A dual regulatory circuit consisting of S-adenosylmethionine decarboxylase protein and its reaction product controls expression of the paralogous activator prozyme in Trypanosoma brucei.

Polyamines are essential for cell growth of eukaryotes including the etiologic agent of human African trypanosomiasis (HAT), Trypanosoma brucei. In trypanosomatids, a key enzyme in the polyamine biosynthetic pathway, S-adenosylmethionine decarboxylase (TbAdoMetDC) heterodimerizes with a unique catalytically-dead paralog called prozyme to form the active enzyme complex. In higher eukaryotes, polyamine metabolism is subject to tight feedback regulation by spermidine-dependent mechanisms that are absent in trypanosomatids. Instead, in T. brucei an alternative regulatory strategy based on TbAdoMetDC prozyme has evolved. We previously demonstrated that prozyme protein levels increase in response to loss of TbAdoMetDC activity. Herein, we show that prozyme levels are under translational control by monitoring incorporation of deuterated leucine into nascent prozyme protein. We furthermore identify pathway factors that regulate prozyme mRNA translation. We find evidence for a regulatory feedback mechanism in which TbAdoMetDC protein and decarboxylated AdoMet (dcAdoMet) act as suppressors of prozyme translation. In TbAdoMetDC null cells expressing the human AdoMetDC enzyme, prozyme levels are constitutively upregulated. Wild-type prozyme levels are restored by complementation with either TbAdoMetDC or an active site mutant, suggesting that TbAdoMetDC possesses an enzyme activity-independent function that inhibits prozyme translation. Depletion of dcAdoMet pools by three independent strategies: inhibition/knockdown of TbAdoMetDC, knockdown of AdoMet synthase, or methionine starvation, each cause prozyme upregulation, providing independent evidence that dcAdoMet functions as a metabolic signal for regulation of the polyamine pathway in T. brucei. These findings highlight a potential regulatory paradigm employing enzymes and pseudoenzymes that may have broad implications in biology.

Synthesis and evaluation of analogs of 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (MDL 73811, or AbeAdo) - An inhibitor of S-adenosylmethionine decarboxylase with antitrypanosomal activity.

We describe our efforts to improve the pharmacokinetic properties of a mechanism-based suicide inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), essential for the survival of the eukaryotic parasite Trypanosoma brucei responsible for Human African Trypanosomiasis (HAT). The lead compound, 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (1, also known as MDL 73811, or AbeAdo), has curative efficacy at a low dosage in a hemolymphatic model of HAT but displayed no demonstrable effect in a mouse model of the CNS stage of HAT due to poor blood-brain barrier permeation. Therefore, we prepared and evaluated an extensive set of analogs with modifications in the aminobutenyl side chain, the 5'-amine, the ribose, and the purine fragments. Although we gained valuable structure-activity insights from this comprehensive dataset, we did not gain traction on improving the prospects for CNS penetration while retaining the potent antiparasitic activity and metabolic stability of the lead compound 1.

Product feedback regulation implicated in translational control of the Trypanosoma brucei S-adenosylmethionine decarboxylase regulatory subunit prozyme.

Human African sleeping sickness (HAT) is caused by the parasitic protozoan Trypanosoma brucei. Polyamine biosynthesis is an important drug target in the treatment of HAT. Previously we showed that trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme for biosynthesis of the polyamine spermidine, is activated by heterodimer formation with an inactive paralogue termed prozyme. Furthermore, prozyme protein levels were regulated in response to reduced AdoMetDC activity. Herein we show that T. brucei encodes three prozyme transcripts. The 3'UTRs of these transcripts were mapped and chloramphenicol acetyltransferase (CAT) reporter constructs were used to identify a 1.2 kb region that contained a 3'UTR prozyme regulatory element sufficient to upregulate CAT protein levels (but not RNA) upon AdoMetDC inhibition, supporting the hypothesis that prozyme expression is regulated translationally. To gain insight into trans-acting factors, genetic rescue of AdoMetDC RNAi knock-down lines with human AdoMetDC was performed leading to rescue of the cell growth block, and restoration of prozyme protein to wild-type levels. Metabolite analysis showed that prozyme protein levels were inversely proportional to intracellular levels of decarboxylated AdoMet (dcAdoMet). These data suggest that prozyme translation may be regulated by dcAdoMet, a metabolite not previously identified to play a regulatory role.

De-novo modeling and ESR validation of a cyanobacterial F(o)F(1)-ATP synthase subunit bb' left-handed coiled coil.

The structure and functional role of the dimeric external stalk of F(o)F(1)-ATP synthases have been very actively researched over the last years. To understand the function, detailed knowledge of the structure and protein packing interactions in the dimer is required. In this paper we describe the application of structural prediction and molecular modeling approaches to elucidate the structural packing interaction of the cyanobacterial ATP synthase external stalk. In addition we present biophysical evidence derived from ESR spectroscopy and site directed spin labeling of stalk proteins that supports the proposed structural model. The use of the heterodimeric bb' dimer from a cyanobacterial ATP synthase (Synechocystis sp. PCC 6803) allowed, by specific introduction of spin labels along each individual subunit, the evaluation of the overall tertiary structure of the subunits by calculating inter-spin distances. At defined positions in both b and b' subunits, reporter groups were inserted to determine and confirm inter-subunit packing. The experiments showed that an approximately 100 residue long section of the cytoplasmic part of the bb'-dimer exists mostly as an elongated alpha-helix. The distant C-terminal end of the dimer, which is thought to interact with the delta-subunit, seemed to be disordered in experiments using soluble bb' proteins. A left-handed coiled coil packing of the dimer suggested from structure prediction studies and shown to be feasible in molecular modeling experiments was used together with the measured inter-spin distances of the inserted reporter groups determined in ESR experiments to support the hypothesis that a significant portion of the bb' structure exists as a left-handed coiled coil.

Structural Basis and Kinetic Pathway of RBM39 Recruitment to DCAF15 by a Sulfonamide Molecular Glue E7820.

E7820 and indisulam are two examples of aryl sulfonamides that recruit RBM39 to Rbx-Cul4-DDA1-DDB1-DCAF15 E3 ligase complex, leading to its ubiquitination and degradation by the proteasome. To understand their mechanism of action, we performed kinetic analysis on the recruitment of RBM39 to DCAF15 and solved a crystal structure of DDA1-DDB1-DCAF15 in complex with E7820 and the RRM2 domain of RBM39. E7820 packs in a shallow pocket on the surface of DCAF15 and the resulting modified interface binds RBM39 through the α1 helix of the RRM2 domain. Our kinetic studies revealed that aryl sulfonamide and RBM39 bind to DCAF15 in a synergistic manner. The structural and kinetic studies confirm aryl sulfonamides as molecular glues in the recruitment of RBM39 and provide a framework for future efforts to utilize DCAF15 to degrade other proteins of interest.

Structure of the cytosolic part of the subunit b-dimer of Escherichia coli F0F1-ATP synthase.

The structure of the external stalk and its function in the catalytic mechanism of the F(0)F(1)-ATP synthase remains one of the important questions in bioenergetics. The external stalk has been proposed to be either a rigid stator that binds F(1) or an elastic structural element that transmits energy from the small rotational steps of subunits c to the F(1) sector during catalysis. We employed proteomics, sequence-based structure prediction, molecular modeling, and electron spin resonance spectroscopy using site-directed spin labeling to understand the structure and interfacial packing of the Escherichia coli b-subunit homodimer external stalk. Comparisons of bacterial, cyanobacterial, and plant b-subunits demonstrated little sequence similarity. Supersecondary structure predictions, however, show that all compared b-sequences have extensive heptad repeats, suggesting that the proteins all are capable of packing as left-handed coiled-coils. Molecular modeling subsequently indicated that b(2) from the E. coli ATP synthase could pack into stable left-handed coiled-coils. Thirty-eight substitutions to cysteine in soluble b-constructs allowed the introduction of spin labels and the determination of intersubunit distances by ESR. These distances correlated well with molecular modeling results and strongly suggest that the E. coli subunit b-dimer can stably exist as a left-handed coiled-coil.

Conformational changes in the Escherichia coli ATP synthase b-dimer upon binding to F(1)-ATPase.

Conformational changes within the subunit b-dimer of the E. coli ATP synthase occur upon binding to the F(1) sector. ESR spectra of spin-labeled b at room temperature indicated a pivotal point in the b-structure at residue 62. Spectra of frozen b +/- F(1) and calculated interspin distances suggested that where contact between b (2) and F(1) occurs (above about residue 80), the structure of the dimer changes minimally. Between b-residues 33 and 64 inter-subunit distances in the F(1)-bound b-dimer were found to be too large to accommodate tightly coiled coil packing and therefore suggest a dissociation and disengagement of the dimer upon F(1)-binding. Mechanistic implications of this "bubble" formation in the tether domain of ATP synthase b ( 2 ) are discussed.

Species-Selective Pyrimidineamine Inhibitors of Trypanosoma brucei S-Adenosylmethionine Decarboxylase.

New therapeutic options are needed for treatment of human African trypanosomiasis (HAT) caused by protozoan parasite Trypanosoma brucei. S-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the polyamine pathway of T. brucei. Previous attempts to target this enzyme were thwarted by the lack of brain penetration of the most advanced series. Herein, we describe a T. brucei AdoMetDC inhibitor series based on a pyrimidineamine pharmacophore that we identified by target-based high-throughput screening. The pyrimidineamines showed selectivity for T. brucei AdoMetDC over the human enzyme, inhibited parasite growth in whole-cell assay, and had good predicted blood-brain barrier penetration. The medicinal chemistry program elucidated structure-activity relationships within the series. Features of the series that were required for binding were revealed by determining the X-ray crystal structure of TbAdoMetDC bound to one analog. The pyrimidineamine series provides a novel starting point for an anti-HAT lead optimization.

Identification of Trypanosoma brucei AdoMetDC Inhibitors Using a High-Throughput Mass Spectrometry-Based Assay.

Human African trypanosomiasis (HAT) is a fatal infectious disease caused by the eukaryotic pathogen Trypanosoma brucei (Tb). Available treatments are difficult to administer and have significant safety issues. S-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the parasite polyamine biosynthetic pathway. Previous attempts to develop TbAdoMetDC inhibitors into anti-HAT therapies failed due to poor brain exposure. Here, we describe a large screening campaign of two small-molecule libraries (∼400,000 compounds) employing a new high-throughput (∼7 s per sample) mass spectrometry-based assay for AdoMetDC activity. As a result of primary screening, followed by hit confirmation and validation, we identified 13 new classes of reversible TbAdoMetDC inhibitors with low-micromolar potency (IC50) against both TbAdoMetDC and T. brucei parasite cells. The majority of these compounds were >10-fold selective against the human enzyme. Importantly, compounds from four classes demonstrated high propensity to cross the blood-brain barrier in a cell monolayer assay. Biochemical analysis demonstrated that compounds from eight classes inhibited intracellular TbAdoMetDC in the parasite, although evidence for a secondary off-target component was also present. The discovery of several new TbAdoMetDC inhibitor chemotypes provides new hits for lead optimization programs aimed to deliver a novel treatment for HAT.

Improved Synthesis of MDL 73811 - a Potent AdoMetDC Inhibitor and Anti-Trypanosomal Compound.

An improved synthesis of MDL 73811 - a potent AdoMetDC (S-adenosylmethionine decarboxylease) inhibitor and anti-trypanosomal compound with in vivo activity has been completed in four steps from commercially available 2',3'-O-isopropylideneadenosine. Utilization of Mitsunobu chemistry was crucial for the reliable and scalable introduction of the 5'-methylamine moiety, which was problematic using traditional activation/displacement chemistry as previously reported. All reactions in this synthesis were run on gram-scale resulting in a five-fold increase in yield over the original synthesis.

Relief of autoinhibition by conformational switch explains enzyme activation by a catalytically dead paralog.

Catalytically inactive enzyme paralogs occur in many genomes. Some regulate their active counterparts but the structural principles of this regulation remain largely unknown. We report X-ray structures of Trypanosoma brucei S-adenosylmethionine decarboxylase alone and in functional complex with its catalytically dead paralogous partner, prozyme. We show monomeric TbAdoMetDC is inactive because of autoinhibition by its N-terminal sequence. Heterodimerization with prozyme displaces this sequence from the active site through a complex mechanism involving a cis-to-trans proline isomerization, reorganization of a ß-sheet, and insertion of the N-terminal α-helix into the heterodimer interface, leading to enzyme activation. We propose that the evolution of this intricate regulatory mechanism was facilitated by the acquisition of the dimerization domain, a single step that can in principle account for the divergence of regulatory schemes in the AdoMetDC enzyme family. These studies elucidate an allosteric mechanism in an enzyme and a plausible scheme by which such complex cooperativity evolved.