The Impact of Terminal Peptide Extensions of Retinal Inosine 5´Monophosphate Dehydrogenase 1 Isoforms on their DNA-binding Activities.

The main structural difference between the mutation-susceptible retinal isoforms of inosine 5´-monophosphate dehydrogenase-1 (IMPDH-1) with the canonical form resides in the C- and N-terminal peptide extensions with unknown structural/functional impacts. In this report, we aimed to experimentally evaluate the functional impact of these extensions on the specific/non-specific single-stranded DNA (ssDNA)-binding activities relative to those of the canonical form. Our in silico findings indicated the possible contribution of the C-terminal segment to the reduced flexibility of the Bateman domain of the enzyme. In addition, the in silico data indicated that the N-terminal tail acts by altering the distance between the tetramers in the concave octamer complex (the native form) of the enzyme. The overall impact of these predicted structural variations became evident, first, through higher Km values with respect to either of the substrates relative to the canonical isoform, as reported previously (Andashti et al. in Mol Cell Biochem 465(1):155-164, 2020). Secondary, the binding of the recombinant mouse retinal isoform IMPDH1 (603) to its specific Rhodopsin target gene was significantly augmented while its binding to non-specific ssDNA was lower than that of the canonical isoform. The DNA-binding activity of the other mouse retinal isoform, IMPDH1(546), to specific and non-specific ssDNA was lower than that of the canonical form most probably due to the in silico predicted rigidity created in the Bateman domain by the C-terminal peptide extension. Furthermore, the DNA binding to the Rhodopsin target gene by each of the IMPDH isoforms influenced in the presence of GTP (Guanosine triphosphate) and ATP (Adenosine triphosphate).

GuaB3, an overlooked enzyme in cyanobacteria's toolbox that sheds light on IMP dehydrogenase evolution.

IMP dehydrogenase and GMP reductase are enzymes from the same protein family with analogous structures and catalytic mechanisms that have gained attention because of their essential roles in nucleotide metabolism and as potential drug targets. This study focusses on GuaB3, a less-explored enzyme within this family. Phylogenetic analysis uncovers GuaB3's independent evolution from other members of the family and it predominantly occurs in Cyanobacteria. Within this group, GuaB3 functions as a unique IMP dehydrogenase, while its counterpart in Actinobacteria has a yet unknown function. Synechocystis sp. PCC6803 GuaB3 structures demonstrate differences in the active site compared to canonical IMP dehydrogenases, despite shared catalytic mechanisms. These findings highlight the essential role of GuaB3 in Cyanobacteria, provide insights into the diversity and evolution of the IMP dehydrogenase protein family, and reveal a distinctive characteristic in nucleotide metabolism, potentially aiding in combating harmful cyanobacterial blooms-a growing concern for humans and wildlife.

Potential of Onchocerca ochengi inosine-5'-monophosphate dehydrogenase (IMPDH) and guanosine-5'-monophosphate oxidoreductase (GMPR) as druggable and vaccine candidates: immunoinformatics screening.

Onchocerciasis is a vector-borne disease caused by the filarial nematode Onchocerca volvulus, which is responsible for most of the visual impairments recorded in Africa, Asia and the Americas. It is known that O. volvulus has similar molecular and biological characteristics as Onchocerca ochengi in cattle. This study was designed to screen for immunogenic epitopes and binding pockets of O. ochengi IMPDH and GMPR ligands using immunoinformatic approaches. In this study, a total of 23 B cell epitopes for IMPDH and 7 B cell epitopes for GMPR were predicted using ABCpred tool, Bepipred 2.0 and Kolaskar and Tongaonkar methods. The CD4+ Th computational results showed 16 antigenic epitopes from IMPDH with strong binding affinity for DRB1_0301, DRB3_0101, DRB1_0103 and DRB1_1501 MHC II alleles while 8 antigenic epitopes from GMPR were predicted to bind DRB1_0101 and DRB1_0401 MHC II alleles, respectively. For the CD8+ CTLs analysis, 8 antigenic epitopes from IMPDH showed strong binding affinity to human leukocyte antigen HLA-A*26:01, HLA-A*03:01, HLA-A*24:02 and HLA-A*01:01 MHC I alleles while 2 antigenic epitopes from GMPR showed strong binding affinity to HLA-A*01:01 allele, respectively. The immunogenic B cell and T cell epitopes were further evaluated for antigenicity, non-alllergernicity, toxicity, IFN-gamma, IL4 and IL10. The docking score revealed favorable binding free energy with IMP and MYD scoring the highest binding affinity at -6.6 kcal/mol with IMPDH and -8.3 kcal/mol with GMPR. This study provides valuable insight on IMPDH and GMPR as potential drug targets and for the development of multiple epitope vaccine candidates.Communicated by Ramaswamy H. Sarma.

IMPDH1 retinal variants control filament architecture to tune allosteric regulation.

Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.

IMPDH forms the cytoophidium in zebrafish.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide biosynthesis. Its activity is negatively regulated by the binding of GTP. IMPDH can form a membraneless subcellular structure termed the cytoophidium in response to certain changes in the metabolic status of the cell. The polymeric form of IMPDH, which is the subunit of the cytoophidium, has been shown to be more resistant to the inhibition by GTP at physiological concentrations, implying a functional correlation between cytoophidium formation and the upregulation of GTP biosynthesis. Herein we demonstrate that zebrafish IMPDH1b and IMPDH2 isoforms can assemble abundant cytoophidium in most of cultured cells under stimuli, while zebrafish IMPDH1a shows distinctive properties of forming the cytoophidium in different cell types. Point mutations that disrupt cytoophidium structure in mammalian models also prevent the aggregation of zebrafish IMPDHs. In addition, we discover the presence of the IMPDH cytoophidium in various tissues of larval and adult fish under normal growth conditions. Our results reveal that polymerization and cytoophidium assembly of IMPDH can be a regulatory machinery conserved among vertebrates, and with specific physiological purposes.

Terminal Peptide Extensions Augment the Retinal IMPDH1 Catalytic Activity and Attenuate the ATP-induced Fibrillation Events.

Defects in inosine monophosphate dehydrogenase-1 (IMPDH1) lead to insufficient biosyntheses of purine nucleotides. In eyes, these defects are believed to cause retinitis pigmentosa (RP). Major retinal isoforms of IMPDH1 are structurally distinct from those in other tissues, by bearing terminal extensions. Using recombinant mouse IMPDH1 (mH1), we evaluated the kinetics and oligomerization states of the retinal isoforms. Moreover, we adopted molecular simulation tools to study the possible effect of terminal tails on the function of major enzyme isoforms with the aim to find structural evidence in favor of contradictory observations on retinal IMPDH1 function. Our findings indicated higher catalytic activity for the major mouse retinal isoform (mH1603) along with lower fibrillation capacity under the influence of ATP. However, higher mass oligomerization products were formed by the mH1 (603) isoform in the presence of the enzyme inhibitors such as GTP and/or MPA. Collectively, our findings demonstrate that the structural differences between the retinal isoforms have led to functional variations possibly to justify the retinal cells' requirements.

Molecular Basis of P131 Cryptosporidial-IMPDH Selectivity-A Structural, Dynamical and Mechanistic Stance.

Cryptosporidiosis accounts for a surge in infant (<5 years) mortality and morbidity. To date, several drug discovery efforts have been put in place to develop effective therapeutic options against the causative parasite. Based on a recent report, P131 spares inosine monophosphate dehydrogenase (IMPDH) in a eukaryotic model (mouse IMPDH (mIMPDH)) while binding selectively to the NAD+ site in Cryptosporidium parvum (CpIMPDH). However, no structural detail exists on the underlining mechanisms of P131-CpIMPDH selective targeting till date. To this effect, we investigate the selective inhibitory dynamics of P131 in CpIMPDH relative to mIMPDH via molecular biocomputation methods. Pairwise sequence alignment revealed prominent variations at the NAD+ binding regions of both proteins that accounted for disparate P131 binding activities. The influence of these variations was further revealed by the MM/PBSA energy estimations coupled with per-residue energy decomposition which monitored the systematic binding of the compound. Furthermore, relative high-affinity interactions occurred at the CpIMPDH NAD+ site which were majorly mediated by SER22, VAL24, PRO26, SER354, GLY357, and TYR358 located on chain D. These residues are unique to the parasite IMPDH form and not in the eukaryotic protein, highlighting variations that account for preferential P131 binding. Molecular insights provided herein corroborate previous experimental reports and further underpin the basis of CpIMPDH inhibitor selectivity. Findings from this study could present attractive prospects toward the design of novel anticryptosporidials with improved selectivity and binding affinity against parasitic targets.

Evaluation of Bronopol and Disulfiram as Potential Candidatus Liberibacter asiaticus Inosine 5'-Monophosphate Dehydrogenase Inhibitors by Using Molecular Docking and Enzyme Kinetic.

Citrus huanglongbing (HLB) is a destructive disease that causes significant damage to many citrus producing areas worldwide. To date, no strategy against this disease has been established. Inosine 5'-monophosphate dehydrogenase (IMPDH) plays crucial roles in the de novo synthesis of guanine nucleotides. This enzyme is used as a potential target to treat bacterial infection. In this study, the crystal structure of a deletion mutant of CLas IMPDHΔ98-201 in the apo form was determined. Eight known bioactive compounds were used as ligands for molecular docking. The results showed that bronopol and disulfiram bound to CLas IMPDHΔ98-201 with high affinity. These compounds were tested for their inhibition against CLas IMPDHΔ98-201 activity. Bronopol and disulfiram showed high inhibition at nanomolar concentrations, and bronopol was found to be the most potent molecule (Ki = 234 nM). The Ki value of disulfiram was 616 nM. These results suggest that bronopol and disulfiram can be considered potential candidate agents for the development of CLas inhibitors.

In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors.

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.

Cryo-EM structures demonstrate human IMPDH2 filament assembly tunes allosteric regulation.

Inosine monophosphate dehydrogenase (IMPDH) mediates the first committed step in guanine nucleotide biosynthesis and plays important roles in cellular proliferation and the immune response. IMPDH reversibly polymerizes in cells and tissues in response to changes in metabolic demand. Self-assembly of metabolic enzymes is increasingly recognized as a general mechanism for regulating activity, typically by stabilizing specific conformations of an enzyme, but the regulatory role of IMPDH filaments has remained unclear. Here, we report a series of human IMPDH2 cryo-EM structures in both active and inactive conformations. The structures define the mechanism of filament assembly, and reveal how filament-dependent allosteric regulation of IMPDH2 makes the enzyme less sensitive to feedback inhibition, explaining why assembly occurs under physiological conditions that require expansion of guanine nucleotide pools. Tuning sensitivity to an allosteric inhibitor distinguishes IMPDH from other metabolic filaments, and highlights the diversity of regulatory outcomes that can emerge from self-assembly.

The functional impact of the C/N-terminal extensions of the mouse retinal IMPDH1 isoforms: a kinetic evaluation.

Mutations in the retinal inosine monophosphate dehydrogenase1 (IMPDH1) gene is believed to be one cause of retinitis pigmentosa (RP). The main structural difference between the mutation-susceptible retinal isoforms with canonical one resides in the C- and N-terminal extensions. There are limited studies on the structure and function of terminal peptide extensions of the IMPDH1 retinal isoforms. Using recombinant murine IMPDH1 (mH1), we evaluated the kinetics of the retinal isoforms along with inhibition by some of the purine nucleotides. Molecular modeling tools were also applied to study the probable effect(s) of the terminal peptide tails on the function of the retinal isoforms. Molecular dynamic simulations indicated the possible impact of the end-terminal segments on the enzyme function through interactions with the enzyme's finger domain, affecting its critical pseudo barrel structure. The higher experimentally-determined Km and Ki values of the retinal mIMPDH1 (546) and mIMPDH1 (603) relative to that of the canonical isoform, mIMPDH1 (514), might clearly be due to these interactions. Furthermore and despite of the canonical isoform, the retinal isoforms of mH1 exhibited no NAD+ substrate inhibition. The resent data would certainly provide the ground for future evaluation of the physiological significance of these variations.

The Bateman domain of IMP dehydrogenase is a binding target for dinucleoside polyphosphates.

IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the de novo guanine nucleotide biosynthetic pathway. Because of its involvement in the control of cell division and proliferation, IMPDH represents a therapeutic for managing several diseases, including microbial infections and cancer. IMPDH must be tightly regulated, but the molecular mechanisms responsible for its physiological regulation remain unknown. To this end, we recently reported an important role of adenine and guanine mononucleotides that bind to the regulatory Bateman domain to allosterically modulate the catalytic activity of eukaryotic IMPDHs. Here, we have used enzyme kinetics, X-ray crystallography, and small-angle X-ray scattering (SAXS) methodologies to demonstrate that adenine/guanine dinucleoside polyphosphates bind to the Bateman domain of IMPDH from the fungus Ashbya gossypii with submicromolar affinities. We found that these dinucleoside polyphosphates modulate the catalytic activity of IMPDHs in vitro by efficiently competing with the adenine/guanine mononucleotides for the allosteric sites. These results suggest that dinucleoside polyphosphates play important physiological roles in the allosteric regulation of IMPDHs by adding an additional mechanism for fine-tuning the activities of these enzymes. We propose that these findings may have important implications for the design of therapeutic strategies to inhibit IMPDHs.

ANKRD9 is a metabolically-controlled regulator of IMPDH2 abundance and macro-assembly.

Members of a large family of Ankyrin Repeat Domain (ANKRD) proteins regulate numerous cellular processes by binding to specific protein targets and modulating their activity, stability, and other properties. The same ANKRD protein may interact with different targets and regulate distinct cellular pathways. The mechanisms responsible for switches in the ANKRDs' behavior are often unknown. We show that cells' metabolic state can markedly alter interactions of an ANKRD protein with its target and the functional outcomes of this interaction. ANKRD9 facilitates degradation of inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in GTP biosynthesis. Under basal conditions ANKRD9 is largely segregated from the cytosolic IMPDH2 in vesicle-like structures. Upon nutrient limitation, ANKRD9 loses its vesicular pattern and assembles with IMPDH2 into rodlike filaments, in which IMPDH2 is stable. Inhibition of IMPDH2 activity with ribavirin favors ANKRD9 binding to IMPDH2 rods. The formation of ANKRD9/IMPDH2 rods is reversed by guanosine, which restores ANKRD9 associations with the vesicle-like structures. The conserved Cys109Cys110 motif in ANKRD9 is required for the vesicle-to-rods transition as well as binding and regulation of IMPDH2. Oppositely to overexpression, ANKRD9 knockdown increases IMPDH2 levels and prevents formation of IMPDH2 rods upon nutrient limitation. Taken together, the results suggest that a guanosine-dependent metabolic switch determines the mode of ANKRD9 action toward IMPDH2.

Synthesis and Structure-Activity relationship of 1-(5-isoquinolinesulfonyl)piperazine analogues as inhibitors of Mycobacterium tuberculosis IMPDH.

Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure-activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation.

Ribavirin induces widespread accumulation of IMP dehydrogenase into rods/rings structures in multiple major mouse organs.

Ribavirin (RBV) is a guanosine analogue triazole most commonly used in the treatment of chronic hepatitis C (HCV) infection. Although its mechanism of action is a matter of debate, several possibilities have been proposed, including depletion of guanine nucleotides through inhibition of inosine monophosphate dehydrogenase (IMPDH). IMPDH has been shown to assemble into micron-scale rod- and ring-shaped structures (rods/rings or RR), also called "IMPDH filaments," both in vitro and in vivo. Formation of RR structures can occur naturally, potentially to influence IMPDH activity, or when de novo guanosine monophosphate biosynthesis or IMPDH itself are inhibited by nutrient deprivation or drugs like RBV. Numerous studies have also reported the occurrence of autoantibodies targeting RR structures (anti-RR) in HCV patients previously treated or under treatment with interferon-α and ribavirin (IFN/RBV) combination therapy. For this brief study, we considered the strong association between RR autoantibodies and IFN/RBV treatment, and the lack of data assessing how RBV affects RR formation in a variety of tissues in vivo. First, RR structures formed in the spleen and pancreas of normal mice without any treatment. Then, in RBV-treated mice, we detected RR structures in a number of tissues, including stomach, liver, spleen, kidney, brain, skin, and cardiac and skeletal muscle. We made several intriguing observations: predominance of RR structures in the mucosa and submucosa layers of the stomach wall; a high proportion of RR-positive cells in the cerebral cortex, suggesting that RBV actually crosses the blood-brain barrier; and a higher ratio of rings to rods in the epidermis compared to the dermis layer of the skin. Screening for RR structures appears to be a useful method to track tissue penetration of RBV and the many RR-inducing drugs previously identified.

Compositional complexity of rods and rings.

Rods and rings (RRs) are large linear- or circular-shaped structures typically described as polymers of IMPDH (inosine monophosphate dehydrogenase). They have been observed across a wide variety of cell types and species and can be induced to form by inhibitors of IMPDH. RRs are thought to play a role in the regulation of de novo guanine nucleotide synthesis; however, the function and regulation of RRs is poorly understood. Here we show that the regulatory GTPase, ARL2, a subset of its binding partners, and several resident proteins at the endoplasmic reticulum (ER) also localize to RRs. We also have identified two new inducers of RR formation: AICAR and glucose deprivation. We demonstrate that RRs can be disassembled if guanine nucleotides can be generated by salvage synthesis regardless of the inducer. Finally, we show that there is an ordered addition of components as RRs mature, with IMPDH first forming aggregates, followed by ARL2, and only later calnexin, a marker of the ER. These findings suggest that RRs are considerably more complex than previously thought and that the function(s) of RRs may include involvement of a regulatory GTPase, its effectors, and potentially contacts with intracellular membranes.

Expanding Benzoxazole-Based Inosine 5'-Monophosphate Dehydrogenase (IMPDH) Inhibitor Structure-Activity As Potential Antituberculosis Agents.

New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. Mycobacterium tuberculosis ( Mtb) inosine 5'-monophosphate dehydrogenase 2 ( MtbIMPDH2) is a promising yet controversial potential target. The inhibition of MtbIMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure-activity relationship (SAR) for the benzoxazole series of MtbIMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 µM can be achieved. The antibacterial activity of the most promising compound, 17b (Q151), is derived from the inhibition of MtbIMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of 17b, alleviating the concern that guanine salvage can protect Mtb in vivo. These findings suggest that MtbIMPDH2 is a vulnerable target for tuberculosis.

Fragment-Based Approach to Targeting Inosine-5'-monophosphate Dehydrogenase (IMPDH) from Mycobacterium tuberculosis.

Tuberculosis (TB) remains a major cause of mortality worldwide, and improved treatments are needed to combat emergence of drug resistance. Inosine 5'-monophosphate dehydrogenase (IMPDH), a crucial enzyme required for de novo synthesis of guanine nucleotides, is an attractive TB drug target. Herein, we describe the identification of potent IMPDH inhibitors using fragment-based screening and structure-based design techniques. Screening of a fragment library for Mycobacterium thermoresistible ( Mth) IMPDH ΔCBS inhibitors identified a low affinity phenylimidazole derivative. X-ray crystallography of the Mth IMPDH ΔCBS-IMP-inhibitor complex revealed that two molecules of the fragment were bound in the NAD binding pocket of IMPDH. Linking the two molecules of the fragment afforded compounds with more than 1000-fold improvement in IMPDH affinity over the initial fragment hit.

Enzymes Regulated via Cystathionine ß-Synthase Domains.

Cystathionine ß-synthase (CBS) domains discovered 20 years ago can bind different adenosine derivatives (AMP, ADP, ATP, S-adenosylmethionine, NAD, diadenosine polyphosphates) and thus regulate the activities of numerous proteins. Mutations in CBS domains of enzymes and membrane transporters are associated with several hereditary diseases. The regulatory unit is a quartet of CBS domains that belong to one or two polypeptides and usually form a conserved disk-like structure. CBS domains function as "internal inhibitors" in enzymes, and their bound ligands either amplify or attenuate the inhibitory effect. Recent studies have opened a way to understanding the structural basis of enzyme regulation via CBS domains and widened the list of their bound ligands.