and single-cell transcriptomic analyses. This integrated approach highlights the value of COs as an emerging model system of human brain development and neurological disease., Highlights • CO electrophysiology can be quantified with a multi-electrode array method • CO electrophysiological trajectories correlate with molecular and cellular development • The neurotrophin/TRK signaling pathway is active in COs by 5 months in culture, Cerebral organoids (COs) have a unique advantage in modeling human-specific features of early brain development. Here, we profile their neuroelectrical activities with an MEA platform over time and correlate these activities with their increasingly complex molecular and cellular features. This integrated approach highlights the value of COs as an emerging model system of human brain development and neurological disease.

Novel bacterial topoisomerase inhibitors (NBTIs) are among the most promising new antibiotics in preclinical/clinical development. We previously reported dioxane-linked NBTIs with potent antistaphylococcal activity and reduced hERG inhibition, a key safety liability. Herein, polarity-focused optimization enabled the delineation of clear structure-property relationships for both microsomal metabolic stability and hERG inhibition, resulting in the identification of lead compound

Mutations exclusively in equilibrative nucleoside transporter 3 (ENT3), the only intracellular nucleoside transporter within the solute carrier 29 (SLC29) gene family, cause an expanding spectrum of human genetic disorders (e.g., H syndrome, PHID syndrome, and SHML/RDD syndrome). Here, we identify adult stem cell deficits that drive ENT3-related abnormalities in mice. ENT3 deficiency alters hematopoietic and mesenchymal stem cell fates; the former leads to stem cell exhaustion, and the latter leads to breaches of mesodermal tissue integrity. The molecular pathogenesis stems from the loss of lysosomal adenosine transport, which impedes autophagy-regulated stem cell differentiation programs via misregulation of the AMPK-mTOR-ULK axis. Furthermore, mass spectrometry-based metabolomics and bioenergetics studies identify defects in fatty acid utilization, and alterations in mitochondrial bioenergetics can additionally propel stem cell deficits. Genetic, pharmacologic and stem cell interventions ameliorate ENT3-disease pathologies and extend the lifespan of ENT3-deficient mice. These findings delineate a primary pathogenic basis for the development of ENT3 spectrum disorders and offer critical mechanistic insights into treating human ENT3-related disorders., Mutations in equilibrative nucleoside transporter 3 (ENT3), encoded by SLC29A3, cause a spectrum of human genetic disorders. Here, the authors show altered haematopoietic stem cell and mesenchymal stem cell fates in ENT3-deficient mice, due to misregulation of the AMPK-mTOR-ULK axis.

Due to the limitations of existing medications, there is a critical need for new drugs to treat visceral leishmaniasis. Since arylimidamides and antifungal azoles both show oral activity in murine visceral leishmaniasis models, a molecular hybridization approach was employed where arylimidamide and azole groups were separated by phenoxyalkyl linkers in an attempt to capitalize on the favorable antileishmanial properties of both series. Among the target compounds synthesized, a greater antileishmanial potency against intracellular Leishmania donovani was observed as the linker length increased from two to eight carbons and when an imidazole ring was employed as the terminal group compared to a 1,2,4-triazole group. Compound 24c (N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)-2-isopropoxyphenyl) picolinimidamide) displayed activity against L. donovani intracellular amastigotes with an IC50 value of 0.53 μM. When tested in a murine visceral leishmaniasis model, compound 24c at a dose of 75 mg/kg/day p.o. for five consecutive days resulted in a modest 33% decrease in liver parasitemia compared to the control group, indicating that further optimization of these molecules is needed. While potent hybrid compounds bearing an imidazole terminal group were also strong inhibitors of recombinant CYP51 from L. donovani, as assessed by a fluorescence-based assay, additional targets are likely to play an important role in the antileishmanial action of these compounds.

Mutations in human equilibrative nucleoside transporter 3 (ENT3) encoded by SLC29A3 results in anemia and erythroid hypoplasia, suggesting that ENT3 may regulate erythropoiesis. Here, we demonstrate that lysosomal ENT3 transport of taurine-conjugated bile acids (TBA) facilitates TBA chemical chaperone function and alleviates endoplasmic reticulum (ER) stress in expanding mouse hematopoietic stem and progenitor cells (HSPCs). Slc29a3−/− HSPCs accumulate less TBA despite elevated levels of TBA in Slc29a3−/− mouse plasma and have elevated basal ER stress, reactive oxygen species (ROS), and radiation-induced apoptosis. Reintroduction of ENT3 allows for increased accumulation of TBA into HSPCs, which results in TBA-mediated alleviation of ER stress and erythroid apoptosis. Transplanting TBA-preconditioned HSPCs expressing ENT3 into Slc29a3−/− mice increase bone marrow repopulation capacity and erythroid pool size and prevent early mortalities. Together, these findings suggest a putative role for a facilitative lysosomal transporter in the bile acid regulation of ER stress in mouse HSPCs which may have implications in erythroid biology, the treatment of anemia observed in ENT3-mutated human genetic disorders, and nucleoside analog drug therapy., Mutations in ENT3, encoded by SLC29A3, result in anaemia and erythroid hypoplasia, suggesting roles in erythropoiesis. Here the authors show that ENT3 acts as a lysosomal bile acid transporter, and mutation compromises taurine conjugated bile acid transport in erythroid progenitors leading to ER stress, and anaemia.

Many neurodegenerative diseases (NDs) such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis and Huntington's disease, are characterized by the progressive accumulation of abnormal proteinaceous assemblies in specific cell types and regions of the brain, leading to cellular dysfunction and brain damage. Although animal- and in vitro-based studies of NDs have provided the field with an extensive understanding of some of the mechanisms underlying these diseases, findings from these studies have not yielded substantial progress in identifying treatment options for patient populations. This necessitates the development of complementary model systems that are better suited to recapitulate human-specific features of ND pathogenesis. Three-dimensional (3D) culture systems, such as cerebral organoids generated from human induced pluripotent stem cells, hold significant potential to model NDs in a complex, tissue-like environment. In this review, we discuss the advantages of 3D culture systems and 3D modeling of NDs, especially AD and FTD. We also provide an overview of the challenges and limitations of the current 3D culture systems. Finally, we propose a few potential future directions in applying state-of-the-art technologies in 3D culture systems to understand the mechanisms of NDs and to accelerate drug discovery. Graphical abstract.

Aza-peptide aldehydes and ketones are a new class of reversible protease inhibitors that are specific for the proteasome and clan CD cysteine proteases. We designed and synthesised aza-Leu derivatives that were specific for the chymotrypsin-like active site of the proteasome, aza-Asp derivatives that were effective inhibitors of caspases-3 and -6, and aza-Asn derivatives that inhibited S. mansoni and I. ricinus legumains. The crystal structure of caspase-3 in complex with our caspase-specific aza-peptide methyl ketone inhibitor with an aza-Asp residue at P1 revealed a covalent linkage between the inhibitor carbonyl carbon and the active site cysteinyl sulphur. Aza-peptide aldehydes and ketones showed no cross-reactivity towards cathepsin B or chymotrypsin. The initial in vitro selectivity of these inhibitors makes them suitable candidates for further development into therapeutic agents to potentially treat multiple myeloma, neurodegenerative diseases, and parasitic infections.

Background: Acute ingestion of ketone supplements alters metabolism and potentially exercise performance. No studies to date have evaluated the impact of co-ingestion of ketone salts with caffeine ...

Aza-peptide aldehydes and ketones are a new class of reversible protease inhibitors that are specific for the proteasome and clan CD cysteine proteases. We designed and synthesised aza-Leu derivatives that were specific for the chymotrypsin-like active site of the proteasome, aza-Asp derivatives that were effective inhibitors of caspases-3 and -6, and aza-Asn derivatives that inhibited S. mansoni and I. ricinus legumains. The crystal structure of caspase-3 in complex with our caspase-specific aza-peptide methyl ketone inhibitor with an aza-Asp residue at P1 revealed a covalent linkage between the inhibitor carbonyl carbon and the active site cysteinyl sulphur. Aza-peptide aldehydes and ketones showed no cross-reactivity towards cathepsin B or chymotrypsin. The initial in vitro selectivity of these inhibitors makes them suitable candidates for further development into therapeutic agents to potentially treat multiple myeloma, neurodegenerative diseases, and parasitic infections.

After the inhibition of acetylcholinesterase (AChE) by organophosphorus (OP) nerve agents, a dealkylation reaction of the phosphylated serine, referred to as aging, can occur. When aged, known reactivators of OP-inhibited AChE are no longer effective. Realkylation of aged AChE may provide a route to reversing aging. We designed and synthesized a library of quinone methide precursors (QMPs) as proposed realkylators of aged AChE. Our lead compound (C8) from an in vitro screen successfully resurrected 32.7 and 20.4% of the activity of methylphosphonate-aged and isopropyl phosphate-aged electric-eel AChE, respectively, after 4 days. C8 displays properties of both resurrection (recovery from the aged to the native state) and reactivation (recovery from the inhibited to the native state). Resurrection of methylphosphonate-aged AChE by C8 was significantly pH-dependent, recovering 21% of activity at 4 mM and pH 9 after only 1 day. C8 is also effective against isopropyl phosphate-aged human AChE.

Acetylcholinesterase (AChE) is an essential enzyme that can be targeted by organophosphorus (OP) compounds, including nerve agents. Following exposure to OPs, AChE becomes phosphylated (inhibited) and undergoes a subsequent aging process where the OP-AChE adduct is dealkylated. The aged AChE is unable to hydrolyze acetylcholine, resulting in accumulation of the neurotransmitter in the central nervous system (CNS) and elsewhere. Current therapeutics are only capable of reactivating inhibited AChE. There are no known therapeutic agents to reverse the aging process or treat aged AChE. Quinone methides (QMs) have been shown to alkylate phosphates under physiological conditions. In this study, a small library of novel quinone methide precursors (QMPs) has been synthesized and examined as potential alkylating agents against model nucleophiles, including a model phosphonate. Computational studies have been performed to evaluate the affinity of QMPs for the aged AChE active site, and preliminary testing with electric eel AChE has been performed.

Chemical warfare nerve agents (CWNAs) present a global threat to both military and civilian populations. The acute toxicity of CWNAs stems from their ability to effectively inhibit acetylcholinesterase (AChE). This inhibition can lead to uncontrolled cholinergic cellular signaling, resulting in cholinergic crisis and, ultimately, death. Although the current FDA-approved standard of care is moderately effective when administered early, development of novel treatment strategies is necessary. Butyrylcholinesterase (BChE) is an enzyme which displays a high degree of structural homology to AChE. Unlike AChE, the roles of BChE are uncertain and possibilities are still being explored. However, BChE appears to primarily serve as a bioscavenger of toxic esters due to its ability to accommodate a wide variety of substrates within its active site. Like AChE, BChE is also readily inhibited by CWNAs. Due to its high affinity for binding CWNAs, and that null-BChE yields no apparent health effects, exogenous BChE has been explored as a candidate therapeutic for CWNA intoxication. Despite years of research, minimal strides have been made to develop a catalytic bioscavenger. Furthermore, BChE is only in early clinical trials as a stoichiometric bioscavenger of CWNAs, and large quantities must be administered to treat CWNA toxicity. Here, we describe previously unidentified mutations to residues within and adjacent to the acyl binding pocket (positions 282–285 were mutagenized from YGTP to NHML) of BChE that confer catalytic degradation of the CWNA, sarin. These mutations, along with corresponding future efforts, may finally lead to a novel therapeutic to combat CWNA intoxication.

Objective: To test the hypothesis that miltefosine (MTF)-polyphenol co-loaded second-generation nano-transfersomes (SGNTs) can be an effective approach for the topical treatment of cutaneous leishmaniasis (CL). Methods: The co-loaded SGNTs with various MTF-polyphenol combinations were developed, evaluated and compared for the entrapment efficiency, vesicle size, deformability index, ex-vivo permeation, cytotoxicity, and anti-leishmanial potential, using both in-vitro and in-vivo models. Results: The co-loaded SGNTs were spherical in shape, with an average size of 119 ± 1.5 nm and a high entrapment efficiency of 73.7 ± 3.7%. The ex-vivo study displayed a 3.2-fold higher permeation of MTF when entrapped in co-loaded SGNTs, whereas cytotoxicity potential of co-loaded SGNTs was 43.2% higher than the MTF solution. A synergistic interaction was observed between MTF and apigenin (APG) among all polyphenols and an 8.0-fold lower IC50 was found against amastigotes of DsRed Leishmania mexicana, compared with the plain MTF solution. Moreover, the in-vivo studies displayed a 9.5-fold reduced parasitic burden in the L. mexicana infected BALB/c mice treated with MTF-APG co-loaded SGNTs gel. Conclusions: The potential of MTF-APG co-loaded SGNTs topical formulation is established for the first time as an effective drug delivery strategy against CL.

Neural circuits are the underlying functional units of the human brain that govern complex behavior and higher-order cognitive processes. Disruptions in neural circuit development have been implicated in the pathogenesis of multiple neurodevelopmental disorders such as autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), and schizophrenia. Until recently, major efforts utilizing neurological disease modeling platforms based on human induced pluripotent stem cells (hiPSCs), investigated disease phenotypes primarily at the single cell level. However, recent advances in brain organoid systems, microfluidic devices, and advanced optical and electrical interfaces, now allow more complex hiPSC-based systems to model neuronal connectivity and investigate the specific brain circuitry implicated in neurodevelopmental disorders. Here we review emerging research advances in studying brain circuitry using in vitro and in vivo disease modeling platforms including microfluidic devices, enhanced functional recording interfaces, and brain organoid systems. Research efforts in these areas have already yielded critical insights into pathophysiological mechanisms and will continue to stimulate innovation in this promising area of translational research.

Two-photon excitable (850 nm) NIR fluorescent turn-on (702 nm) probe TP-NIR was synthesized for selective detection of cysteine (Cys). The probe itself shows is turned on by reaction with Cys in aqueous buffer. In addition, the probe displays greater selectivity for Cys over other thiols, including glutathione (GSH) and homocysteine (Hcy). Moreover, the large Stokes shift, NIR excitation, and NIR emission make this probe suitable for biological imaging.

Aging is a dealkylation reaction of organophosphorus (OP)-inhibited acetylcholinesterase (AChE). Despite many studies to date, aged AChE cannot be reactivated directly by traditional pyridinium oximes. This review summarizes strategies that are potentially valuable in the treatment against aging in OP poisoning. Among them, retardation of aging seeks to lower the rate of aging through the use of AChE effectors. These drugs should be administered before AChE is completely aged. For postaging treatment, realkylation of aged AChE by appropriate alkylators may pave the way for oxime treatment by neutralizing the oxyanion at the active site of aged AChE. The other two strategies, upregulation of AChE expression and introduction of exogenous AChE, cannot resurrect aged AChE but may compensate for lowered active AChE levels by in situ production or external introduction of active AChE. Upregulation of AChE expression can be triggered by some peptides. Sources of exogenous AChE can be whole blood or purified AChE, either from human or nonhuman species.

Cyanine compounds have previously shown excellent in vitro and promising in vivo antileishmanial efficacy, but the potential toxicity of these agents is a concern. A series of 22 analogs of thiazole orange ((Z)-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium salt), a commercial cyanine dye with antileishmanial activity, were synthesized in an effort to increase the selectivity of such compounds while maintaining efficacy. Cyanines possessing substitutions on the quinolinium ring system displayed potency against Leishmania donovani axenic amastigotes that differed little from the parent compound (IC50 12–42 nM), while ring disjunction analogs were both less potent and less toxic. Changes in DNA melting temperature were modest when synthetic oligonucleotides were incubated with selected analogs (ΔTm ≤ 5 °C), with ring disjunction analogs showing the least effect on this parameter. Despite the high antileishmanial potency of the target compounds, their toxicity and relatively flat SAR suggests that further information regarding the target(s) of these molecules is needed to aid their development as antileishmanials.

An analytical method was developed for measuring the effect of OATP1B2 deficiency on plasma levels of the kinase inhibitor regorafenib and its metabolites regorafenib-N-oxide, N-desmethyl-regorafenib-N-oxide, and regorafenib-N-β-glucuronide (RG) in mice. Compounds were separated by liquid chromatography and monitored by a triple quadrupole mass spectrometer in the selected reaction monitoring mode after positive electrospray ionization. All calibration curves were linear in the selected concentration range (R2 ≥ 0.99). The lower limit of quantification was 5 ng/mL for the four analytes. Within-day precisions, between-day precisions, and accuracies were 2.59–6.82%, 3.97–11.3%, and 94.5–111%, respectively. The identification and structure elucidation of RG, isolated from human urine, was performed by NMR. Compared with wild-type mice given regorafenib (10 mg/kg), deficiency of the drug transporter OATP1B2 in vivo had minimal effects on plasma levels of parent drug and the metabolite regorafenib-N-oxide, and N-desmethyl-regorafenib-N-oxide. However, the area under the curve and peak levels of RG were increased by 5.6-fold and 5.1-fold, respectively, in OATP1B2-knockout mice. In conclusion, our analytical method allowed accurate and precise quantitation of regorafenib and its main metabolites in mouse plasma, and is suitable for evaluation of transporter-dependent pharmacokinetic properties of these agents in vivo.

The current standard of care for treatment of organophosphate (OP) poisoning includes pretreatment with the weak reversible acetylcholinesterase (AChE) inhibitor pyridostigmine bromide. Because this drug is an AChE inhibitor, similar side effects exist as with OP poisoning. In an attempt to provide a therapeutic capable of mitigating AChE inhibition without such side effects, high-throughput screening was performed to identify a compound capable of increasing the catalytic activity of AChE. Herein, two such novel positive allosteric modulators (PAMs) of AChE are presented. These PAMs increase AChE activity threefold, but they fail to upshift the apparent IC 50 of a variety of OPs. Further development and optimization of these compounds may lead to pre- and/or postexposure therapeutics with broad-spectrum efficacy against pesticide and nerve agent poisoning. In addition, they could be used to complement the current therapeutic standard of care to increase the activity of uninhibited AChE, potentially increasing the efficacy of current therapeutics in addition to altering the therapeutic window.

The trp RNA-binding attenuation protein (TRAP) assembles into an 11-fold symmetric ring that regulates transcription and translation of trp-mRNA in bacilli via heterotropic allosteric activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic resonance studies have revealed that Trp-induced activation coincides with both microsecond to millisecond rigidification and local structural changes in TRAP, the pathway of binding of the 11 Trp ligands to the TRAP ring remains unclear. Moreover, because each of 11 bound Trp molecules is completely surrounded by protein, its release requires flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence to study the kinetics of Trp binding by Bacillus stearothermophilus TRAP over a range of temperatures and observed well-separated kinetic steps. These data were analyzed using nonlinear least-squares fitting of several two- and three-step models. We found that a model with two binding steps best describes the data, although the structural...

Four different libraries of overall twenty three N3-substituted thymidine (dThd) analogues, including eleven 3-carboranyl thymidine analogues (3CTAs), were synthesized. The latter are potential agents for Boron Neutron Capture Therapy (BNCT) of cancer. Linker between the dThd scaffold and the m-carborane cluster at the N3-position of the 3CTAs contained amidinyl-(3e and 3f), guanidyl-(7e-7g), tetrazolylmethyl-(9b1/2-9d1/2), or tetrazolyl groups (11b1/2-11d1/2) to improve human thymidine kinase 1 (hTK1) substrate characteristics and water solubilities compared with 1st generation 3CTAs, such as N5 and N5-2OH. The amidinyl- and guanidyl-type N3-substitued dThd analogues (3a-3f and 7a-7g) had hTK1 phosphorylation rates of30% relative to that of dThd, the endogenous hTK1 substrate, whereas the tetrazolyl-type N3-substitued dThd analogues (9a, 9b1/2-9d1/2 and 11a, 11b1/2-11d1/2) had relative phosphorylation rates (rPRs) of40%. Compounds 9a, 9b1/2-9d1/2 and 11a, 11b1/2-11d1/2 were subjected to in-depth enzyme kinetics studies and the obtained rk(cat)/K(m) (k(cat)/K(m) relative to that of dThd) ranged from 2.5 to 26%. The tetrazolyl-type N3-substitued dThd analogues 9b1/2 and 11d1/2 were the best substrates of hTK1 with rPRs of 52.4% and 42.5% and rk(cat)/K(m) values of 14.9% and 19.7% respectively. In comparison, the rPR and rk(cat)/K(m) values of N5-2OH in this specific study were 41.5% and 10.8%, respectively. Compounds 3e and 3f were1900 and1500 times, respectively, better soluble in PBS (pH 7.4) than N5-2OH whereas solubilities for 9b1/2-9d1/2 and 11b1/2-11d1/2 were only 1.3-13 times better.

Aging is a dealkylation reaction of organophosphorus (OP)-inhibited acetylcholinesterase (AChE). Despite many studies to date, aged AChE cannot be reactivated directly by traditional pyridinium oximes. This review summarizes strategies that are potentially valuable in the treatment against aging in OP poisoning. Among them, retardation of aging seeks to lower the rate of aging through the use of AChE effectors. These drugs should be administered before AChE is completely aged. For postaging treatment, realkylation of aged AChE by appropriate alkylators may pave the way for oxime treatment by neutralizing the oxyanion at the active site of aged AChE. The other two strategies, upregulation of AChE expression and introduction of exogenous AChE, cannot resurrect aged AChE but may compensate for lowered active AChE levels by in situ production or external introduction of active AChE. Upregulation of AChE expression can be triggered by some peptides. Sources of exogenous AChE can be whole blood or purified AChE, either from human or nonhuman species.

We report here an unliganded receptor structure in the common gamma-chain (γ c ) family of receptors and cytokines. The crystal structure of the unliganded form of the interleukin-7 alpha receptor (IL-7Rα) extracellular domain (ECD) at 2.15 Å resolution reveals a homodimer forming an “X” geometry looking down onto the cell surface with the C termini of the two chains separated by 110 Å and the dimer interface comprising residues critical for IL-7 binding. Further biophysical studies indicate a weak association of the IL-7Rα ECDs but a stronger association between the γ c /IL-7Rα ECDs, similar to previous studies of the full-length receptors on CD4 + T cells. Based on these and previous results, we propose a molecular mechanism detailing the progression from the inactive IL-7Rα homodimer and IL-7Rα–γ c heterodimer to the active IL-7–IL-7Rα–γ c ternary complex whereby the two receptors undergo at least a 90° rotation away from the cell surface, moving the C termini of IL-7Rα and γ c from a distance of 110 Å to less than 30 Å at the cell surface. This molecular mechanism can be used to explain recently discovered IL-7– and γ c -independent gain-of-function mutations in IL-7Rα from B- and T-cell acute lymphoblastic leukemia patients. The mechanism may also be applicable to other γ c receptors that form inactive homodimers and heterodimers independent of their cytokines.

Identifying potential ligand binding sites on a protein surface is an important first step for targeted structure-based drug discovery. While performing control experiments with Escherichia coli peptide deformylase (PDF), we noted that the organic solvents used to solubilize some ligands perturbed many of the same resonances in PDF as the small molecule inhibitors. To further explore this observation, we recorded (15)N HSQC spectra of E. coli peptide deformylase (PDF) in the presence of trace quantities of several simple organic solvents (acetone, DMSO, ethanol, isopropanol) and identified their sites of interaction from local perturbation of amide chemical shifts. Analysis of the protein surface structure revealed that the ligand-induced shift perturbations map to the active site and one additional surface pocket. The correlation between sites of solvent and inhibitor binding highlights the utility of organic solvents to rapidly and effectively validate and characterize binding sites on proteins prior to designing a drug discovery screen. Further, the solvent-induced perturbations have implications for the use of organic solvents to dissolve candidate ligands in NMR-based screens.

The trp RNA-binding attenuation protein (TRAP) functions in many Bacilli to control the expression of the tryptophan biosynthesis genes. Transcription of the trp operon is controlled by TRAP through an attenuation mechanism, in which competition between two alternative secondary structural elements in the 5′ leader sequence of the nascent mRNA is influenced by tryptophan-dependent binding of TRAP to the RNA. Previously, NMR studies of the undecamer (11-mer) suggested that tryptophan-dependent control of RNA binding by TRAP is accomplished through ligand-induced changes in protein dynamics. We now present further insights into this ligand-coupled event from hydrogen/deuterium (H/D) exchange analysis, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Scanning calorimetry showed tryptophan dissociation to be independent of global protein unfolding, while analysis of the temperature dependence of the binding enthalpy by ITC revealed a negative heat capacity change larger than expected from surface burial, a hallmark of binding-coupled processes. Analysis of this excess heat capacity change using parameters derived from protein folding studies, corresponds to the ordering of 17-24 residues per monomer of TRAP upon tryptophan binding. This result is in agreement with qualitative analysis of residue-specific broadening observed in TROSY NMR spectra of the 91 kDa oligomer. Implications for the mechanism of ligand-mediated TRAP activation through a shift in a preexisting conformational equilibrium and an induced fit conformational change are discussed.

The tryptophan biosynthesis genes of several Bacilli are controlled through terminator/anti-terminator transcriptional attenuation. This process is regulated by tryptophan-dependent binding of the trp RNA-binding attenuation protein (TRAP) to the leader region of the trp operon mRNA, precluding formation of the antiterminator RNA hairpin, and allowing formation of the less stable terminator hairpin. Crystal structures are available of TRAP in complex with tryptophan and in ternary complex with tryptophan and RNA. However, no structure of TRAP in the absence of tryptophan is available; thus, the mechanism of allostery remains unclear. We have used transverse relaxation optimized spectroscopy (TROSY)-based NMR experiments to study the mechanism of ligand-mediated allosteric regulation in the 90.6kDa 11-mer TRAP. By recording a series of two-dimensional 15N-edited TROSY NMR spectra of TRAP from the thermophile Bacillus stearothermophilus over an extended range of temperatures, we have found tryptophan binding to be temperature-dependent, in agreement with the previously observed temperature-dependent RNA binding. Triple-resonance TROSY-based NMR spectra recorded at 55 degrees C have allowed us to obtain backbone resonance assignments for uniformly 2H,13C,15N-labeled TRAP in the inactive form and in the active form (free and bound to tryptophan). On the basis of ligand-dependent differential line-broadening and chemical shift perturbations, coupled with the results of proteolytic sensitivity measurements, we infer that tryptophan-modulated protein flexibility (dynamics) plays a central role in TRAP function by altering its RNA-binding affinity. Furthermore, because the crystal structures show that the ligand is buried completely in the bound state, we speculate that such dynamic behavior may be important to enable rapid response to changes in intracellular tryptophan levels. Thus, we propose that allosteric control of TRAP is accomplished by ligand-altered protein dynamics.

The control of tryptophan production in Bacillus is a paradigmatic example of gene regulation involving the interplay of multiple protein and nucleic acid components. Central to this combinatorial mechanism are the homo-oligomeric proteins TRAP ( trp RNA-binding attenuation protein) and anti-TRAP (AT). TRAP forms undecameric rings, and AT assembles into triskelion-shaped trimers. Upon activation by tryptophan, the outer circumference of the TRAP ring binds specifically to a series of tandem sequences present in the 5′ UTR of RNA transcripts encoding several tryptophan metabolism genes, leading to their silencing. AT, whose expression is up-regulated upon tryptophan depletion to concentrations not exceeding a ratio of one AT trimer per TRAP 11-mer, restores tryptophan production by binding activated TRAP and preventing RNA binding. How the smaller AT inhibitor prevents RNA binding at such low stoichiometries has remained a puzzle, in part because of the large RNA-binding surface on the tryptophan-activated TRAP ring and its high affinity for RNA. Using X-ray scattering, hydrodynamic, and mass spectrometric data, we show that the polydentate action of AT trimers can condense multiple intact TRAP rings into large heterocomplexes, effectively reducing the available contiguous RNA-binding surfaces. This finding reveals an unprecedented mechanism for substoichiometric inhibition of a gene-regulatory protein, which may be a widespread but underappreciated regulatory mechanism in pathways that involve homo-oligomeric or polyvalent components.

Peptide deformylase (PDF) is an enzyme that is responsible for removing the formyl group from nascently synthesized polypeptides in bacteria, attracting much attention as a potential target for novel antibacterial agents. Efforts to develop potent inhibitors of the enzyme have progressed on the basis of classical medicinal chemistry, combinatorial chemistry, and structural approaches, yet the validity of PDF as an antibacterial target hangs, in part, on the ability of inhibitors to selectively target this enzyme in favor of structurally related metallohydrolases. We have used (15)N NMR spectroscopy and isothermal titration calorimetry to investigate the high-affinity interaction of EcPDF with actinonin, a naturally occurring potent EcPDF inhibitor. Backbone amide chemical shifts, residual dipolar couplings, hydrogen-deuterium exchange, and (15)N relaxation reveal structural and dynamic effects of ligand binding in the immediate vicinity of the ligand-binding site as well as at remote sites. A comparison of the crystal structures of free and actinonin-bound EcPDF with the solution data suggests that most of the consequences of the ligand binding to the protein are lost or obscured during crystallization. The results of these studies improve our understanding of the thermodynamic global minimum and have important implications for structure-based drug design.

Solution NMR spectroscopy represents a powerful tool for examining the structure and function of biological macromolecules. The advent of multidimensional (2D-4D) NMR, together with the widespread use of uniform isotopic labeling of proteins and RNA with the NMR-active isotopes, 15N and 13C, opened the door to detailed analyses of macromolecular structure, dynamics, and interactions of smaller macromolecules (< approximately 25 kDa). Over the past 10 years, advances in NMR and isotope labeling methods have expanded the range of NMR-tractable targets by at least an order of magnitude. Here we briefly describe the methodological advances that allow NMR spectroscopy of large macromolecules and their complexes and provide a perspective on the wide range of applications of NMR to biochemical problems.

We have determined the solution structure of Mth11 ( Mth Rpp29), an essential subunit of the RNase P enzyme from the archaebacterium Methanothermobacter thermoautotrophicus (Mth ). RNase P is a ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving the 5′ leader sequence during maturation of tRNAs in all three domains of life. In eubacteria, this enzyme is made up of two subunits: a large RNA (≈120 kDa) responsible for mediating catalysis, and a small protein cofactor (≈15 kDa) that modulates substrate recognition and is required for efficient in vivo catalysis. In contrast, multiple proteins are associated with eukaryotic and archaeal RNase P, and these proteins exhibit no recognizable homology to the conserved bacterial protein subunit. In reconstitution experiments with recombinantly expressed and purified protein subunits, we found that Mth Rpp29, a homolog of the Rpp29 protein subunit from eukaryotic RNase P, is an essential protein component of the archaeal holoenzyme. Consistent with its role in mediating protein–RNA interactions, we report that Mth Rpp29 is a member of the oligonucleotide/oligosaccharide binding fold family. In addition to a structured β-barrel core, it possesses unstructured N- and C-terminal extensions bearing several highly conserved amino acid residues. To identify possible RNA contacts in the protein–RNA complex, we examined the interaction of the 11-kDa protein with the full 100-kDa Mth RNA subunit by using NMR chemical shift perturbation. Our findings represent a critical step toward a structural model of the RNase P holoenzyme from archaebacteria and higher organisms.

IL-7 and IL-7Ralpha bind the gamma(c) receptor, forming a complex crucial to several signaling cascades leading to the development and homeostasis of T and B cells. We report that the IL-7Ralpha ectodomain uses glycosylation to modulate its binding constants to IL-7, unlike the other receptors in the gamma(c) family. IL-7 binds glycosylated IL-7Ralpha 300-fold more tightly than unglycosylated IL-7Ralpha, and the enhanced affinity is attributed primarily to an accelerated on rate. Structural comparison of IL-7 in complex to both forms of IL-7Ralpha reveals that glycosylation does not participate directly in the binding interface. The SCID mutations of IL-7Ralpha locate outside the binding interface with IL-7, suggesting that the expressed mutations cause protein folding defects in IL-7Ralpha. The IL-7/IL-7Ralpha structures provide a window into the molecular recognition events of the IL-7 signaling cascade and provide sites to target for designing new therapeutics to treat IL-7-related diseases.