Hexapeptides from a mammalian inhibitory hormone activate and inactivate nematode reproduction

Background Biopurification has been used to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. A (rat) bioassay-guided physicochemical fractionation using ovine materials yielded via Edman degradation a 14-residue amino acid (aa) sequence. As a 14mer synthetic peptide (EPL001) this displayed antiproliferative and reproduction-modulating activity, while representing only a part of the native polypeptide. Even more unexpectedly, a scrambled-sequence control peptide (EPL030) did likewise. Methods Reproduction has been investigated in the nematode Steinernema siamkayai, using a fermentation system supplemented with different concentrations of exogenous hexapeptides. Peptide structure-activity relationships have also been studied using prostate cancer and other mammalian cells in vitro, with peptides in solution or immobilized, and via the use of mammalian assays in vivo and through molecular modelling. Results Reproduction increased (x3) in the entomopathogenic nematode Steinernema siamkayai after exposure to one synthetic peptide (IEPVFT), while fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose-dependent. These hexamers are opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030). Bioactivity is unexpected as EPL030 is a control compound, based on a scrambled sequence of the test peptide MKPLTGKVKEFNNI (EPL001). EPL030 and EPL001 are both bioinformatically obscure, having no convincing matches to aa sequences in the protein databases. EPL001 has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nematode Caenorhabditis elegans. Peptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro, with reproductive upregulation demonstrated previously in fish and frogs, as well as nematodes. EPL001 encodes the sheep neuroendocrine prohormone secretogranin II (sSgII), as deduced on the basis of immunoprecipitation using an anti-EPL001 antibody, with bespoke bioinformatics. Six sSgII residues are key to EPL001’s bioactivity : MKPLTGKVKEFNNI. A stereospecific bimodular tri-residue signature is described involving simultaneous accessibility for binding of the side chains of two specific trios of amino acids, MKP & VFN. An evolutionarily conserved receptor is conceptualised having dimeric binding sites, each with ligand-matching bimodular stereocentres. The bioactivity of the 14mer control peptide EPL030 and its hexapeptide progeny is due to the fortuitous assembly of subsets of the novel hormonal motif, MKPVFN, a default reproductive and tissue-building OFF signal. NOTE Please see the end of the paper for links to ten files of supplementary information and for an independent peer review. KEY POINTS Synthetic hexapeptides have upregulated and downregulated nematode reproduction, following similar work in other nematode species and in fish and frogs. Peptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro and influence circulating levels of ovine pituitary hormones in vivo via intracerebroventricular peptide infusion. The amino acid sequence of the 14mer master peptide arose from a (rat) bioassay- guided fractionation using ovine materials, whose aim was to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. The bioactive peptides mimic the non-contiguous six-residue receptor-binding active face of a polypeptide hormone, which in mammals is a derivative of the secretory vesicle prohormone secretogranin II (SgII), taking the form of a novel, structually complex 70mer dubbed ‘SgII-70’. The ability has been gained to reinforce or block a default reproductive and tissue- building OFF signal, using peptide mimetics.

During a hunt for a postulated novel hormone that is reproductively related and tissue mass 123 reducing (Hart, 2014), ovarian follicular fluid and systemic blood plasma from sheep (Ovis 124 aries) were fractionated on the basis of bioassays in vivo (organ weight reduction) (Hart, 1999) 125 and in vitro (suppressed proliferation in bone marrow stem cells) in the rat (Rattus norvegicus) 126 (Hart, 2000). Edman degradation of an active fraction of sheep plasma yielded as the cleanest 127 N-terminal aa sequence MKPLTGKVKEFNNI (Hart, 2008), with variants on six other 128 occasions (Supplementary Information 1, Sequencing & Purification = S1 of 10). The sequence 129 was synthesized as a 14mer peptide with the proprietary designation EPL001 (which 130 designation will be used hereafter interchangeably to denote both the synthetic peptide and its 131 sequence). Resistant to conventional bioinformatic analysis (e.g. BLAST searches) and 132 molecular biology, EPL001 is more than an incomprehensible singularity: it is an 133 incomprehensible plurality, having been detected multiple times using Edman degradation (S1). 134 A logo plot is shown in Fig. 1  for the bioactivity of EPL001. Lower left is a minimized structural prediction (omitting general form as the grid path for sSgII-14, suggesting that the grid overall is structurally 159 predictive. 160 The 14-residue N-terminal Edman sequence EPL001 was determined in the context of a 161 polypeptide candidate of m/z ~7,500 ('Candidate 7500') in MALDI-TOF mass spectrometry 162 (MS) and a corresponding band in SDS polyacrylamide gel electrophoresis (Hart, 2008), 163 implying a chain of ~70 aa for the native molecule. In spite of comprising only 20% of 164 Candidate 7500, EPL001 surprised by being anti-organotrophic in its own right in an assay in 165 vivo of rat compensatory renal growth (Haylor et al, 2009)as indeed were Candidate 7500 166 fractions (MS validated) of ovine ovarian follicular fluid purified by spin filtration, gel filtration 167 and anion exchange chromatography (Hart, 2008) and by modulating reproduction in the 168 nematode Caenorhabditis elegans (Davies & Hart, 2008). Both these activities are aspects of 169 the inhibitory hormone hypothesis (Hart, 2014). The likelihood of a project-unrelated entity 170 having both the sought-for activities is low, especially when effects are evident in the cross-171 phylum manner characteristic of endocrinology. Additionally, anti-EPL001 antisera (hereafter 172 'antibodies') have provided localisation staining in mammals and in the fruit fly Drosophila 173 melanogaster in immunohistochemistry (IHC) of seeming neuroendocrine significance (Hart et 174 al, 2017). Immunostaining with anti-EPL001 antibodies was apparent in individual neurons in 175 the ovine lateral and ventromedial hypothalamus and preoptic area, for example, with heavy 176 staining in the palisade (neuroendocrine) region of the median eminence, with axonal beading 177 betokening transport. The findings overall were highly reminiscent of granin protein IHC, 178 particularly relating to secretogranin II (SgII: UniProt WEQEU8 in sheep), which is involved in 179 the biogenesis of neuroendocrine secretory granules and is a prohormone giving rise to 180 bioactive peptides. Further circumstantial evidence in support of the factor's granin identity is 181 target molecule acidity disclosed during purification (anion exchange chromatography), 182 apparent thermostability and stoichiometric effects of semi-purified factor in assays 183 (Bartolomucci et al, 2011). There is also a multi-way molecular weight concordance in gel 184 electrophoresis (Hart et al, 2017). Briefly, western blots of sheep plasma deliver a single band established prohormone, SgII (UniProt W5QEU8), the other candidates (e.g. dynein, titan) 211 being dismissable as unlikely to deliver a secreted derivative. SgII has two main sorting 212 domains in the form of stretches of aa directing newly synthesized SgII into intracellular 213 secretory vesicles (Courel et al, 2008). NNI is predicted to be physically adjacent to hSgII's 214 first sorting domain (AlphaFold, in the absence of an experimentally determined structure in 215 and share proximity in 3D space, as suggested by the sequence grid of Fig. 1. The prolinaceous 286 sextets were derived empirically, as bioactive, having a position-3 proline in common. Yet 287 preliminary molecular modelling in silico (RPN) drew the eye to EPL001's C-terminal FNNI as 288 a biologically relevant motif, as alluded to elsewhere (Hart, 2008), suggesting the involvement 289 of the Beale 4's FN. EPL001's C-terminal KEFNNI is the epitope of the anti-EPL001 antibody 290 (G530) and also probably the SgII-related endogenous epitope (Howlett et al, 2019), indicating 291 surface accessibility for the antibody in both cases. The epitope residues within EPL001 are 292 ranged in the correct order across the foot of the sequence grid. Why? Meanwhile, the proposed 293 active face residues are ranged across the top of the grid, in the form of MKP, dipping down to 294 VFN, with the sequence logo ( Fig. 1, lower panel) portraying these six residues as among the 295 landmarks in the physicochemical purification campaign. Layered across the middle of the 296 sequence grid are LTG, residues deemed uninvolved in antibody or receptor binding. Besides 297 matching up residues exogenous and endogenous, the sequence grid appears to offer a 298 contoured functional landscape. The results of the present peptide structure-activity work 299 arguably illuminate an evolutionarily conserved biological motif relating to a polypeptide 300 product of SgII, of relevance to reproductive status and tissue-mass determination. 301

MATERIALS AND METHODS 303
Peptides 304 The aa sequences of 21 chemically synthetized peptides are given in Table 1 for deployment is the production of 'infective juveniles'. These forms penetrate the larvae of 344 insect pests, releasing a bacterium, Xenorhabdus spp., which ultimately kills the larvae. Stock 345 solutions (5 mg mL -1 ) of EPL036 (IEPVFT) and EPL630 (KLKMNG) were made up and stored 346 at −20 o C. Lipid Agar plates were prepared by pouring 5 ml media in 60 mm plastic Petri dishes. 347 The plates were inoculated with an overnight-grown bacterial culture of Xenorhabdus spp. (10 9 348 cfu/ml) at 100 µl/plate and incubated at 28 o C for 24h. Xenorhabdus bacteria are symbiotically 349 associated with S. siamkayai. While multiplying, Xenorhabdus metabolises the constituents of 350 the lipid agar and the resulting by-products are the food supplement for the nematodes that 351 triggers the worms to grow and multiply. From the stock solutions, peptides were introduced 352 individually into the centre of the Lipid Agar plates with Xenorhabdus lawn. Two 353 concentrations of peptide were used, 4 and 8 µL of the 5 mg mL -1 stock peptide solutions 354 (making 20 and 40 µg of peptide per plate in total, respectively, i.e. ~6 and ~12 µM). Each 355 peptide solution was uniformly spread using an L shaped spreader and allowed to absorb into 356 the agar plate for 10 minutes. Nematodes were harvested beforehand as follows. Third-stage 357 infective juveniles (IJs) of S. siamkayai were used to infect fourth instar Galleria larvae. 358 maintained at 37℃ in a humidified incubator with 5%/95% CO2/O2. Incorporation of 3 H-380 thymidine was used to assess DNA synthesis. Cells were seeded overnight at 0.5 x 10 5 cells per 381 well in 24-well tissue culture plates in DMEM culture medium (supplemented as above with 382 10% FCS, penicillin and streptomycin). Cell growth was synchronised by culturing cells in 383 DMEM containing 0.1% bovine serum albumin (supplemented with penicillin and 384 streptomycin, but without FCS) for 24h. Cells were then exposed via fresh medium (with FCS) 385 to EPL001 or EPL030 to achieve a final concentration of 0.06 or 6 M, or to the aqueous 386 vehicle (controls), for 24h, before being pulsed for 48h with 1 Ci/ml 3 H-thymidine administered to groups of 2 mice intraperitoneally (i.p.) daily on Days 0-4 at 500 mg/kg/dose 458 and then 1000 mg/kg/dose. Following treatment, body weight was measured on a regular basis, 459 and behaviour and general appearance monitored visually to assess for deleterious effects (e.g. 460 dehydration, impaired mobility, hunched posture, low body temperature, ulceration and significant body weight loss), with any effects during the study recorded. If body weight loss 462 was >15% over a 72-hour period or if animal behaviour and appearance were significantly 463 altered, then mice were immediately sacrificed by cervical dislocation. If no deleterious effects 464 were seen after at least 16 days of study, then the animals were sacrificed, with the dose 465 considered non-toxic. 466

467
The MCF-7 human breast adenocarcinoma model was selected for xenograft studies. EPL001 468 inhibits the proliferation in vitro of MCF-7 cells stimulated to divide by insulin-like growth 469 factor 1 (IGF-1) (Hart, 2008). An oestrogen pellet was implanted subcutaneously in the dorsal Osteogenic peptides in one experiment in vitro were markedly more active in surface-485 immobilised form than when provided in solution (Lee et al, 2010), presumably due to a more enduring exposure of the cells. In the first study EPL001 was immobilised by either its N 487 terminus or C terminus and human stem cells exposed accordingly. This followed a 488 confirmation of dissolved EPL001's antiproliferative activity in the same laboratory (JAH). In 489 the second study, aa sections of EPL001 were substituted with alanine strings prior to tethering, 490 to define a functional biological motif. Into the base of each well of a 24-well plate was inserted  Table 1 were constructed by sequential additions of amino acid residues. Each 520 model was adjusted in conformation to minimize energy levels: energy minimization was 521 carried out in 1-fs time steps, to a total of 10,000 fs, with 100 equilibrium steps per iteration. 522 Iterations were continued until six repeat iterations yielded no change in energy gradient. In the 523 first instance analysis of interatomic distances involved identifying 21 atoms in the amino acid 524 side chains and the peptide backbone of the master test peptide EPL001 and designating these 525 atoms a-u. A subset of six of these was then selectedwithin the hypothesis of SgII-relatedness 526 and granted that protein interactions primarily involve aa side chainsrelating to atoms in the 527 side chains of the proposed active site residues M (= m), K (= a), P (= p), V (= q), F (= e) and N 528 (= i). These comprise the carbon, nitrogen or sulphur atoms in the relevant amino acid side 529 chains most remote from the peptide bond. As such, these atoms would be free to show the 530 most intramolecular movement thence variation in subsequent interatomic distances after 531 energy minimization. The distances of each of these atoms was measured from the others, 532 yielding 15 measurements in all for EPL001, in angstrom units (Å). These distances were then 533 measured in each of the other peptides of Table 1, as appropriate, given differing aa 534 compositions. Distances between pairs of atoms were computed automatically after atoms were 535 selected manually on-screen. Each measurement was repeated twice more after closing the model and reloading to verify the initial measurement. Twenty peptides provided the observed 537 (O) distances for a chi-squared hypothesis test of SgII-relatedness, with the expected (E) 538 interatomic distances from EPL143, a contiguous version of sSgII-14 (Fig. 1). The χ 2 formula is 539 Σ (O -E) 2 /E. This was solved online (https://goodcalculators.com/chi-square-calculator/), with 540 double-checking online (https://www.danielsoper.com/statcalc/calculator.aspx?id=11) and 541 manually. Complementing the dimensional comparison is an analysis of tri-residue topology: 542 tri-residue, because stereospecificity requires a minimum of three points of attachment (Easson 543 & Stedman, 1933;Ogston, 1948), a number conferring analytical tractability in the present 544 context; and topology, used here to describe structural arrangements in space in relation to side 545 chain accessibility for receptor binding. In brief, molecular models in silico were rotated to 546 provide 3D views of sets of three aa residues (notably MKP & VFN), read in the order of the 547 primary peptide sequence and viewed in clockwise and counter-clockwise directions. Each of 548 the tri-residue views was then assessed visually as to whether, for all three specified aa 549 residues, there was collective availability for receptor binding or if access was blocked by other 550 residues. In full, energy-minimized complete ball and stick models were subject to hydrogen 551 atom deletion (for ease of visualization), with retention of overall 3D peptide configuration. 552 Tri-residues were read in the order in which they appeared in the peptide primary structure and 553 viewed either clockwise or counter-clockwise. Models were slowly rotated so that two of the aa 554 side chains in a tri-residue module were visible: the model was then further rotated to determine 555 whether all of the atoms of the third tri-residue aa side chain could be visualised, or only 556 partially obscured by its own atoms or those of the other two tri-residue side chains, while 557 retaining visibility of the first two tri-residue side chains. If all three side chains could be so 558 visualised, the module was designated accessible for receptor binding. In the event of 559 obstruction by a non-tri-residue aa of a tri-residue aa, the module was deemed inaccessible. 560 Each assessment of accessibility was carried out three times (replicated twice, with model identities concealed), commencing from the original minimized complete ball and stick model. 562 Other parameters examined were point charges (Del Re: Huckel), electrostatic surface maps, 563 hydrophilic surface areas, solubility, partition coefficients, dihedral angles of side chains around 564 peptide bonds, triatomic bond angles relating to the six active face residues and lipophilicity, as 565 well as molecular volumes, surface areas and overall dimensions. dependently. At 4 days the majority of the first-generation EPL036-exposed IJs had 616 transformed into mature adults which subsequently mated and went on to produce second-617 generation IJs (J1, J2, J3 and pre-adults), with adults barely represented, implying a die-off 618 among these forms after an accelerated life cycle. In contrast, EPL630 suppressed to a 619 significant degree the development of second-generation IJs, with adults at 26-29% of the 620 population at termination, compared to controls at ~23%. IJs in the higher dose groups at both 621 inoculum levels were more than three times as numerous for EPL036 than controls at 622 termination and less than half as numerous than controls for EPL630. 623 624 Observationally, infective juveniles (~0.8 mm in length) when released on EPL036 grew and 625 matured at a faster rate than worms in the other groups and within 24h they had transformed 626 into pre-adults (males and females). In another 12h (i.e. 36h after incubation) they had 627 developed into fully grown males (~1.5 mm) and females (~5.0 mm) and mating had 628 commenced. In comparison, infective juveniles exposed to EPL630 or exposed to no peptide 629 (i.e. untreated controls) transformed into pre-adults in about 36h and developed into males and 630 females in the next 12h (i.e. 48h after incubation). With differences in development and 631 maturation rates came differences in size. EPL036 females were bigger than those in the other 632 two groups. They were visibly bloated with eggs compared with untreated controls, whereas 633 egg production in EPL630-exposed females was markedly below that of controls (Fig. 3). Apart 634 from dual inocula and dual dose levels, a duplication is provided by a pilot study, which gave 635 similar outcomes (S3). Both the 14mer peptide EPL001 and its scrambled-sequence control, EPL030, were 670 antiproliferative (Fig. 4). A dose-response relationship was more clearly evinced by EPL030 671 than EPL001. In the assays involving different sized peptides the results were similar for PC3 672 and PNT2 cells, with the smaller items associated with lower ODs than the 14mers (Fig. 5) Assays involved a range of peptides from 14mers down to 6mers (Table 1), with viable cells 706 assessed using a 96-well plate MTS colorimetric assay. Absorbance measured at 492 nm. Cells 707 were seeded at 8,000 per well and grown for 27h. Peptides were administered at the outset to a 708 final concentration of 30 µM (in 2 µl) to samples in triplicate. Controls received buffer. Data 709 are OD mean +/-SEM, with statistical analysis by two tailed t-test. * p<0.05; **p<0.025. 710

Bone marrow cell assays 711
Whereas EPL001 was antiproliferative in this system, EPL030 was additionally pro-apoptotic 712 ( Fig. 6) as, to a more remarkable extent still, was EPL120, the 20mer extension of EPL001. The 713 relative potencies were reversed when proline-containing hexamers from the 14mers were tried: 714 MKPLTG (EPL016) from MKPLTGKVKEFNNI (EPL001) was a more potent inhibitor than 715 IEPVFT (EPL036) from KLKMNGKNIEPVFT (EPL030) (Fig. 7). 716 718 719 Figure 6. Bone marrow cells in culture exposed to long peptides. Cells were counted over 720 24h after exposure to the 14mer peptide EPL001 or its scrambled-sequence control EPL030 at 721 6µM final concentration or to the 20mer EPL120 at 5µM final concentration, compared to 722 peptide-untreated controls. 723 items. Assuming each arrangement is equally likely the probability of selecting GH>LH >PRL 741 for EPL001 is 1/6. Similarly, the probability of selecting the same order for EPL030 is also 1/6. 742 The probability of selecting GH>LH>PRL for both peptides by chance is therefore 1/6 x 1/6 = 743 1/36 = 0.028 or 2.8%. Given that P = 0.028 the observed ranking arrangement GH>LH>PRL 744 is unlikely to have occurred by chance. The consistency of these results from a pair of 745 anagrammatical 14mer peptides argues against non-specific effects, though that was the initial In the evaluation of toxicity over 14 days in mice, both EPL001 and EPL030 were well-750 tolerated at relatively high doses (See S5, Murine Studies). No discernible toxicity was seen 751 with either EPL001 or EPL030 at 500mg/kg/day ip Days 0 to 4, with a non-significant 752 maximum weight loss (compared to the initial starting weight on Day 0) of 5.0% and 4.0% 753 respectively on Day 2. A higher dose of 1000mg/kg/day ip on Days 0 to 4 was then tried for both peptides. Again, no discernible signs of toxicity were seen, however in this case weight 755 loss was sustained for longer with EPL030 (maximum weight loss of 7.0% on Day 3) than 756 EPL001 (maximum weight loss of 3.5% on Day 1). The foregoing reinforces a positive safety 757 perception in regard to the peptides under investigation (Davies et al, 2015). 758

759
In the MCF-7 tumour xenograft study, no statistically significant tumour growth delay was seen 760 with either EPL001 or EPL030 compared to controls, although there was evidence of a slight 761 reduction in the rate of tumour growth as the experiment progressed, more obviously with 762 EPL030 (−20.2% at termination compared with PBS-only controls, ns)(S5 Fig. 3 Tucking in these aa to make EPL601, by eliminating EPL143 residues posited to be uninvolved 864 in binding (xLKTGExxxxKxNI), potentiates activity (Fig. 5). 865 866 Why is the EPL001 scrambled control peptide EPL030 active, for example in a bone marrow 867 cell assay (Fig. 6), more so indeed than EPL001? There is a significant probability of structural 868 dissimilarity with the SgII benchmark, as we have seen: P = 0.024. This implies that the activity 869  6.01 (1.17). The possession of PVF presumably accounts for the bioactivity 880 of EPL036 in a mammalian cell assay (Fig. 7) and for its pro-fecundity activity in S. siamkayai (Fig. 2). PVF in EPL036 is +/− in terms of tri-residue topology, the same co-accessibility as is 882 seen for the PVF in the hexamer benchmark EPL601. Looking at the other end of EPL030, why 883 is its N-terminal hexamer EPL630 anti-fecundity in S. siamkayai? The chi-squared analysis says 884 that EPL630 is not significantly dissimilar to EPL143: P = 0.088. The active face residues in 885 949 EPL120 was markedly more inhibitory than EPL001 (Fig. 6). Why? Even though EPL120 is 950 half a dozen residues longer than EPL001, there are only relatively minor differences between 951 these peptides in terms of the 15 measured interatomic distances, with none differing by more 952 than an angstrom. For both EPL001 and EPL120 the side chains of MKP are accessible for 953 binding when this tri-residue is viewed in either clockwise or counter-clockwise orientations 954 (Table 1). VFN is available for binding in both peptides when this tri-residue is in a clockwise orientation but whereas VFN is unavailable in EPL001 when viewed counter-clockwise, it is 956 available in EPL120. Extending EPL001 to EPL120 seemingly induces a positive 957 conformational change in terms of receptor binding. 958

959
The inactive EPL801 is EPL601 with EPL001's C-terminal doubleton tacked on: MKPVFNNI. 960 The addition was not predicted to reduce activity as EPL120 has NI C-terminally and 6 other 961  (Table 1). For comparison, EPL120 is + +/+ + and EPL143 is + −/+ +. The 971 two trios in EPL601 are arranged with complementary accessibility, hinged at proline. The 972 accessibility for EPL801's MKP is again clockwise, but VFN is inaccessible this time in both 973 orientations: + −/− −. Unlike in EPL601's VFN counter-clockwise orientation, EPL801's N6 is 974 not co-accessible (Fig.9) An aspiration in biology is control of fundamentals. One fundamental is reproduction. The 993 results described here confirm that a hexapeptide of aa sequence IEPVFT (EPL036 in Table 1)  994 is a reproductive activator, providing among Steinernema siamkayai entomopathogenic 995 nematodes a superabundant monoculture of the economically important infective juveniles, 996 while another hexamer, KLKMNG (EPL630), is revealed to be a novel reproductive 997 inactivator. The peptides were provided at commencement ambiently via an agar substrate. The 998 effects were dependent on dose but not on the number of worms present at the start of the study. 999 In a study in C. elegans, genital tract localisation was observed after ambient exposure to a 1000 19mer extended version of the 14mer master peptide EPL001, C-terminally labelled with 1001 fluorescein (Davies & Hart, 2008). Ingestion can be assumed to explain this uptake, rather than 1002 cuticular diffusion, and the same can be assumed here for S. siamkayai, but route of ingress has 1003 not been investigated and neither has the molecular mode of action. The present work is a 1004 replication study for EPL036. With two peptide dose levels and two worm population levels, there was a double duplication inherent in a study that was in any case prefigured by a pilot 1006 study conveying the same result (S3) by a new principal investigator (SM) in a new species of 1007 nematode using peptide from a different supplier. yet EPL001 displays on its own in vivo the two crucial features of the sought-for hormone: anti-prior assay using the same system, cells exposed to EPL001 were significantly fewer than Peptide studies frequently in fact involve generating a scrambled-sequence (anagrammatical) 1103 peptidethat is, a control peptide of the same aa composition and MW but devoid of relevant 1104 sequence-related activity, though possibly confounding for other reasons (e.g. nutrient effect, fortuitous assembly of an unrelated biological motif etc.). This is the provision of a negative 1106 control (Moser, 2020). How best to scramble aa sequences? Rule-based randomisation is 1107 suggested, rather than unregulated randomisation used here. NIEPVFT. Referring to the proposed active face residues, MKP is present in that tri-residue 1125 order, variously spaced, in all three control peptides -EPL010, EPL030 and EPL040and 1126 V•F•N is to be found additionally in EPL010 (Table 1). 1127 1128 A failure of randomisation is available for inspection in the form of EPL630. This hexamer of 1129 sequence KLKMNG was anticipated to be an inactive control in nematode breeding studies, but turned out to be a reproductive inactivator. The KM in EPL630 is a reversal of the MK in distances. EPL630's KMN, a tight trimer within KLKMNG, paradoxically displays wide half-endogenous inhibitor). In this scheme EPL036 acts as a binding-accessible (+/−), narrowly 1181 bimodular (xxP-VFx) antagonist, as per a prediction of antagonistic activity from prior 1182 molecular modelling (Davies et al, 2015), to counteract an endogenous inhibitory hormone. assuming that less than a full complement of aa is sufficient for activity. Each of the three 1190 alanine-substituted EPL001 peptides has at least half of the six active face residues, explaining 1191 retention of antiproliferative activity within the bimodularity concept. The minimum necessary 1192 for stereospecificity is three points of attachment (Easson & Stedman, 1933;Ogston, 1948). 1193 Compared with EPL601 (MKPVFN), EPL001 has a similar MKP and independently a similar 1194 V•FN, the distances between these two modules in the 14mer bearing no relationship to those in 1195 the 6mer. In the peptide immobilisation study in vitro, N-terminally immobilised EPL001 was 1196 less inhibitory of stem cell proliferation than C-terminally tethered EPL001. The probable 1197 reason for this is that the two effector modules have differential binding affinities to cell 1198 membrane receptors in this system, with the forward module MKP being more avid than the 1199 rearward module V•FN. Bimodularity as between MKP and VFN is an implicit prediction of 1200 the Fig. 1 sequence grid: in spite of P and V being covalently linked in the sSgII scheme they 1201 are in different sectors of the grid. EPL601, MKPVFN, is posited to be a biomodular effector 1202 hinged at P. The EPL601 both-modules-together binding is compromised by elongating the 1203 hexamer to provide EPL801: MKPVFNNI. EPL801's N6 departs the VFN space (Fig. 9) and 1204 intrudes into the MKP space, providing steric hindrance. EPL120, the 20mer extended form of the 14mer EPL001, and EPL601, the 6mer minimisation of EPL001, are both more anti-1206 proliferative than EPL001 in mammalian cell assays, as is the 14mer sSgII benchmark EPL143. 1207 In the tri-residue topological analysis only three ovine test peptides have the stereospecific 1208 signature 'MKP clockwise, VFN counter-clockwise': EPL120, EPL143 and EPL601 (Table 1). 1209 As between its two ligand modules, the unpredictable master test peptide EPL001 has adverse 1210 topology, 'MKP clockwise, VFN clockwise', while the inactive EPL801 merely boasts 'MKP 1211 clockwise'. At issue with these ligands is simultaneous accessibility for binding, or the lack 1212 thereof, of two trios of aa side chains. 1213  Table 1 this  1218 is 'VFN '. The viewing starts at V (VAL 4), moving counter-clockwise to F (PHE 5) then N 1219 (ASN 6). The VFN side-chains are in the forward plane in EPL601 and accessible together for 1220 binding (+). In EPL801 they are not co-accessible (−), as ASN 6 projects rearwards. 1221 available single stereocentre in the two receptor sites: M'K'P', V'F'N', M"K"P" or V"F"N". The 1247 binding to a single stereocentre might impinge on other EPL001 bindings through steric 1248 hindrance, prejudicing dual occupancy, accounting for the unpredictable activity of EPL001. 1249 The EPL030 control peptide would bind to two stereocentres within a single receptor site, as it 1250 boasts a bimodular 2/3 rd residue complement of K•PVF rather than either of EPL001's 1251 unimodular half complements, MKP and V•FN. A second EPL030 molecule might well be able 1252 to bind with the other receptor site. These differences explain the bizarre finding that 1253 randomising EPL001 yields, in EPL030, a stronger agonist cell inhibitor in mammalian 1254 systems. EPL030 was deduced to be acting to anti-fecundity effect in nematodes as an agonist. 1255 Within the inhibitory hormone hypothesis, this peptide, appreciated now as a crypto-test 1256 compound, would be expected to be an agonist inhibitor in mammalian cell assaysand it is. 1257 Why is EPL001 also inhibitory in these systems when it is a pro-fecundity antagonist in 1258 nematodes, blocking an endogenous brake? (This question is also relevant to the pro-fecundity 1259 peptides EPL016 and EPL036.) This is presumably because mammalian cell cultures lack the 1260 endogenous hormonal inhibitor, so EPL001 behaves as a weak agonist to inhibitory effect (ditto 1261 As to receptor location, a hint is available from nematode histology and pharmacology. 1273 Concentration in the genital tract of C. elegans was seen after exposure to a fluorescent 1274 analogue of EPL001 (antagonist, pro-fecundity) but not with an analogue of EPL030 (agonist, 1275 anti-fecundity), from which an uninformative generalised picture was obtained (Davies & Hart, 12762008. 1277

1278
The suggestion has been made that the body has a reproductive hormonal brake against tissue 1279 overgrowth, which brake is lifted to initiate puberty and the assumption of adult stature and 1280 whose action wanes with age, leading to a rise in cancers and the enlargement of the prostate 1281 (Hart, 2014). The factor hunt that arose from this suggestion has progressed as follows: 1282 Inhibitory Hormone Hypothesis  Candidate 7500  EPL001  SgII Relatedness. The 1283 final step is supported by neuroendocrine IHC, together with MW and other circumstantial 1284 evidence of granin identity (see Introduction); an anti-EPL001 antibody immunoprecipitation 1285 leading to mammalian 'likely SgII relatedness' and a fruit fly homologue of SgII; 1286 unconventional bioinformatics (the ovine NNI-ome) identifying the potential relevance to 1287 EPL001 of the second sorting domain of SgII, consideration of which opened up the possibility 1288 of SgII-based epitope and active face analyses and led to the sequence grid of Fig. 1; and the 1289 14mer constructs EPL141 and EPL143 (representing h/sSgII-9 + EPL001's F, K & NNI) being 1290 more cell-inhibitory than straight EPL001 itself, while sSgII-9 (MLKTGEKPV = EPL901) on 1291 its own is also active (Fig. 5). Gene expression has been studied in human bone marrow stem 1292 cells in vitro using RT-qPCR (Rogers, 2018). This is in regard to the core circadian clock genes 1293  Table  1325 1, Column 2. The grid predicts that after sSgII-14's K11, the string loops off and comes back as 1326 a C-terminal NNI. This would facilitate the involvement of both ends of sSgII-70 in (i) aberrant 1327 Edman sequencing, (ii) antibody binding and (iii) the assembly of a non-contiguous active 1328 hormonal face for receptor binding. Explained by this hairpin structure is why EPL001, 1329 representing just 20% of Candidate 7500, has the activity of the sought-for inhibitory hormone. 1330 EPL001 is the aberrant reading of the two ends of the hairpin, which together contribute to the 1331 active face. Cross-linking can be suspected, as effecting the hairpin and bringing FK and NNI 1332 together, though the absence in sSgII-70 of cysteine residues (a secretogranin feature), to 1333 provide stabilising disulphide bonds, makes the chemical character of this conjectural. The 1334 EPL001 sequencing zigzag in Fig. 1 suggests the reading of a spiral: so, a cross-linked, 1335 spiralised polypeptide hairpinwith C-terminal polyanionic clustering for good measure: 1336 48ExxDEExxxxxDDEDD63.

1338
The Edman reagent, phenyl isothiocyanate, reacts with the α-amino groups of peptide 1339 molecules anchored to a solid phase, to liberate a cyclic derivative of the N-terminal residue for 1340 chromatographic identification (Smith, 2001). The process then repeats itself. The assumption 1341 here is that in automated sequencing the Edman reagent behaved faithfully in latching on to α-1342 amines and that the non-provision of a database-meaningful N-terminal sequence is due to 1343 target molecule chemistry. Nothing like Edman Nonsequentialism, as it might be called, has 1344 been reported since the method was first described (Edman, 1950). Yet Edman 1345 Nonsequentialism is not a conjecture, it must be emphasized, but an observation, given the 1346 variant sequences obtained from the physicochemical purification campaign. The available 1347 seven sequences have much in common with one another in terms of residue incidence and suggesting picks around an amphipathic spiral, while the last two, FN, imply physical 1374 proximity; and there is the speculation of cross-linkage to explain the structural hairpin 1375 presumed necessary to bring together FK and NNI. 1376 residues comprise two functional groups of three: MKP and V/NFN. Proteins interact with one 1423 another primarily via the side chains of surface amino acids. There is enrichment of certain aa at 1424 protein interaction sites as compared with their incidence across protein surfaces generally 1425 (Sillerud & Larson, 2005). In the enrichment league's third and fourth places are M and F, 1426 respectively, the former the most prevalent of the aliphatics at interaction sites and the latter an 1427 aromatic, all of which are over-represented. No enrichment is seen with the other active face 1428 residues, K, P, V and N, yet a bimodularity analysis of bioactive ovine peptides emphasized the 1429 importance of KP in the forward module and V in the rearward module. 1430 1431 Peptide structure influences ligand binding for sure, but in complex systems in vitro and in vivo 1432 also impacts solubility, permeation, half-life, resistance to hydrolysis and other factors 1433 unconsidered here, including altered substrate specificity for enzymes such as proprotein 1434 convertase and carboxypeptidase, meaning that changes in peptide effects may reflect a factor 1435 independent of its biological activity. Limitations of the research further include the 1436 consideration that the peptides have not all been tested together in the same assay system. 1437 sSgII-70 has been used to make sense of analytical findings and peptide bioactivity, but its 1438 existence is a deduction. The attempt to develop peptide mimetics of the active face of a 1439 deduced entity might appear quixotic, were it not for the rewards on offer (tissue mass 1440 downregulation and reproductive modulation) and the apparent success of the journey from 1441 fortuitous 14mer (EPL001) to honed hexamers (including EPL036 and EPL601), for other 1442 researchers to explore. The SgII-70 model that has emerged from peptide mimetics is 'a subset 1443 of the six active face residues in appropriate topological conformity will work'. In terms of a 1444 ligand-receptor lock-and-key model what is envisaged is a ligand 'key' with six ridges, 1445 organised in two stereospecific groups of three (MKP & VFN). Two of these keys at the same 1446 time access a receptor 'double lock' with plenty of give in it. The lock is possibly a granin-style

INDEPENDENT PEER REVIEW (reprinted with permission) 1708
Reviewer (Joseph Banoub) Basic reporting In this manuscript, the authors present an extensive biopurification study aimed to reveal an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. For this reason, they examined the reproduction of the nematode Steinernema siamkayai following fermentation in a system supplemented with different concentrations of exogenous hexapeptides.
The authors used a (rat) bioassay which permitted the physicochemical fractionation (using ovine materials) and Edman degradation, which established the presence of a 14-residue amino acid peptide.

Authors' response
No. The presence was established of an ~70-residue amino acid peptide. This was subject to Edman degradation, providing a 14-residue N-terminal sequence. This was synthesized as the 14-mer synthetic peptide EPL001. This distinction between the Edman sequence result (14 residues) and the much longer native molecule (~70 residues) has now been clarified, below.
Also, they found that the 14-mer synthetic peptide (EPL001), MKPLTGKVKEFNNI, displayed anti-proliferative and reproduction-modulating activity. Moreover, they unexpectedly found that the scrambled-sequence control peptide (EPL030) did likewise.
In addition, they also studied a series of hexapeptides for potential uses for the agonists. Furthermore, they established that reproduction increased (x3) after exposure to one synthetic peptide (IEPVFT), while fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose dependent. They also used hexamers with opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030).
Additionally, they established that EPL001 possessed anti-proliferative effects on human prostate cancer cells and on the rat bone marrow cells. Also, the intra-cerebroventricular infusion of EPL001 in sheep showed elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless had similar biological effects in common in mammalian systems, whereas divergently anti-fecundity respectively in the nematode Caenorhabditis elegans.
Peptides up to a 20mer were shown to inhibit the proliferation of human cancer and other mammalian cells in vitro, with reproductive upregulation demonstrated previously in fish and frogs, as well as nematodes.
EPL001was also found to encodes the sheep neuroendocrine prohormone secretogranin II (sSgII). This was deduced on the basis of immunoprecipitation using an anti-EPL001 antibody. And six sSgII residues were responsible for the EPL001bioactivity.
Finally, the authors describe a stereospecific bimodular tri-residue signature involving simultaneous accessibility for binding of the side chains of two specific trios of amino acids, MKP & VFN. An evolutionarily conserved receptor was proposed as having dimeric binding sites, each with ligand-matching bimodular stereocentres.
The bioactivity of the 14mer control peptide EPL030 and its hexapeptide progeny is due to the fortuitous assembly of subsets of the novel hormonal motif, MKPVFN, a default reproductive and tissue building OFF signal.
This manuscript is very well written in Literarily English. It was so well written that this referee found himself wondering what the authors were trying to say. The Introduction part provides an adequate scientific background to pinpoint the aim of this work, the literature references are sufficient.
Although, this referee finds this manuscript long, this is not necessarily a fault, as this length is due to the very comprehensive body of work presented. The article structure, figures, tables and raw data shared are first class.
A quick perusal of the KEY POINTS presented with the manuscript indicate that they are relevant, meaningful and performed to a high technical standard.
The following are some examples that this referee found obscure.
1. In your Abstract, you have written the following: Background. A biopurification campaign aimed to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination.
Query: What was written makes no sense. I presume the biopurification campaign was fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose dependent. They also used hexamers with opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030).

Validity of the findings
This manuscript validity can be judged by the results obtained from this research.
Indeed, the results obtained are accurate according to the researcher explanation, and prediction. These could be surmised as follows: The purified ovine materials, the 14-residue aa sequence designated EPL001, MKPLTGKVKEFNNI, has proved to be deeply encoded within the mammalian genome.
The hexapeptides could be used in reproductive activation (EPL036: IEPVFT an antagonist of a hormonal OFF signal, delivering reproductive disinhibition) and Although, this referee finds this manuscript long, this is not necessarily a fault, as this length is due to the very comprehensive body of work presented. The article structure, figures, tables and raw data shared are first class.