A NOVEL PEPTIDE, A MICROBIOLOGICAL PROCESS FOR ITS PRODUCTION AND USE THEEOF AS AN ANTIBIOTIC, CYTOTOXIC AND PHYTHOTOXIC AGENT

Abstract: A novel peptide isolated from submerged cultures of fungus Paecilomyces marquandii (syn. Spicaria violacea) showing an antibiotic, cytotoxic and phythotoxic activity.

Description: "A novel peptide, a Microbiological process for its production and use thereof as an antibiotic, cytotoxic and phythotoxic agent" SPECIFICATION The present invention relates to a novel peptide compound, a microbiological process for its production and the use thereof as an antibiotic, cytotoxic and phythotoxic agent. Description of the prior art Previous studies on submerged cultures of Fungus paecilomyces marquandii (syn. Spicaria violacea) have shown the presence of a number of substances having an antibiotic, cytotoxic and phythotoxic activity. From said substances the main components of formulas I and II have been isolated and analyzed (PhythopatoLogi a Mediterranea - Vol. XXII, 1983, pages 103-106 and 209- 211) MeEse-MePro-AIMOD-Hyleu-Aib-Leu-Leu-Aib-Aib- βAla-DAP (I) MeEse-MePro-AIMOD-Hyleu-Aib-Leu-Leu-Aib-Aib- |3Ala-MAP (II) wherein the abbreviations have the following meaning: MeEse 3 o<-methyIhexenoi c acid MePro ci s-4-methyIproline AIM0D 2-amino-6-hydroxy-4-methyl- 8-ketodecanoic acid HyLeu -treo- (-■* -hydroxyleuci ne Aib c -aminoisobutyric acid Leu leuci ne (3-Ala β -alanine DAP dimethyla i no-2-ami nopropane MPD methylami no-2-ami nopropane Peptide compounds I and II have resulted identical with Leucinostatins A and B ndependently isolated by a different group of researchers from Paecilomyces lilacinus CCA, 1983, Vol. 98, 15460x). Fungus Paecilomyces marquandii (syn. Spicaria violacea) has been deposited at CBS (Centraalbureau voor Schimmelcutures, 3740 A6 Baarn, The Netherlands) under the deposit number CBS 141.85, dated 5 February 1985. Supaary of the invention Upon further studies on isolation and characterization of the components in the mixture of substances obtained from submerged cultures, according to the invention another peptide substance not described in the prior art was separated from the culture filtrates obtained by benzene extraction. The novel substances has the following formula III: MeEse-MePro-Leu-HyLeu-Aib-Leu-Leu-Aib-Aib-β Ala-DAP (III) wherein the abbreviations have the same meaning as indicated above, having an antibiotic, cytotoxic and phythotoxic activity. An object of the present invention is a novel peptide * compound of formula III MeEse-MePro-Leu-HyLeu-Aib-Leu-Leu-Aib-Aib-j3 Ala-DAP (III) A Further object of the present invention is a microbiological process for the production of the novel peptide, characterized in that Fungus Paeci lomyces marquandi i (syn. Spi cari a vi olacea) is treated in submerged cultures, comprising: a) a first inoculation step, b) a second multiplication step, c) a third production step in a sterile, aerated and pressurized environment, and d) a final step of solvent extraction of the substances so obtained and chromatographi c separation for obtaining the pep ide compound III. The t ird step can be carried out by using the sparger and antivortex system, or simpl fied vortex system, or an air-lift system. Solvents which can be used in the extraction step are benzene, butyl acetate, chloroform, butanol and si ilar solvents of fats. Benzene is particularly preferred for its higher extraction yield. Culture media which can be used are semi syntheti c media (for example media in which the organic source is yeast autolysate) or complex organic media based upon by-products of food industry (flour from peanut and soy extraction, corn steep liquor, potato and bean broth and the like). A further object of the present invention is the use of the peptide as an antibiotic, cytotoxic and phythotoxic agent. o Example The compound according to the present invention is obtained in a mixture with the other leuci nostati ns (10% of the whole) at the third step in submerged cultures: first inoculation step in flasks containing 3% potato-glucose broth at pH 6 for 5-6 days on a , rotating stirrer (220 rpm) at 27°C; second multiplication step in a 5 liter Erlenmeyer flask containing 900 ml of the same medium, by incubation for three days in similar conditions; third production step in Czapek- Dox fer entors of effective 50 liters with 0.1% yeast autolysate, 3% glucose at pH 6, in conventional conditions (propeller 130 mm, 200 rpm, sparger, 3 antivortexes, 27°C, 1 atm pressure, 50 liters/min outlet air). The toxin extraction is generally effected on the fourth fermentation day, 24 hours after glucose exhaustion. The culture broth, after mycelium filtration was repeatedly extracted ith benzene at the fermentation pH (7.6 - 7.8); after solvent evaporation, a light yellow oily residue is obtained (about 7 g/100 liters) comprising at least about ten substances of basic character as shown by paper e lect rophoresi s and by the fact that they can be detected with the Dragendorff reagent . After continuous ch romotographi es on silica gel column with suitable eluent mixtures, the practically pure compound III was obtained from the residue, besides the compounds I and II. Compound III FAB MS m/z 1118 (MH + ), m.p. 182-184°C L • 6 (c 1.05 CHCl 3 ), UV(EtOH) λ max 202 nm C £ =27930), 220 ( 6 =18380), IR (CHCI3) V max 3310, 1650 cm -1 , elemental analysis: found: C, 59.91; H, 9.50; N, 13.62; 0, 16.78; for C57H-103 N 11 °11 - H 2° calc. %: C, 60.26; H, 9.25; N, 13.56; 0, 16.91. The peptide nature of compound III is self-evident from the chemical-physical data and from examination of the H and 13 C RMN spectra. Compound III gave a negative reaction to the ninhydrine test and to any methylation attempt and a positive reaction to the ' Rei nde l-Hoppe reagent. These reactions suggest the presence of a peptide having blocked terminal C and N atoms. Compound III was hydrolized with HCl 6N at 110°C for 24 hours in a sealed tube and conventionally analyzed by an automatic analysis apparatus for aminoacids. The results show that it consists of one mole of hydroxy leucine (HyLeu), one mole ci s-4-methyIproli ne (HePro), three moles < ■ •*- ■ -aminoi sobutyri c acid ( Aib), three moles of leucine (Leu) and one mole of -alanine ( β -AIa) listed in the order of their increasing retention times; said proportions are also easily deduced from the 1 H RMN spectra (see Table I). By extraction of the acid hydrolysate with ethtyl ether an oily residue was obtained, w ich, after purification, showed to be 3- <-•*. -methyIhexenoi c acid (MeEse). L 1 H RMN (90 MHz; CDCl 3 ) : 0.83 (3H, t, J=7 Hz), 1.05 ( 3H, d, J=7 Hz, 1.43 ( 2H, m), 2.24 (1H, m), 5.73 (1H, d, J=16 Hz), 6.93 (1H, dd, J=16 and 8 Hz; 13 C RMN (25 MHz; CDCl 3 ) S : 171.23 (s), 156.68 (d), 118.79 (d), 37.72 (d), 28.29 (q), 18.29 Cq)]. The acid hydrolysate, after careful a Ika li ni zati on with NaOH aqueous solution, was continuously extracted with ethyl ether. The strongly basic oily residue after purification, resulted corresponding to dimethylamino- 2-aminopropane [_DAP_| . |~ 1 H RMN (90 MHz; CDCI3) S : 1.34 (3H, d, J=7 Hz), 3.07 and 3.09 (3H each, s), 3.47 (2H, m) and 3.90 (1H, m; 13 C RMN (25 MHz; CDCI3) 6 - 18.7 (q), 42.6 (d), 44.76 (2xq), 63.6 (t)| . The complete attribution of the proton spectrum as listed in Table I has been made possible by using mono and bidimensi ona I spectra (COSY) at 400 MHz. The 57 signals (see Table II) which appear in the 13 C spectrum (CDCI3; 50 MHz) show chemical shifts and multiplicities in accordance with the allotted structure. TABLE I 1 H RMN spectrum ETHYLHEXENOIC UNITS C<-AMINOISOBUTYRIC UNITS 2-H 6.30 dd J=15.4; 1 .25 2-NH 8.16 s f 7.99 s, 7.65 s 3-H 6.67 dd J=15.4; 7 .3 2-CH 1.54 s; 1.53 s, 1.48 s, 1.46 s, — 3 • 4-H 2.25 m 1.43 s, 1.42 s. METHYLPROLINE UNITS 3-H 2 1.60 m 4-H 2-H 4.26 dd J=6.5; 11 .5 4-CH„ 0.87 d J=7 — 3 3-H 2.25 m 4-CH 0.88 d J=7 — . * . 1.85 m 4-H 1.80 m LEUCINE C or B UNITS 5-H 3.92 m 4-CH 3 1.05 d J=6.7 2-NH 7.22 d J=5.7 2-H 3.83 m LEUCINE A UNITS 3-H 2 1.90 m - 4-H ~ I 2-NH 8.75 d J=6.5 4-CH 0.89 d J=6.7 — 3 i 2 ~__ 4.92 ddd J=6.5; 6 .5; 8.5 4-CH 0.94 d J=6.3 — 3 I 3-H 1.99 m 1.70 m - ALANINE UNITS I -b 4-H 1.62 m i 5 -^3 0.85 d J=6.7 3-NH 8.22 dd J=3.7; 9.5 0.86 d J=6.2 3-H 3.96 m i J -a 3.17 m HYDROXYLEUCI E UNITS 2.93 ddd J=14.0; 12.5; 2.5 2.23 m 2-NH 8.57 d J=4.5 2-H 3.93 m DIMETHYLAMINO-2-AMINOPROPANE UNITS 3-H 3.67 lq 3 4 4 4 TABLE II Chemical shifts of *• 3 C RMN listed in a decreasing order. 187.86; 176.54; 175.58; 175.47; 175.37; 174.62; 173.76; 173.28; 172.74; 167.05; 152.2 120.17; 74.89; 63.51; 63.05; 60.82; 56.76; 56.59; 56.44; 56.33; 55.35; 54.89; 54.79; 44.88; 42.38; 41.31; 39.68; 39.34; 37.96; 37.42; 33.58; 30.52; 28.89; 27.33; 27.22; 27.06; 26.72; 25.03; 24.86; 24.67; 23.13; 23.10; 22.79; 22.51; 22.41; 21.87; 21.71; 21.55; 21.32; 21.22; 19.57; 19.46; 18.70; 18.58; 17.45; 16.52; 11.48. The sequence of the units forming the peptide compound III was deduced by FAB mass spectrum based upon two main fragmentation schemes +CA and +CAlk, as hereinafter referred : MeEse MePro Leu HyLeu AIB Leu Leu AIB 1118 > 1008 ■ 897 -^ 784 ^ 655 ^ 570 - 457 -> 344 -> (882) (769) . (640) (555) ' (442) (328) ( 945 m/z Sequence = MH + -HNCH CH CO-NHCH(CH )CH N(CH )„ 2 2 3 2 3 2 Leu HyLeu AIB Leu Leu*^ AIB AIB- 222 —335 - 464 <- 549 < 662 <- 775 <• 860 -ζ 945 The foregoing evidences the structure of formula III for the peptide according to the present invention. Farmacolog cal activity The "in vitro" antibacterial acti ity of the peptide compound according to the present invention was verified by carrying out a test on staphy lococci recently isolated in hospital, which are resistant to some antibiotics. The results are referred in Table III. The results of additional tests on antibacterial and antimycotic activity are referred in Tables IV and V. The minimum inhibiting concentra on (MIC) for determining the antibacterial and antimycotic activity was determined as follows. 5 mg of the compound of the invention were dissolved into 0.5 ml dimethylsulfo ide and diluted to 10 ml with sterile distilled water; portions of the above starting solution are subsequently diluted with sterile di sti I led water . Table III compares the activity of compound III with cefazoline on different resistent staphylococci isolated in different hospitals in Lombardy (Italy) Table III "In vitro" antibacterial activity on recently in hospital - isolated resistant stafi lococci of compound III in coimparison with |3 -lactamic cefazoline. Inhibiting concentrations C jm/ml). STAPHILOCOCCUS AUREUS COMPOUND III CEFAZOLINE RHO 5734 6.2 0.2 RHO 58299 12.5 0.2 N 280 6.2 0.2 N 417 ' 25 0.8 N 667 6.2 0.4 N 611 6.2 0.8 N 612 12.5 Q.8 Sacco 623 6.2 0.8 N 038 6.2 1.6 a N 625 6.2 100 Como 658 b 12.5 6.2 N 585 6.2 100 N 844 6.2 100 I.I.M. P b 6.2 100 C I.I.M. D 6.2 100 c N 102 6.2 100 c N 081 12.5 >100 d N 589 6.2 100 d N 766 6.2 100 d I.I.M. A 6.2 >100 d I.I.M. F 25 100 a = penici llin G resistant b = metici lliπ resistant c = metici llin and erythromycin resistant d = meti ci llin, erythromicin and spi ra ycin resistant TABLE I V "In vitro" antibacterial activity. Minimal inhibiting concentration (yum/ml) in sensitivity test Agar (oxoi d) Strains Compound III 5 Streptococcus pneumoniae ATCC 6301 12.5 St reptococuccus j3 -haemolyti cus C 203 12.5 Streptococcus faecalis ATCC 6057 25.0 Staphylococcus aureus Smith 6.2 Staphylococcus aureus 39/2 (penicillin resistant) 6.2 10 Escherichia coli 0147/ISS 100.0 Pseudomonas aeruginosa 100.0 Salmonella typhi 100.0 Shigella sonnei B68 100.0 Proteus vulgaris 100.0 15 TABLE V "In vitro" antimycotic activity. Minimal inhibiting concentration ( jm/ l) in Agar Sabouraud. Strains Compound III Microsporum gypseu ATCC 14683 >100.0 •* ' υ Microsporum cookey Negroni >100.0 Microsporum canis Pavia 3.1 Trichophyton mentagrophytes I. dC 6.2 Trichophyton mentagrophytes F 0.8 " Trichophyton tonsurans L 0.8 Candida albicans CBS 562 10.0 Candida albicans L 100.0 Candida utilis 93/ISS 25.0 Torulopsis famata 4077/ISS 2.0 30 Torulopsis stellata 4122/ISS 25.0 Cryptococcus neoformans 4710/ISS 4.0 Saccharomyces cerevisiae 92/ISS 25.0 Microsporum canis 2155/ISS 4.0 Trichophyton verrucosum 1466/ISS 2.0 35 Tricosporon undulatom 6805/ISS 10.0 Tricosporon cutaneum 6742/ISS 45.0 Cytotoxic activity The cytotoxic activity of the peptide according to the present invention was evaluated on some cell Lines, such as HeLA, KB, and the results of the concentration inhibiting a 50% cell growth are referred in Table VI. Table VI "In vitro" cytotoxic activity. Concentration I 50) inhibiting a 50% cell growth ( ug/ml). Cell line II> 50 HeLA 820.00 KB 0.95 P.388/F < 1.00 P.388 adriamycin resistant from 100.00 to 1000.00 Phythotoxic activity The phythotoxic activity evaluated by the modified Gaumann test (Ann. 1st. Super. Sanita, 4., 317-332. (1968)) on Supermarmande tomato cuttings is 2 /jg/ml. Said concentration produces, after a 2 days inhibition, an irreversible and progressive withering of the tested cuttings. DL50 calculated with white Swiss mice resulted practically identical both intravenously and int raperi toneal ly (0.7 mg/kg body weight). International Application No: PCT/ MICROORGANISMS Optional Sheet In connection with In* microorganism referred tβ on pap* J . Una J -__ Q ol th* daacrlptlon > A. IDINTII.CATION Of OEPOSIT • Further depβalte ara Idantiflad on an additional aheet Q' Nam* ol dapoellary Inatltutlon « Centraalbureau voor Schimmelcultures Addraaa *l dapoaltaηr Inatltutlon (Including poatal coda and country) * 3740 AG Baarn, The Netherlands Data ol depoaK * Accaaalon Number • 5 February 1985 CBS 141.85 ADDITIONAL INDICATIONS ' (laav* blank II not applicable). Thla Information la continued on a aaparat* attached *h*et Q C. DESIONATCD STATIS FOR WHICH INDICATIONS AM MADE • (II the Indication* at* not lor all dealgnatod 5._i*a) O. SEPARATE FURHISHINO OF INDICATIONS • (leave blank H not applicable) The Indication* Hated below wlH be aubmltted to the International Bureau later* (Specify the genersl nature ol th* Indication* e.p-, ** Acceaaion Number ol DepoaM**) E. Q Thla i m waa received with the international application whan filed (to be checked by th* receiving Office) (Authorited Officer) [ I The del* ol receipt (Irom the applicant) by the International Bureeu >• wee (Authorised Officer) fott-t PCf HO <J» . «->u«'r ">')

Claims: C lai ms 1. The peptide compound of formula (III) MeEse-MePro-Leu-HyLeu-Aib- eu-Leu-Aib-S Ala-DAP (III) 2. A microbiological process for producing a peptide compound of formula (III) as recited in claim 1, characterized in that the fungus Paecilomyces marquandii (syn. Spicaria violacea) is treated in submerged cultures by the steps of 0 a) inoculation; b) multiplication; c) fermentative production in sterile, aerated and pressurized environment followed by separation by solvent extraction of a mixture of substances 15 and insulation by chromatographic separation of the compounds of formula (III). 3. An antibiotic against gram-positive microorganisms, anti ycotic, cytotoxic and phythotoxic active agent consisting of the peptide cor un of formula (III), -^ as defined in claim 1. 4. Composition having an antibiotic against gram- positive microorganisms, and an antimycotic, cytoto ic and phythotoxic activity, comprising the peptide compound of formula (III) as recited in claim 1, and compatible 25 excipient and carriers. J5MBOTT1JTE § HWS

Inventors: CASINOVI CARLO GIULIO, ROSSI CARLO, TUTTOBELLO LORENZO, CASINOVI, CARLO, GIULIO, ROSSI, CARLO, TUTTOBELLO, LORENZO
Applicants: CONSIGLIO NAZIONALE RICERCHE, IST SUPERIORE SANITA, SANITA IST SUPERIORE
Application year: 1986
Main classification: C07K7
IPC codes: C07K7