Effects of a long-acting secretin peptide analog alone and in combination with a GLP-1R agonist in a diet-induced obesity mouse model.

OBJECTIVE: Obesity is a major global health problem which can be targeted with new mechanistic diverse pharmacological interventions. Here we evaluate a new long-acting secretin receptor agonist as a potential treatment for obesity. METHODS: BI-3434 was designed as a secretin analog with stabilized peptide backbone and attached fatty acid-based half-life extension group. The peptide was evaluated in vitro for its ability to stimulate cAMP accumulation in a cell line stably expressing recombinant secretin receptor. On the functional level, stimulation of lipolysis in primary adipocytes after treatment with BI-3434 was determined. The ability of BI-3434 to activate secretin receptor in vivo was assessed in a cAMP reporter CRE-Luc mouse model. Furthermore, a diet-induced obesity mouse model was used to test the effects of BI-3434 on body weight and food intake following repeated daily subcutaneous administration alone and in combination with a GLP-1R agonist. RESULTS: BI-3434 potently activated human secretin receptor. However, lipolysis was only weakly induced in primary murine adipocytes. BI-3434 had an extended half-life compared to endogenous secretin and activated target tissues like pancreas, adipose tissue, and stomach in vivo. BI-3434 did not lower food intake in lean or diet-induced obese mice, but it increased energy expenditure after daily administration. This led to a loss of fat mass, which did not translate in a significant effect on body weight. However, treatment in combination with a GLP-1R agonist led to a synergistic effect on body weight loss. CONCLUSIONS: BI-3434 is a highly potent and selective agonist of secretin receptor with an extended pharmacokinetic (PK) profile. Increased energy expenditure after daily treatment with BI-3434 suggests that secretin receptor is involved in metabolic regulation and energy homeostasis. Targeting secretin receptor alone may not be an efficient anti-obesity treatment, but could be combined with anorectic principles like GLP-1R agonists.

BI 456906: Discovery and preclinical pharmacology of a novel GCGR/GLP-1R dual agonist with robust anti-obesity efficacy.

OBJECTIVE: Obesity and its associated comorbidities represent a global health challenge with a need for well-tolerated, effective, and mechanistically diverse pharmaceutical interventions. Oxyntomodulin is a gut peptide that activates the glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) and reduces bodyweight by increasing energy expenditure and reducing energy intake in humans. Here we describe the pharmacological profile of the novel glucagon receptor (GCGR)/GLP-1 receptor (GLP-1R) dual agonist BI 456906. METHODS: BI 456906 was characterized using cell-based in vitro assays to determine functional agonism. In vivo pharmacological studies were performed using acute and subchronic dosing regimens to demonstrate target engagement for the GCGR and GLP-1R, and weight lowering efficacy. RESULTS: BI 456906 is a potent, acylated peptide containing a C18 fatty acid as a half-life extending principle to support once-weekly dosing in humans. Pharmacological doses of BI 456906 provided greater bodyweight reductions in mice compared with maximally effective doses of the GLP-1R agonist semaglutide. BI 456906's superior efficacy is the consequence of increased energy expenditure and reduced food intake. Engagement of both receptors in vivo was demonstrated via glucose tolerance, food intake, and gastric emptying tests for the GLP-1R, and liver nicotinamide N-methyltransferase mRNA expression and circulating biomarkers (amino acids, fibroblast growth factor-21) for the GCGR. The dual activity of BI 456906 at the GLP-1R and GCGR was supported using GLP-1R knockout and transgenic reporter mice, and an ex vivo bioactivity assay. CONCLUSIONS: BI 456906 is a potent GCGR/GLP-1R dual agonist with robust anti-obesity efficacy achieved by increasing energy expenditure and decreasing food intake.

BILN: A Human-Readable Line Notation for Complex Peptides.

We present an easy, human-readable line notation to describe even complex peptides.

Characterization of combined linagliptin and Y2R agonist treatment in diet-induced obese mice.

Dipeptidyl peptidase IV (DPP-IV) inhibitors improve glycemic control by prolonging the action of glucagon-like peptide-1 (GLP-1). In contrast to GLP-1 analogues, DPP-IV inhibitors are weight-neutral. DPP-IV cleavage of PYY and NPY gives rise to PYY3-36 and NPY3-36 which exert potent anorectic action by stimulating Y2 receptor (Y2R) function. This invites the possibility that DPP-IV inhibitors could be weight-neutral by preventing conversion of PYY/NPY to Y2R-selective peptide agonists. We therefore investigated whether co-administration of an Y2R-selective agonist could unmask potential weight lowering effects of the DDP-IV inhibitor linagliptin. Male diet-induced obese (DIO) mice received once daily subcutaneous treatment with linagliptin (3 mg/kg), a Y2R-selective PYY3-36 analogue (3 or 30 nmol/kg) or combination therapy for 14 days. While linagliptin promoted marginal weight loss without influencing food intake, the PYY3-36 analogue induced significant weight loss and transient suppression of food intake. Both compounds significantly improved oral glucose tolerance. Because combination treatment did not further improve weight loss and glucose tolerance in DIO mice, this suggests that potential negative modulatory effects of DPP-IV inhibitors on endogenous Y2R peptide agonist activity is likely insufficient to influence weight homeostasis. Weight-neutrality of DPP-IV inhibitors may therefore not be explained by counter-regulatory effects on PYY/NPY responses.

SAR matrices: automated extraction of information-rich SAR tables from large compound data sets.

We introduce the SAR matrix data structure that is designed to elucidate SAR patterns produced by groups of structurally related active compounds, which are extracted from large data sets. SAR matrices are systematically generated and sorted on the basis of SAR information content. Matrix generation is computationally efficient and enables processing of large compound sets. The matrix format is reminiscent of SAR tables, and SAR patterns revealed by different categories of matrices are easily interpretable. The structural organization underlying matrix formation is more flexible than standard R-group decomposition schemes. Hence, the resulting matrices capture SAR information in a comprehensive manner.

The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses.

Baculoviruses are ubiquitous insect viruses well known for their use as bioinsecticides, gene therapy vectors, and protein expression systems. Overexpression of recombinant proteins in insect cell culture utilizes the strong promoter of the polyhedrin gene. In infected larvae, the polyhedrin protein forms robust intracellular crystals called polyhedra, which protect encased virions for prolonged periods in the environment. Polyhedra are produced by two unrelated families of insect viruses, baculoviruses and cypoviruses. The atomic structure of cypovirus polyhedra revealed an intricate packing of trimers, which are interconnected by a projecting N-terminal helical arm of the polyhedrin molecule. Baculovirus and cypovirus polyhedra share nearly identical lattices, and the N-terminal region of the otherwise unrelated baculovirus polyhedrin protein sequence is also predicted to be alpha-helical. These results suggest homology between the proteins and a common structural basis for viral polyhedra. Here, we present the 2.2-A structure of baculovirus polyhedra determined by x-ray crystallography from microcrystals produced in vivo. We show that the underlying molecular organization is, in fact, very different. Although both polyhedra have nearly identical unit cell dimensions and share I23 symmetry, the polyhedrin molecules are structurally unrelated and pack differently in the crystals. In particular, disulfide bonds and domain-swapped N-terminal domains stabilize the building blocks of baculovirus polyhedra and interlocking C-terminal arms join unit cells together. We show that the N-terminal projecting helical arms have different structural roles in baculovirus and cypovirus polyhedra and conclude that there is no structural evidence for a common evolutionary origin for both classes of polyhedra.

Development of benzophenone-based farnesyltransferase inhibitors as novel antimalarials.

The development of farnesyltransferase inhibitors directed against Plasmodium falciparum is a strategy towards new drugs against malaria. Previously, we described benzophenone-based farnesyltransferase inhibitors with high in vitro antimalarial activity but no in vivo activity. Through the introduction of a methylpiperazinyl moiety, farnesyltransferase inhibitors with in vivo antimalarial activity were obtained. Subsequently, a structure-based design approach was chosen to further improve the antimalarial activity of this type of inhibitor. As no crystal structure of the farnesyltransferase of the target organism is available, homology modeling was used to reveal differences between the active sites of the rat/human and the P. falciparum farnesyltransferase. Based on flexible docking data, the piperazinyl moiety was replaced by a N,N,N'-trimethylethylenediamine moiety. This resulted in an inhibitor with significantly improved in vitro and in vivo antimalarial activity. Furthermore, this inhibitor displayed a notable increase in selectivity towards malaria parasites relative to human cells.

Sulfate acts as phosphate analog on the monomeric catalytic fragment of the CPx-ATPase CopB from Sulfolobus solfataricus.

The crystal structure of the catalytic fragment of a Sulfolobus solfataricus P-type ATPase, CopB-B, was determined with a 2.6 A resolution. CopB-B is the major soluble fragment of the archaeal CPx-ATPase CopB and is comprized of a nucleotide and a phosphorylation domain. In the crystalline state two molecules of CopB-B are in close contact to each other, although the presence of dimers in free solution could be ruled out by analytical ultracentrifugation. The overall architecture of CopB-B is similar to that of other P-type ATPases such as Ca-ATPase. Short peptide segments are linking the nucleotide binding to the phosphorylation domain. CopB-B exhibits 33% sequence identity (of 216 aligned residues) with the respective fragment of the Archaeoglobus fulgidus ATPase CopA. The CopB-B nucleotide-binding domain has the most primitive fold yet identified for this enzyme class. It is 24% identical to the nucleotide-binding domain of the disease-related Wilson ATPase ATP7B (80 structurally aligned residues). Structural superposition with Ca-ATPase suggests a putative nucleotide-binding site in CopB-B. The phosphorylation domain of CopB-B is structurally related to the corresponding part of Ca-ATPase in the anion-bound E2 state. In CopB-B crystals, a bound sulfate anion was identified at the phosphate-binding location. In solution state, the potential binding of CopB-B to phosphate was probed with (32)P(i). Bound phosphate could be readily displaced by orthovanadate at submillimolar concentration as well as by sulfate at millimolar concentration. It is possible therefore to assign the structure of the sulfate-bound phosphorylation domain of CopB-B to a state related to the E2.P(i) intermediate state of the catalytic cycle.

The molecular organization of cypovirus polyhedra.

Cypoviruses and baculoviruses are notoriously difficult to eradicate because the virus particles are embedded in micrometre-sized protein crystals called polyhedra. The remarkable stability of polyhedra means that, like bacterial spores, these insect viruses remain infectious for years in soil. The environmental persistence of polyhedra is the cause of significant losses in silkworm cocoon harvests but has also been exploited against pests in biological alternatives to chemical insecticides. Although polyhedra have been extensively characterized since the early 1900s, their atomic organization remains elusive. Here we describe the 2 A crystal structure of both recombinant and infectious silkworm cypovirus polyhedra determined using crystals 5-12 micrometres in diameter purified from insect cells. These are the smallest crystals yet used for de novo X-ray protein structure determination. We found that polyhedra are made of trimers of the viral polyhedrin protein and contain nucleotides. Although the shape of these building blocks is reminiscent of some capsid trimers, polyhedrin has a new fold and has evolved to assemble in vivo into three-dimensional cubic crystals rather than icosahedral shells. The polyhedrin trimers are extensively cross-linked in polyhedra by non-covalent interactions and pack with an exquisite molecular complementarity similar to that of antigen-antibody complexes. The resulting ultrastable and sealed crystals shield the virus particles from environmental damage. The structure suggests that polyhedra can serve as the basis for the development of robust and versatile nanoparticles for biotechnological applications such as microarrays and biopesticides.

Crystal structure of Bacillus subtilis S-adenosylmethionine:tRNA ribosyltransferase-isomerase.

The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine. It is unprecedented in nature as it uses the cofactor S-adenosylmethionine as the donor of a ribosyl group. We have determined the crystal structure of Bacillus subtilis QueA at a resolution of 2.9A. The structure reveals two domains representing a 6-stranded beta-barrel and an alpha beta alpha-sandwich, respectively. All amino acid residues invariant in the QueA enzymes of known sequence cluster at the interface of the two domains indicating the localization of the substrate binding region and active center. Comparison of the B. subtilis QueA structure with the structure of QueA from Thermotoga maritima suggests a high domain flexibility of this enzyme.

Benzophenone-based farnesyltransferase inhibitors with high activity against Trypanosoma cruzi.

Less toxic drugs are needed to combat the human parasite Trypanosoma cruzi (Chagas's disease). One novel target for antitrypanosomal drug design is farnesyltransferase. Several farnesyltransferase inhibitors based on the benzophenone scaffold were assayed in vitro and in vivo with the parasite. The common structural feature of all inhibitors is an amino function which can be protonated. Best in vitro activity (LC50 values 1 and 10 nM, respectively) was recorded for the R-phenylalanine derivative 4a and for the N-propylpiperazinyl derivative 2f. These inhibitors showed no cytotoxicity to cells. When tested in vivo, the survival rates of infected animals receiving the inhibitors at 7 mg/kg body weight/day were 80 and 60% at day 115 postinfection, respectively.

Non-thiol farnesyltransferase inhibitors: N-(4-aminoacylamino-3-benzoylphenyl)-3-[5-(4-nitrophenyl)-2 furyl]acrylic acid amides and their antimalarial activity.

Water solubility was previously found to be essential for in vivo-antimalarial activity of a novel type of benzophenone-based farnesyltransferase inhibitors. Introduction of a alpha-amino group into the phenylacetic acid substructure provided more soluble compounds with high farnesyltransferase inhibitory activity. The in vitro-antimalarial activity was detrimentally influenced by this structural modification.

Non-thiol farnesyltransferase inhibitors: N-(4-tolylacetylamino-3-benzoylphenyl)-3-arylfurylacrylic acid amides.

We have designed arylfurylacryl-substituted benzophenones as non-thiol farnesyltransferase inhibitors utilizing a novel aryl binding site of farnesyltransferase. These compounds display activity in the low nanomolar range.

Dial tm for rescue: tmRNA engages ribosomes stalled on defective mRNAs.

Ribosomes translate genetic information encoded by mRNAs into protein chains with high fidelity. Truncated mRNAs lacking a stop codon will cause the synthesis of incomplete peptide chains and stall translating ribosomes. In bacteria, a ribonucleoprotein complex composed of tmRNA, a molecule that combines the functions of tRNAs and mRNAs, and small protein B (SmpB) rescues stalled ribosomes. The SmpB-tmRNA complex binds to the stalled ribosome, allowing translation to resume at a short internal tmRNA open reading frame that encodes a protein degradation tag. The aberrant protein is released when the ribosome reaches the stop codon at the end of the tmRNA open reading frame and the fused peptide tag targets it for degradation by cellular proteases. The recently determined NMR structures of SmpB, the crystal structure of the SmpB-tmRNA complex and the cryo-EM structure of the SmpB-tmRNA-EF-Tu-ribosome complex have provided first detailed insights into the intricate mechanisms involved in ribosome rescue.

Structure and functional analysis of the fungal galectin CGL2.

Recognition of and discrimination between potential glyco-substrates is central to the function of galectins. Here we dissect the fundamental parameters responsible for such selectivity by the fungal representative, CGL2. The 2.1 A crystal structure of CGL2 and five substrate complexes reveal that this prototype galectin achieves increased substrate specificity by accommodating substituted oligosaccharides of the mammalian blood group A/B type in an extended binding cleft. Kinetic studies on wild-type and mutant CGL2 proteins demonstrate that the tetrameric organization is essential for functionality. The geometric constraints due to the orthogonal orientation of the four binding sites have important consequences on substrate binding and selectivity.

Crystal structure of the transfer-RNA domain of transfer-messenger RNA in complex with SmpB.

Accurate translation of genetic information into protein sequence depends on complete messenger RNA molecules. Truncated mRNAs cause synthesis of defective proteins, and arrest ribosomes at the end of their incomplete message. In bacteria, a hybrid RNA molecule that combines the functions of both transfer and messenger RNAs (called tmRNA) rescues stalled ribosomes, and targets aberrant, partially synthesized, proteins for proteolytic degradation. Here we report the 3.2-A-resolution structure of the tRNA-like domain of tmRNA (tmRNA(Delta)) in complex with small protein B (SmpB), a protein essential for biological functions of tmRNA. We find that the flexible RNA molecule adopts an open L-shaped conformation and SmpB binds to its elbow region, stabilizing the single-stranded D-loop in an extended conformation. The most striking feature of the structure of tmRNA(Delta) is a 90 degrees rotation of the TPsiC-arm around the helical axis. Owing to this unusual conformation, the SmpB-tmRNA(Delta) complex positioned into the A-site of the ribosome orients SmpB towards the small ribosomal subunit, and directs tmRNA towards the elongation-factor binding region of the ribosome. On the basis of this structure, we propose a model for the binding of tmRNA on the ribosome.

The disulfide bond isomerase DsbC is activated by an immunoglobulin-fold thiol oxidoreductase: crystal structure of the DsbC-DsbDalpha complex.

The Escherichia coli disulfide bond isomerase DsbC rearranges incorrect disulfide bonds during oxidative protein folding. It is specifically activated by the periplasmic N-terminal domain (DsbDalpha) of the transmembrane electron transporter DsbD. An intermediate of the electron transport reaction was trapped, yielding a covalent DsbC-DsbDalpha complex. The 2.3 A crystal structure of the complex shows for the first time the specific interactions between two thiol oxidoreductases. DsbDalpha is a novel thiol oxidoreductase with the active site cysteines embedded in an immunoglobulin fold. It binds into the central cleft of the V-shaped DsbC dimer, which assumes a closed conformation on complex formation. Comparison of the complex with oxidized DsbDalpha reveals major conformational changes in a cap structure that regulates the accessibility of the DsbDalpha active site. Our results explain how DsbC is selectively activated by DsbD using electrons derived from the cytoplasm.