Short bioactive peptides and methods for their use

[0001] The present application claims priority to U.S. Provisional Patent Application Serial No. 60/279,505 filed Mar. 28, 2001, and to pending U.S. patent application Ser. No. 09/820,053 filed Mar. 28, 2001.

[0002] The invention relates to short length peptides containing phenylalanine, leucine, alanine, and lysine amino acid residues (F, L, A, and K; FLAK peptides) in their primary sequence. In particular, FLAK peptides having desirable antimicrobial, antifungal, anticancer, and other biological activities are disclosed.

[0003] Various bioactive peptides have been reported in both the scientific literature and in issued patents. Peptides historically have been isolated from natural sources, and have recently been the subject of structure-function relationship studies. Additionally, natural peptides have served as starting points for the design of synthetic peptide analogs.

[0004] A review of peptide antibiotics was published by R. E. W. Hancock in 1997 (Lancet 349: 418-422). The structure, function, and clinical applications of various classes of peptides were discussed. An additional review of cationic peptide antibiotics was published in 1998 (Hancock, R. E. W. and Lehrer, R. Trends Biotechnol. 16: 82-88). The peptides are typically cationic amphipathic molecules of 12 to 45 amino acids in length. The peptides permeabilize cell membranes leading to the control of microbial agents. The clinical potential of host defense cationic peptides was discussed by R. E. W. Hancock in 1999 (Drugs 57(4): 469-473; Antimicrobial Agents and Chemotherapy 43(6): 1317-1323). The antibacterial, antifungal, antiviral, anticancer, and wound healing properties of the class of peptides are discussed.

[0005] Reviews of the structural features of helical antimicrobial peptides, and their presumed mechanisms of action have been published (see, for example, Dathe, M. and Wieprecht, T. Biochimica et Biophysica Acta 1462: 71-87 (1999); Epand, R. M. and Vogel H. J. Biochimica et Biophysica Acta 1462: 11-28 (1999)). Structural parameters believed to be capable of modulating activity and selectivity include helicity, hydrophobic moment, hydrophobicity, angle subtended by the hydrophilic/hydrophobic helix surfaces, and charge.

[0006] A wide array of naturally occurring alpha helical peptides have been reported. The following are representative of the many references in the field.

[0007] Cecropins are a family of -helical peptides isolated from insects. Cecropins are known for their antibacterial properties, as described in U.S. Pat. Nos. 4,355,104 and 4,520,016. The cecropins were generally found to have activity against gram-negative bacteria, but not against all gram-negative bacteria. Cecropins were found not to have activity against eucaryotic cells (Andreu, et al., Biochemistry 24: 163-188 (1985); Boman, et al., Developmental and Comparative Immunol. 9: 551-558 (1985); Steiner et al., Nature 292: 246-248 (1981)). Cecropins from Drosophila and Hyalphora were presented as having activity against various strains of fungi (Ekengren, S. and Hultmark, D., Insect Biochem. and Molec. Biol. 29: 965-972 (1999)). Cecropin A from mosquito Aedes aegypti is reportedly different from most insect cecropins in that it lacks tryptophan and C-terminal amidation (Lowenberger, C. et al., J. Biol. Chem. 274(29): 20092-20097 (1999)).

[0008] Frogs from the genus Rana produce a wide array of antimicrobial peptides in their skin (Goraya, J. et al., Eur. J. Biochem. 267: 894-900 (2000)). Peptides as short as 13 amino acids were reported, and were grouped into structural families. The sequences showed little or no sequence identity to peptides isolated from frogs of other genera, such as the magainin and dermaseptin peptides.

[0009] U.S. Pat. No. 5,962,410 disclosed the inhibition of eucaryotic pathogens, and the stimulation of lymphocytes and fibroblasts with lytic peptides such as cecropins and sarcotoxins. Various peptides presented include Cecropin B, Cecropin SB-37, Cecropin A, Cecropin D, Shiva-1, Lepidopteran, Sarcotoxin 1A, Sarcotoxin 1B, and Sarcotoxin 1C.

[0010] Transgenic mice producing the Shiva-1 cecropin class lytic peptide were reported by Reed, W. A. et al., Transgenic Res. 6: 337-347 (1997). Infection of the transgenic mice with a Brucella abortus challenge resulted in a reduction of the number of bacteria relative to infection of non-transgenic mice.

[0011] Magainin is an -helical 23 amino acid peptide isolated from the skin of the African frog Xenopus laevis (Zasloff, M. Proc. Natl. Acad. Sci. U.S.A. 84: 5449-5453 (1987).

[0012] Cathelin associated -helical peptides of 23 to 38 amino acids are found in the blood cells of sheep, humans, cattle, pigs, mice, and rabbits (Zanetti, M. et al., FEBS Lett. 374: 1-5 (1995)).

[0013] The antimicrobial activities of buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin were reported by Giacomette, A. et al. (Peptides 20: 1265-1273 (1999)). The peptides showed variable activities against bacteria and yeast.

[0014] Various synthetic peptides have been prepared and assayed both in vitro and in vivo.

[0015] U.S. Pat. No. 5,861,478 disclosed synthetic lytic peptides of about 20 to 40 amino acids which adopt an -helical conformation. The peptides are effective in the treatment of microbial infections, wounds, and cancer. The peptides disclosed include cecropin B, SB-37*, LSB-37, SB-37, Shiva 1 and 10-12, -fibrin signal peptide, Manitou 1-2, Hecate 1-3, Anubis 1-5 and 8, and Vishnu 1-3 and 8.

[0016] Hecate was described as a synthetic peptide analog of melittin by Baghian, A. et al. (Peptides 18(2): 177-183 (1997)). The peptides differ in their charge distribution, but not in their amphipathic alpha helical conformation. Hecate inhibited herpes simplex virus (HSV-1) while not adversely affecting cell growth and protein synthesis.

[0017] Synthetic peptides D2A21, D4E1, D2A22, D5C, D5C1, D4E, and D4B were described in Schwab, U. et al., Antimicrob. Agents and Chemotherapy 43(6): 1435-1440 (1999). Activities against various bacterial strains were presented.

[0018] Hybrid peptides made of cecropin and melittin peptides were reportedly prepared and assayed by Juvvadi, P. et al. (J. Peptide Res. 53: 244-251 (1999)). Hybrids were synthesized to investigate the effects of sequence, amide bond direction (helix dipole), charge, amphipathicity, and hydrophobicity on channel forming ability and on antibacterial activity. Sequence and amide bond direction were suggested to be important structural requirements for the activity of the hybrids.

[0019] A 26 amino acid insect cecropinbee melittin hybrid, and analogs thereof, were described in a study of salt resistance (Friedrich, C. et al., Antimicrobial Agents and Chemotherapy 43(7): 1542-1548 (1999)). A tryptophan residue in the second position was found to be critical for activity. Modest changes in sequence were found to lead to substantial changes in the properties of the peptides.

[0020] The effects of proline residues on the antibacterial properties of -helical peptides has been published (Zhang, L. et al., Biochem. 38: 8102-8111 (1999)). The addition of prolines was reported to change the membrane insertion properties, and the replacement of a single proline may change an antimicrobial peptide into a toxin.

[0021] A series of peptides having between 18 and 30 amino acids were prepared in order to test the effects of changes in sequence and charge on antibacterial properties (Scott, M. G., et al., Infect. Immun. 67(4): 2005-2009 (1999)). No significant correlation was found between length, charge, or hydrophobicity and the antimicrobial activity of the peptides. A general trend was found that shorter peptides were less active than longer peptides, although the authors expressed that this effect would probably be sequence dependent.

[0022] Modellins, a group of synthetic peptides were prepared and assayed to compare sequence and structure relationships (Bessalle, R. et al. J. Med. Chem. 36: 1203-1209 (1993)). Peptides of 16 and 17 amino acids having hydrophobic and hydrophilic opposite faces were highly hemolytic and antibacterial. Smaller peptides tended to have lower biological activities.

[0023] A cecropin-melittin hybrid peptide and an amidated flounder peptide were found to protect salmon from Vibrio anguillarum infections in vivo (Jia, X. et al., Appl. Environ. Microbiol. 66(5): 1928-1932 (2000)). Osmotic pumps were used to deliver a continuous dose of either peptide to the fish.

[0024] Amphipathic peptides have been reported as being capable of enhancing wound healing and stimulating fibroblast and keratinocyte growth in vivo (U.S. Pat. Nos. 6,001,805 and 5,561,107). Transgenic plants have been reportedly prepared expressing lytic peptides as a fusion protein with ubiquitin (U.S. Pat. No. 6,084,156). Methylated lysine rich lytic peptides were reportedly prepared, displaying improved proteolytic resistance (U.S. Pat. No. 5,717,064).

[0025] While a number of natural and synthetic peptides exist, there exists a need for improved bioactive peptides and methods for their use.

[0026] Short (i.e. no more than 23 amino acids in length) peptides containing phenylalanine, leucine, alanine, and lysine amino acid residues in their primary sequence are disclosed. The peptides display desirable antibacterial, antifungal, anticancer biological activities, and also cause stimulation and proliferation of human fibroblasts and lymphocytes.

[0027] The following sequence listings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these sequences in combination with the detailed description of specific embodiments presented herein.

TABLE 1
SEQ ID		P-
NO:	Name	No.	Primary sequence
1	Hecate AC 10101	1	FALALKALKKALKKLKKALKKAL-COOH
2	Hecate AM	2	FALALKALKKALKKLKKALKKAL-NH2
3	SB-37 AC 1018	5	MPKWKVFKKIEKVGRNIRNGIVKAGPAIAVLGEAKALG-
			COOH
4	Shiva 10 AM	11	FAKKLAKKLKKLAKKLAKLALAL-NH2
5	SB-37 AM	12	MPKWKVFKKIEKVGRNIRNGIVKAGPAIAVLGEAKALG-
			NH2
6	Shiva 10 AC 1015	13	FAKKLAKKLKKLAKKLAKLALAL-COOH
7	Magainin 2	16	GIGKFLHSAKKFGKAFVGGIMNS-NH2
8	FLAK01 AM	23	FALAAKALKKLAKKLKKLAKKAL-NH2
9	FLAK03 AM	24	FALALKALKKLLKKLKKLAKKAL-NH2
10	FLAK04 AM	25	FALALKALKKLAKKLKKLAKKAL-NH2
11	FLAK05 AM	26	FALAKLAKKAKAKLKKALKAL-NH2
12	FLAK06 AM	27	FALALKALKKLKKALKKAL-NH2
13	FLAK06 AC	28	FALALKALKKLKKALKKAL-COOH
14	FLAK06 R-AC	29	FAKKLAKKLKKLAKLALAL-COOH
15	KAL V	30	VALALKALKKALKKLKKALKKAL-NH2
16	FLAK 17 AM	34	FALALKKALKALKKAL-NH2
17	FLAK 26 AM	35	FAKKLAKLAKKLAKLAL-NH2
18	FLAK 25 AM	36	FAKKLAKLAKKLAKLALAL-NH2
19	Hecate 2DAc	37	FALALKALKKAL-(D)-K-(D)-KLKKALKKAL-COOH
20	FLAK43 AM	38	FAKKLAKLAKKLLAL-NH2
21	FLAK44 AM	39	FAKKLAKLAKKALAL-NH2
22	FLAK62 AM	40	FALAKKALKKAKKAL-NH2
23	FLAK06R-AM	41	FAKKLAKKLKKLAKLALAK-NH2
24	MSI-78 AM	42	GIGKFLKKAKKFGKAFVKILKK-NH2
25	FLAK50	43	FAKLLAKLAKKLL-NH2
26	FLAK51	44	FAKKLAKLALKLAKL-NH2
27	FLAK57	45	FAKKLAKKLAKLAL-NH2
28	FLAK71	46	FAKKLKKLAKLAKKL-NH2
29	FLAK77	47	FAKKALKALKKL-NH2
30	FLAK50V	48	VAKLLAKLAKKLL-NH2
31	FLAK50F	49	FAKLLAKLAKKL-NH2
32	FLAK26V AM	50	VAKKLAKLAKKLAKLAL-NH2
33	CAME-15	53	KWKLFKKIGAVLKVL-NH2
34	FLAK50C	54	FAKLLAKLAKKAL-NH2
35	FLAK50D	55	FAKLLAKALKKLL-NH2
36	FLAK50F	56	FAKLLKLAAKKLL-NH2
37	FLAK80	57	FAKLLAKKLL-NH2
38	FLAK81	58	FAKKLAKALL-NH2
39	FLAK82	59	FAKKLAKKLL-NH2
40	FLAK83M	60	FAKLAKKLL-NH2
41	FLAK 26 Ac	61	FAKKLAKLAKKLAKLAL-COOH
42	Indolicidin	63	ILPWKWPWWPWRR-NH2
43	FLAK 17C	64	FAKALKALLKALKAL-NH2
44	FLAK 50H	65	FAKLLAKLAKAKL-NH2
45	FLAK 50G	66	FAKLLAKLAKLKL-NH2
46	Shiva Deriv	70	FAKKLAKKLKKLAKKLAKKWKL-NH2
	P69 KWKL
47	Shiva 10 (1-18 AC)	71	FAKKLAKKLKKLAKKLAK-COOH
48	Shiva 10 peptide	72	FAKKLAKKLKKLAKKLAKKWKL-COOH
	71 KWKL
49	CA(1-7)Shiva10(1-	73	KWKLFKKKTKLFKKFAKKLAKKL-NH2
	16)
50	FLAK 54	74	FAKKLAKKLAKAL-NH2
51	FLAK 56	75	FAKKLAKKLAKLL-NH2
52	FLAK 58	76	FAKKLAKKLAKAAL-NH2
53	FLAK 72	77	FAKKLAKKAKLAKKL-NH2
54	FLAK 75	79	FAKKLKKLAKKL-NH2
55	Shiva 10 (1-16) Ac	80	KTKLFKKFAKKLAKKLKKLAKKL-COOH
56	CA(1-7)Shiva10	81	KWKLFKKKTKLFKKFAKKLAKKL-COOH
	(1-16)-COOH
57	Indolocidin-ac	91	ILPWKWPWWPWRR-COOH
58	FLAK50B	92	FAKALAKLAKKLL-NH2
59	FLAK50J	93	FAKLLAKLAKKAA-NH2
60	FLAK50I	94	FAKLLALALKLKL-NH2
61	FLAK50K	95	FAKLLAKLAKAKA-NH2
62	FLAK50L	96	FAKLLAKLAKAKG-NH2
63	Shiva-11	98	FAKKLAKKLKKLAKKLAKLALALKALALKAL-NH2
64	Shiva 11	99	FAKKLAKKLKKLAKKLIGAVLKV-COOH
	(1-16)ME(2-9-
	COOH
65	FLAK 50N	101	FAKLLAKALKLKL-NH2
66	FLAK 50O	102	FAKLLAKALKKAL-NH2
67	FLAK 50P	103	FAKLLAKALKKL-NH2
68	CA(1-	104	KWKLFKKALKKLKKALKKAL-NH2
	& Hecate(11/23)
69	PYL-ME	105	KIAKVALAKLGIGAVLKVLTTGL-NH2
70	FLAG26-D1	106	FAKKLAKLAKKL-NH2
71	Vishnu3	107	MPKEKVFLKIEKMGRNIRN-NH2
72	Melittin	108	GIGAVLKVLTTGLPALISWIKRKRQQ-NH2
73	FLAK26-D2	109	FAKKLAKLAKKLAKAL-NH2
74	FLAG26-D3	110	FAKKLLAKALKL-NH2
75	FLAK50 Q1	111	FAKFLAKFLKKAL-NH2
76	FLAK50 Q2	112	FAKLLFKALKKAL-NH2
77	FLAK50 Q3	113	FAKLLAKFLKKAL-NH2
78	FLAK50 Q4	114	FAKLLAKAFKKAL-NH2
79	FLAK50 Q5	117	FAKLFAKAFKKAL-NH2
80	FLAK50 Q6	118	FAKLLAKALKKFL-NH2
81	FLAK50 Q7	119	FAKLLAKALKKFAL-NH2
82	FLAK50 Q8	120	FAKLLAKLAKKFAL-NH2
83	FLAK50 Q9	121	FAKLFAKLAKKFAL-NH2
84	FLAK50 Q10	122	FKLAFKLAKKAFL-NH2
85	FLAK50 T1	123	FAKLLAKLAK-NH2
86	FLAK50 T2	124	FAKLLAKLAKKVL-NH2
87	FLAK50 T3	125	FAKLLAKLAKKIL-NH2
88	FLAK50 T4	126	FAKLLAKLAKKEL-NH2
89	FLAK50 T5	127	FAKLLAKLAKKSL-NH2
90	FLAK90	128	FAKLA-NH2
91	FLAK91	129	FAKLF-NH2
92	FLAK92	130	KAKLF-NH2
93	FLAK93	131	KWKLF-NH2
94	FLAK50 Z1	132	FGKGIGKVGKKLL-NH2
95	FLAK50 Z2	133	FAFGKGLGKVGKKLL-NH2
96	FLAK50 Z3	134	FAKAIAKIAFGKGIGKVGKKLL-NH2
97	FLAK50 Z4	135	FAKLWAKLAFGKGIGKVGKKLL-NH2
98	FLAK50 Z5	136	FAKLWAKLAKKL-NH2
99	FLAK50 Z6	137	FAKGVGKVGKKAL-NH2
100	FLAK50 Z7	138	FAFGKGIGKIGKKGL-NH2
101	FLAK50 Z8	139	FAKIIAKIAKIAKKIL-NH2
102	FLAK50 Z9	140	FAFAKIIAKIAKKII-NH2
103	FLAK94	141	FALALKA-NH2
104	FLAK93B	142	KWKLAKKALALL-NH2
105	FLAK50 Z10	143	FAKIIAKIAKKI-NH2
106	FLAK96	144	FALALKALKKAL-NH2
107	FLAK97	145	FALKALKK-NH2
108	FLAK98	146	KYKKALKKLAKLL-NH2
109	FKRLA	147	FKRLAKIKVLRLAKIKR-NH2
110	FLAK91B	148	FAKLAKKALAKLL-NH2
111	FLAK92B	149	KAKLAKKALAKLL-NH2
112	FLAK99	150	KLALKLALKALKAAKLA-NH2
113	FLAK50T6	151	FAKLLAKLAKK-NH2
114	FLAK50T7	152	FAKLLAKLAKKGL-NH2
115	FLAK95	153	FALKALKKLKKALKKAL-NH2
116	FLAK50T8	154	VAKLLAKLAKKVL-NH2
117	FLAK50T9	155	YAKLLAKLAKKAL-NH2
118	FLAK100-CO2H	156	KLLKLLLKLYKKLLKLL-COOH
119	FAGVL	157	FAVGLRAIKRALKKLRRGVRKVAKDL-NH2
120	Modelin-5	159	KLAKKLAKLAKLAKAL-NH2
121	Modelin-5-CO2H	160	KLAKKLAKLAKLAKAL-COOH
122	Modelin-8	161	KWKKLAKKW-NH2
123	Modelin-8-CO2H	162	KWKKLAKKW-COOH
124	Modelin-1	163	KLWKKWAKKWLKLWKAW-NH2
125	Modelin-1-CO2H	164	KLWKKWAKKWLKLWKA-COOH
126	FLAK120	165	FALALKALKKL-NH2
127	FLAK121	166	FALAKALKKAL-NH2
128	FLAK96B	167	FALALKLAKKAL-NH2
129	FLAK96G	168	FALLKL-NH2
130	FLAK96F	169	FALALKALKK-NH2
131	FLAK96C	170	FALKALKKAL-NH2
132	FLAK96D	171	FALLKALKKAL-NH2
133	Modelin-8B	172	KWKK-NH2
134	Modelin-8C	173	KWKKL-NH2
135	Modelin-8D	174	KFKKLAKKF-NH2
136	Modelin-8E	175	KFKKLAKKW-NH2
137	Flak 96	176	FALALKALKKA-NH2
138	Flak 96I	177	FALLKALLKKAL-NH2
139	Flak 96J	178	FALALKLAKKL-NH2
140	Flak 96L	179	LKKLAKLALAF-NH2
141	FLAK-120G	180	VALALKALKKL-NH2
142	FLAK-120D	181	FALALKLKKL-NH2
143	FLAK-120C	182	FALALKAKKL-NH2
144	FLAK-120B	183	FALA-NH2
145	FLAK-120F	184	WALAL-NH2
146	Magainin2wisc	300	GIGKFLHAAKKFAKAFVAEIMNS-NH2
147	D2A21	301	FAKKFAKKFKKFAKKFAKFAFAF-NH2
148	KSL-1	302	KKVVFKVKFK-NH2
149	KSL-7	303	FKVKFKVKVK-NH2
150	LSB-37	306	LPKWKVFKKIEKVGRNIRNGIVKAGPAIAVLGEAKALG-
			NH2
151	Anubis-2	307	FAKKLAKKLKKLAKKLAKLAKKL-NH2
152	FLAK17CV	501	VAKALKALLKALKAL-NH2
153	FLAK50Q1V	502	VAKFLAKFLKKAL-NH2
154	D2A21V	503	VAKKFAKKFKKFAKKFAKFAFAF-NH2
155	FLAK25AMV	504	VAKKLAKLAKKLAKLALAL-NH2
156	FLAK43AMV	505	VAKKLAKLAKKLLAL-NH2
157	FLAK50DV	506	VAKLLAKALKKLL-NH2
158	HECATE AMV	507	VALALKALKKALKKLKKALKKAL-NH2
159	HECATE ACV	508	VALALKALKKALKKLKKALKKAL-COOH
160	FLAK04AMV	509	VALALKALKKLAKKLKKLAKKAL-NH2
161	FLAK03AMV	510	VALALKALKKLLKKLKKLAKKAL-NH2
162	D-Shiva 10 AC	67	(D)-FAKKLAKKLKKLAKKLAKLALAL-COOH
163	Shiva 11 AC	100	FAKKLAKKLKKLAKKLAKLALALKALALKA-COOH
164	Shiva 10 (1-18)AM	69	FAKKLAKKLKKLAKKLAK-NH2
165	FLAK 50M	97	FAKLLALALKKAL-NH2

[0028] The invention is generally directed towards peptides having desirable biological properties, and their use. It is surprising that the peptides are efficacious due to their short length as compared to other peptides described in the art.

[0029] Peptides

[0030] One embodiment of the invention is directed towards an isolated peptide comprising phenylalanine, leucine, alanine, and lysine residues, wherein the peptide is about 5 to about 23 amino acids in length. The peptide can have a minimum length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or about 18 amino acids. The peptide can have a maximum length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or about 23 amino acids. The peptide can be about 5 to about 20 amino acids in length. The peptide can consist essentially of, or consist of phenylalanine, leucine, alanine, and lysine residues. The peptide can have a percent amino acid composition of phenylalanine, leucine, alanine, and lysine residues of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. The peptide can generally be any of the listed SEQ ID NOS which fall within these various guidelines, and more preferably is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:152, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, and SEQ ID NO:165. The peptide is preferably not hecate-1, anubis-1, anubis-2, anubis-5, anubis-8, vishnu-1, vishnu-2, vishnu-3, vishnu-8, or shiva-10.

[0031] The peptide can be similar to any of the above described peptides, and preferably is similar to SEQ ID NO:2 (or SEQ ID NO:16 or SEQ ID NO:126), SEQ ID NO:4 (or SEQ ID NO:14 or SEQ ID NO:17), SEQ ID NO:25, SEQ ID NO:43, SEQ ID NO:75, SEQ ID NO:84, SEQ ID NO:115, SEQ ID NO:126, or SEQ ID NO:132 as determined by percent identity. The percent identity between the peptides is preferably at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. Percent identity is determined using a sequence alignment by the commercial product CLUSTALW. The number of aligned amino acids are divided by the length of the shorter peptide, and the result is multiplied by 100% to determine percent identity. If the length of the shorter peptide is less than 10 amino acids, the number of aligned amino acids are divided by 10, and the result is multiplied by 100% to determine percent identity.

[0032] The peptides can comprise D- or L-amino acids. The peptides can comprise all D-amino acids. The peptides can have an acid C-terminus (CO₂H) or an amide C-terminus (CONH₂, CONHR, or CONR₂).

[0033] Methods of Use

[0034] An additional embodiment of the invention is directed towards methods of using the above described peptides. The methods of use preferably do not cause injury or kill normal uninfected mammalian cells. The methods of use at therapeutic dose levels preferably do not cause injury to or kill normal uninfected or non-neoplastic mammalian cells. The methods of use may involve the use of a single peptide, or may involve the use of multiple peptides.

[0035] An embodiment of the invention is the use of the above described peptides to inhibit or kill microbial cells (microorganisms). The microorganisms may be bacterial cells, fungal cells, protozoa, viruses, or eucaryotic cells infected with pathogenic microorganisms. The method generally is directed towards the contacting of microorganisms with the peptide. The contacting step can be performed in vivo, in vitro, topically, orally, transdermally, systemically, or by any other method known to those of skill in the art. The contacting step is preferably performed at a concentration sufficient to inhibit or kill the microorganisms. The concentration of the peptide can be at least about 0.1 M, at least about 0.5 M, at least about 1 M, at least about 10 M, at least about 20 M, at least about 50 M, or at least about 100 M. The methods of use can be directed towards the inhibition or killing of microorganisms such as bacteria, gram positive bacteria, gram negative bacteria, mycobacteria, yeast, fungus, algae, protozoa, viruses, and intracellular organisms. Specific examples include, but are not limited to, Staphylococcus, Staphylococcus aureus, Pseudomonas, Pseudomonas aeruginosa, Escherichia coli, Chlamydia, Candida albicans, Saccharomyces, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trypanosoma cruzi, or Plasmodium falciparum. The contacting step can be performed by systemic injection, oral, subcutaneous, IP, IM, IV injection, or by topical application. For injection, the dosage can be between any of the following concentrations: about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 25 mg/kg, about 50 mg/kg, about 75 mg/kg, and about 100 mg/kg. The contacting step can be performed on a mammal, a cat, a dog, a cow, a horse, a pig, a bird, a chicken, a plant, a fish, or a human.

[0036] Presently preferred peptides for antibacterial applications include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:93, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, and SEQ ID NO:165.

[0037] Presently preferred peptides for antifungal applications include SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:58, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:143, SEQ ID NO:163, and SEQ ID NO:165.

[0038] An additional embodiment of the invention is the use of any of the above described peptides to inhibit or kill cancer cells. The method generally is directed towards the contacting of cancer cells with the peptide. The contacting step can be performed in vivo, in vitro, topically, orally, transdermally, systemically, or by any other method known to those of skill in the art. The contacting step is preferably performed at a concentration sufficient to inhibit or kill the cancer cells. The concentration of the peptide can be at least about at least about 0.1 M, at least about 0.5 M, at least about 1 M, at least about 10 M, at least about 20 M, at least about 50 M, or at least about 100 M. The cancer cells can generally be any type of cancer cells. The cancer cells can be sarcomas, lymphomas, carcinomas, leukemias, breast cancer cells, colon cancer cells, skin cancer cells, ovarian cancer cells, cervical cancer cells, testicular cancer cells, lung cancer cells, prostate cancer cells, and skin cancer cells. The contacting step can be performed by subcutaneous, IP injection, IM injection, IV injection, direct tumor injection, or topical application. For injection, the dosage can be between any of the following concentrations: about 0.1 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 25 mg/kg, about 50 mg/kg, about 75 mg/kg, and about 100 mg/kg. The contacting step can be performed on a mammal, a cat, a dog, a cow, a horse, a pig, a bird, a chicken, a plant, a fish, a goat, a sheep, or a human. The inhibition of cancer cells can generally be any inhibition of growth of the cancer cells as compared to the cancer cells without peptide treatment. The inhibition is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, and ideally 100% inhibition of growth. The inhibition may be achieved by lysis of the cancer cells or by other means. The cancer inhibiting peptide can be used synergistically with other cancer chemotherapeutic agents.

[0039] Presently preferred peptides for anticancer applications include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:68, SEQ ID NO:75, SEQ ID NO:86, SEQ ID NO:152, and SEQ ID NO:162.

[0040] An additional embodiment of the invention is directed towards a method for promoting the stimulation and/or proliferation of cells. The method can comprise contacting the cells and a composition, wherein the composition comprises a peptide. The peptide can be any of the above described peptides. The concentration of the peptide in the composition can be about 0.01 M to about 500 M, about 0.1 M to about 100 M, about 1 M to about 50 M, or about 1 M to about 10 M. The cells can generally be any type of cells, and preferably are mammalian cells, specifically including, but not limited to fibroblast and leukocyte cells, including lymphocyte and phagocytic cells. The metabolic stimulation and/or proliferation of the cells is preferably increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, or 200% relative to the same cells not contacted with the composition. The composition can further comprise a growth factor. The stimulatory and proliferative properties of some of the FLAK peptides hold promise for their application in skin care, wound healing, and in immunomodulation of compromised mammalian immune systems.

[0041] Presently preferred peptides for stimulation and proliferation applications include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:108, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:164, and SEQ ID NO:165.

[0042] An additional embodiment of the invention is directed towards a method for promoting wound healing of skin or ocular and internal body tissues damaged by normal aging, disease, injury, or by surgery or other medical procedures. The method can comprise administering to the wound of an animal a composition, wherein the composition comprises any of the above described peptides. The concentration of the peptide in the composition can be about 0.01 M to about 500 M, about 0.1 M to about 100 M, about 1 M to about 50 M, or about 1 M to about 10 M. The composition can be administered to the wound topically or by systemic delivery. The animal can generally be any kind of animal, preferably is a mammal, and more preferably is a human, cow, horse, cat, dog, pig, goat, or sheep. The promotion of wound healing is preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, or 200% relative to the same wound not contacted with the composition.

[0043] Presently preferred peptides for wound healing applications include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:159, SEQ ID NO:162, and SEQ ID NO:164.

[0044] A further embodiment of the invention is directed towards methods for the additive or synergistic enhancement of the activity of a therapeutic agent. The method can comprise preparing a composition, wherein the composition comprises a peptide and a therapeutic agent. Alternatively, the method may comprise co-therapy treatment with a peptide (or peptides) used in conjunction with other therapeutic agents. The peptide can be any of the above described peptides. The therapeutic agent can generally be any therapeutic agent, and preferably is an antibiotic, an antimicrobial agent, a growth factor, a chemotherapy agent, an antimicrobial agent, lysozyme, a chelating agent, or EDTA. Preferably, the activity of the composition is higher than the activity of the same composition containing the therapeutic agent but lacking the peptide. The composition or co-therapy can be used in in vitro, in vivo, topical, oral, IV, IM, IP, and transdermal applications. The enhancement of the activity of the composition containing the therapeutic agent and the peptide is preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, or 200% relative to the activity of the therapeutic agent alone.

[0045] Generally, any peptide which is active on a stand-alone basis against a target is preferred for use to increase either additively or synergistically the activity of another therapeutic agent against that target. If several peptides are candidates for a given synergy application, then the less toxic peptides would be more favorably considered.

[0046] A further additional embodiment of the invention is directed towards methods for the treatment of patients diagnosed with Cystic Fibrosis (CF). CF causes, among other effects, inflammation and infection in the lungs. The above described peptides of the instant invention can be used in treating such lung infections, which are often caused by P. aeruginosa. The inventive peptides may possess anti-inflammatory properties, making them further useful for the treatment of lung infections in CF patients. The peptide can be administered to the CF patient by any acceptable method including inhalation or systemic delivery. The peptide can be administered in a single dose, in multiple doses, or as a continuous delivery.

[0047] An additional embodiment of the invention is directed towards methods of treating sexually transmitted diseases (STDs). Many of the fungal species responsible for STDs are inhibited or killed by the inventive peptides described above. Examples of such species include C. albicans, C. glabrata, and C. tropicalis. The inventive peptides may additionally be used against other agents responsible for STDs including viruses and bacteria. The peptides can be administered to an STD patient by any acceptable method, such as topical, oral, or systemic delivery. The peptide can be administered in a single dose, in multiple doses, or as a continuous delivery. The peptide can be administered in any acceptable form, such as a cream, gel, or liquid.

[0048] A further additional embodiment of the invention is directed towards methods for the treatment of acne. The inventive peptides have activity against the bacteria isolated from acne sores, Propionibacterium acnes, and may further possess anti-inflamatory properties. The peptide can be present in a clinical therapeutic composition or in a cosmeceutical composition. The peptide can be administered in any acceptable form, such as a cream, gel, or liquid. The peptide can be administered in any acceptable manner, such as topical administration. The peptide can be used in a treatment method, or in a preventative manner to reduce or eliminate future outbreaks of acne.

[0049] Yet a further embodiment is directed towards cosmetic compositions. The inventive peptides have been shown to stimulate collagen and fibroblasts, and to promote wound healing. The inclusion of the inventive peptides in cosmetic formulations may be useful in the anti-aging and rejuvination markets.

[0050] An additional embodiment of the invention is directed towards the use of peptides in promoting wound healing. The inventive peptides have high potency against the bacteria most associated with wound infections: S. aureus, S. pyogenes, and P. aeruginosa. The peptides also promote wound healing and reducing of inflammation. The peptide can be administered in any acceptable form, such as a cream, gel, or liquid. The peptide can be administered in any acceptable manner, such as topical administration or systemic administration.

[0051] The following Examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0052] The following table lists the various microorganisms used throughout the Examples.

TABLE 2
Microorganism	Reference or source
Escherichia coli	ATCC25922
Staphylococcus aureus	ATCC6538 and ATCC25923
Pseudomonas aeruginosa	ATCC9027 and ATCC27853
Staphylococcus intermedius	ATCC19930 and ATCC20034
Candida albicans	ATCC10231
Escherichia coli UB1005	D. Clark, FEMS Microb. Lett. 21: 189-195, 1984
Salmonella typhimurium 14028S	Fields et al, Science 243: 1059-1062, 1989
Staphylococcus aureus SAP0017	Methicillin resistant clinical isolate from Prof. T.
	Chow, Vancouver General hospital
Staphylococcus epidermidis C621	clinical isolate from David. Speer
Streptococcus pyogenes	ATCC19615
Streptococcus pyogenes M76	From Prof. R. Gallo (UCSD)
Streptococcus pneumoniae	ATCC6305-C718
Streptococcus pneumoniae	ATCC49619-C719
Pseudomonas aeruginosa H187	Angus, et al., AAC 21: 299-309, 1982
Pseudomonas aeruginosa H374	Masuda, N., et al., AAC, 36: 1847-1851, 1992
(nfxB efflux mutant)
Pseudomonas aeruginosa H744	Poole, K., et al. J. Bacteriol. 175-7363-7372, 1993
nalB multiple resistant efflux
mutant
Pseudomonas aeruginosa 100609	Tobramycin resistant strain from Prof. D. Woods
	(U. Calgary)
Pseudomonas aeruginosa 105663	Tobramycin resistant strain from Prof. D. Woods
	(U. Calgary)
Candida albicans 105	From Prof Barbara Dill (UBC)
Candida guilliermondii	ATCC8492
Candida tropicalis	ATCC13803
Candida glabrata	ATCC15126
Propionibacterium acnes	ATCC6919
Propionibacterium acnes	ATCC11827
Acinetobacter baumannii	ATCC19606

[0053] The data for the following antimicrobial assay of the peptides have been obtained by making OD measurements in in vitro cell culture experiments with and without added peptide. The protocol used is as follows.

[0054] Cell lines included Staphylococcus aureus ATCC 6538 or 25923, Pseudomonas aeruginosa ATCC 9027 or 27853. Medium used were Antibiotic Medium 3 (Difco), Antibiotic Medium 2 (Difco), and 0.85% saline. Controls used were physiological saline, and gentamycin at 50, 25, 10, 5, 1, and 0.1 ppm.

[0055] The preparation of all media, stock solutions, and dilutions took place in a laminar flow hood to prevent contamination. Bacterial cells were freshly grown on antibiotic medium 2 agar slants (pH 7.0 at 25 C.). Bacteria were suspended and diluted in antibiotic medium 3 to about 104 cfu/ml and used as the inoculum. Sample solutions (100 l/well) were added to plates according to the plate layout. Inoculum (100 l/well) was added to achieve a final concentration of 510³ cfu/ml. Negative controls received 100 l saline and 100 l growth medium. Positive controls received 100 l saline and 100 l inoculum. Bacterial plates were incubated at 37 C. for 24 hours.

[0056] Absorbance was read at 620 nm after shaking to resuspend cells. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of peptide that completely inhibits the growth of the test organism.

[0057] The yeast assay was performed in RPMI 1640 media (pH 7.0 at 25 C.).

[0058] The data presented in Table 3 were obtained using the above protocol. However, the data for Table 4 were obtained with a modified protocol wherein the medium was tryptic soy broth, inocolum strength was approximately 10⁴ CFU per ml, and values determined were minimum bactericidal concentrations (MBC) or minimum fungicidal concentrations (MFC).

[0059] The following Table 3 describes the antimicrobial properties of the peptides measured as MIC or MFC values in g/mL. Staph6538 is Staphylococcus aureus ATCC accession number 6538; paerug9027 is Pseudomonas aeruginosa ATCC accession number 9027, yeast is Saccharomyces cerevisiae.

TABLE 3
	SEQ ID	P
Name	NO:	Number	staph6538	paerug9027	yeast
Hecate AC 1010	1	1	5	10	>
Hecate AM	2	2	25	100	25
SB-37 AC 1018	3	5	100	50	>
SB-37 AM	5	12	>	100	>
Shiva 10 AC	6	13	10	>	>
1015
FLAK01 AM	8	23	5	50	100
FLAK04 AM	10	25	10	5	25
FLAK05 AM	11	26	10	15	>
FLAK06 AM	12	27	10	10	25
KAL V	15	30	>	>	ND
FLAK 17 AM	16	34	5	50	25
FLAK 26 AM	17	35	5	200	25
Hecate 2D Ac	19	37	5	100	50
FLAK43 AM	20	38	5	50	50
FLAK44 AM	21	39	100	25	100
FLAK62 AM	22	40	100	25	100
FLAK 06R-AM	23	41	10	10	ND
MSI-78 AM	24	42	10	>	200
FLAK50	25	43	5	100	25
FLAK51	26	44	5	5	50
FLAK57	27	45	5	100	100
FLAK71	28	46	10	5	50
FLAK77	29	47	200	100	50
FLAK50V	30	48	5	5	25
FLAK50F	31	49	10	200	50
FLAK26V AM	32	50	5	15	50
CAME-15	33	53	5	15	50
FLAK50C	34	54	5	50	50
FLAK50D	35	55	5	5	25
FLAK 50E	36	56	200	5	50
FLAK80	37	57	100	200	200
FLAK81	38	58	100	100	200
FLAK82	39	59	>	>	>
FLAK83M	40	60	200	100	200
FLAK 17 C	43	64	5	>	200
FLAK 50H	44	65	15	50	200
FLAK 50G	45	66	5	50	100
Shiva deriv	46	70	10	>	100
P69 KWKL
Shiva 10 (1-18	47	71	15	15	200
AC
CA(1-7)Shiva	49	73	50	15	100
10(1-16)
FLAK 54	50	74	15	5	100
FLAK 56	51	75	5	5	50
FLAK 58	52	76	10	100	200
FLAK 72	53	77	200	100	200
FLAK 75	54	79	100	200	100
Shiva 10 (1-16) Ac	55	80	10	100	100
CA(1-7)Shiva10 (1-	56	81	10	>	>
16)-COOH
Indolocidin-ac	57	91	10	>	>
FLAK50B	58	92	5	5	50
FLAK50I	60	94	10	>	>
FLAK50K	61	95	100	200	>
FLAK50L	62	96	>	>	>
Shiva-11	63	98	>	>	>
Shiva 11(1-	64	99	100	>	>
16)ME(2-9)-
COOH
FLAK 50N	65	101	10	25	100
FLAK50O	66	102	5	10	50
FLAK 50P	67	103	10	25	100
CA(1-	68	104	10	10	200
&Hecate(11/23)
PYL-ME	69	105	200	200	>
FLAG26-D1	70	106	100	25	100
Vishnu3	71	107	>	>	>
Melittin	72	108	5	>	25
FLAK26-D2	73	109	>	200	200
FLAG26-D3	74	110	>	200	200
FLAK50 Q1	75	111	5	100	200
FLAK50 Q2	76	112	50	200	100
FLAK50 Q3	77	113	10	200	200
FLAK50 Q4	78	114	50	15	100
FLAK50 Q5	79	117	100	200	200
FLAK50 Q6	80	118	10	100	100
FLAK50 Q7	81	119	50	25	50
FLAK50 Q8	82	120	50	200	200
FLAK50 Q9	83	121	50	>	100
FLAK50 T1	85	123	50	200	100
FLAK50 T2	86	124	5	100	100
FLAK50 T3	87	125	10	100	50
FLAK50 T4	88	126	>	>	>
FLAK50 T5	89	127	100	25	100
FLAK90	90	128	>	100	200
FLAK91	91	129	100	25	100
FLAK92	92	130	200	200	200
FLAK93	93	131	25	10	100
FLAK50 Z1	94	132	>	100	>
FLAK50 Z2	95	133	>	>	>
FLAK50 Z3	96	134	100	>	200
FLAK50 Z4	97	135	15	10	50
FLAK50 Z5	98	136	100	50	100
FLAK50 Z6	99	137	>	>	>
FLAK50 Z7	100	138	>	>	>
FLAK50 Z8	101	139	50	25	200
FLAK50 Z9	102	140	>	>	>
FLAK94	103	141	15	50	200
FLAK93B	104	142	100	50	100
FLAK50 Z10	105	143	100	50	200
FLAK96	106	144	5	50	50
FLAK97	107	145	200	100	200
FLAK98	108	146	10	10	50
FKRLA	109	147	5	5	200
FLAK91B	110	148	>	200	200
FLAK92B	111	149	50	100	200
FLAK99	112	150	100	10	>
FLAK50T6	113	151	>	>	200
FLAK50T7	114	152	100	50	100
FLAK95	115	153	5	25	100
FLAK50T8	116	154	100	100	50
FLAK50T9	117	155	>	>	>
FLAK100-CO2H	118	156	15	>	>
FAGVL	119	157	200	>	>
FLAK120	126	165	10	25	25
FLAK121	127	166	>	>	>
FLAK96B	128	167	10	25	100
FLAK96G	129	168	50	100	>
FLAK96F	130	169	100	100	100
FLAK96C	131	170	200	100	100
FLAK96D	132	171	25	50	100
FLAK96	137	176	>	>	>
FLAK96J	139	178	200	100	>
FLAK96L	140	179	50	50	100
FLAK-120G	141	180	200	>	>
FLAK-120D	142	181	100	200	100
FLAK-120C	143	182	>	>	>
FLAK-120B	144	183	200	100	200
FLAK-120F	145	184	25	100	100
FLAK 50M	165	97	5	50	50
> indicates greater than 200 g/mL;
ND not determined.

[0060] The following Table 4 describes describes the antimicrobial properties of the peptides measured as minimum bactericidal or minimum fungicidal (Candida) concentrations. MBC or MFC values are in g/mL. E. coli is Escherichia coli ATCC accession number 25922; P. aerug is Pseudomonas aeruginosa ATCC accession number 27853, S. aur. is Stapholococcus aureus ATCC accession number 25923; Candida is Candida albicans ATCC accession number 10231.

TABLE 4
		E. coli	P. aerug	S. aur	Candida
SEQ ID NO:	P	A.25922	A.27853	A.25923	A.10231
1	1	25	30	25	>50
2	2	25	10	25	>50
3	5	50	>60	40	ND
4	11	40	25	25	>50
5	12	50	>60	75	ND
6	13	8	15	30	>50
8	23	15	25	30	>50
9	24	>80	30	>40	>50
10	25	40	30	40	>50
11	26	>80	>40	>40	>50
12	27	10	8	8	>50
13	27B	40	10	>40	>40
14	27C	10	4	>40	>40
15	30	10	15	40	>50
16	34	15	15	40	>40
17	35	8	8	10	>40
18	36	30	15	10	>40
19	37	8	8	40	>50
20	38	15	30	15	ND
21	39	>40	>40	>40	ND
22	40	30	40	>40	ND
23	41	40	40	40	ND
24	42	10	30	10	ND
25	43	8	15	4	15
26	44	10	55	30	>50
27	45	30	40	80	>50
29	47	>50	>50	>50	>50
30	48	8	25	4	10
31	49	40	30	50	30
32	50	50	25	25	>50
33	53	15	15	10	30
34	54	15	40	15	30
35	55	4	10	4	25
36	56	50	10	55	30
37	57	>50	>50	>50	>50
38	58	>50	>50	>50	>50
39	59	>50	>50	>50	>50
40	60	>50	>50	>50	>50
41	61	4	50	>80	>40
42	63	10	50	15	60
43	64	10	30	4	>50
44	65	>55	>50	>55	>50
45	66	40	50	30	40
46	70	40	30	40	>50
47	71	50	40	>50	>50
48	72	>50	40	>50	>50
50	74	>55	50	>55	>55
51	75	40	30	>55	30
52	76	40	>55	>55	>50
53	77	>50	>50	>50	>50
54	79	>50	>50	>50	>50
55	80	30	15	>50	>50
58	92	40	25	15	25
59	93	>50	>50	>50	>50
60	94	>50	>50	>50	>50
61	95	>50	>50	>50	>50
62	96	>50	>50	>50	>50
65	101	300	>50	>50	40
66	102	25	30	25	15
67	103	30	30	>50	25
69	105	25	>50	ND	>50
70	106	50	>50	ND	>50
71	107	ND	>50	>50	>50
72	108	>50	>50	25	>50
73	109	ND	ND	80	>50
74	110	8	>50	>50	>50
75	111	30	ND	40	INACT
76	112	30	INACT	INACT	INACT
77	113	INACT	INACT	INACT	40
79	117	INACT	INACT	INACT	INACT
80	118	8	25	10	25
81	119	15	30	4	25
82	120	INACT	INACT	INACT	INACT
83	121	INACT	INACT	INACT	50
84	122	30	30	25	15
85	123	40	INACT	INACT	25
86	124	10	40	8	15
87	125	40	40	INACT	40
88	126	INACT	INACT	INACT	INACT
89	127	INACT	INACT	INACT	INACT
90	128	INACT	INACT	INACT	INACT
91	129	INACT	INACT	INACT	INACT
92	130	INACT	INACT	INACT	INACT
93	131	INACT	INACT	INACT	INACT
94	132	INACT	INACT	INACT	INACT
95	133	INACT	INACT	INACT	INACT
96	134	INACT	INACT	INACT	INACT
97	135	INACT	40	INACT	25
98	136	INACT	INACT	INACT	INACT
99	137	INACT	INACT	INACT	INACT
100	138	INACT	INACT	INACT	INACT
101	139	INACT	INACT	INACT	INACT
102	140	INACT	INACT	INACT	INACT
103	141	INACT	INACT	INACT	INACT
104	142	INACT	INACT	INACT	INACT
105	143	INACT	INACT	INACT	INACT
106	144	10	25	25	25
107	145	INACT	INACT	INACT	100
108	146	10	>250	75	10
109	147	25	75	>250	>250
110	148	150	>250	>250	100
111	149	150	>250	>250	100
112	150	75	>250	>250	50
113	151	>250	>250	>250	100
114	152	150	150	>250	50
115	153	10	25	5	25
116	154	50	100	>250	25
117	155	>250	>250	>250	>250
118	156	100	>250	>250	>250
119	157	75	>250	>250	>250
120	159	10	10	>250	50
121	160	>250	>250	>250	>250
122	161	150	>250	>250	25
123	162	50	>250	>250	100
124	163	25	50	25	25
125	164	25	25	25	25
126	165	10	25	25	10
127	166	>250	>250	>250	>250
128	167	25	>250	10	25
129	168	75	100	>250	150
130	169	200	>250	>250	75
131	170	25	>250	150	25
132	171	75	100	>250	50
133	172	>250	>250	>250	>250
134	173	>250	>250	>250	150
162	67	25	30	30	>50
165	97	25	>50	25	25
INACT refers to no detectable activity.
ND indicates no data available.

[0061] Anti-microbial activity against a broader range of pathogens (including clinical strains) than were tested in Example 2. It should be noted that somewhat different protocols were employed for the assays in Example 2 and Example 3.

[0062] MICs were determined for this Example using a slightly modified version of the NCCLS (National Committee for Clinical Laboratory Standards) broth microdilution method as described previously (Steinberg et al., AAC 41: 1738, 1997). Briefly, antimicrobial agents were prepared as 10 concentrates in the most appropriate solvent. For the peptide, 0.01% acetic acid containing 0.2% bovine serum albumin as a carrier protein was used. Inocula were prepared by resuspending colonies from a BAP in medium and adjusting the suspension to match that of a 0.5 McFarland standard. The suspension was diluted into fresh medium (as recommended by NCCLS for the organism) to give 210⁵ to 710⁵ CFU/ml for bacteria or 210³ to 710³ CFU/ml for Candida. After dispensing 100 l aliquots of the microbial suspension into each well of a 96-well polypropylene microtiter plate, 11 l of test compound was added. The MIC was defined as the lowest concentration of drug which prevented visible turbidity after 16 to 20 hours (bacteria) or 46 to 50 hours (Candida) at 35 C. For facultative anaerobes incubation was performed in 7% carbon dioxide and for strict anaerobes in an oxygen free environment maintained using a standard anaerobic jar. All MICs were performed three times and the mean value determined.

TABLE 5
Activity against gram positive bacteria
	S. aureus	S. epidermidis	S. pyogenes
Peptide (SEQ ID NO:)	(MRS A)	C621	M76
P23 (8)	32	16	16
P25 (10)	16	4	8
P26 (11)	32	4	4
P27 (12)	16	4	4
P34 (16)	16	8	4
P35 (17)	8	4	4
P37 (19)	8	4	8
P41 (23)	64	4	8
P42 (24)	16	2	4
P43 (25)	4	2	2
P44 (26)	8	4	4
P46 (28)	64	8	8
P49 (31)	64	8	8
P50 (32)	4	4	8
P54 (34)	16	8	8
P55 (35)	4	2	4
P59 (39)	8	8	2
P60 (40)	32	4	8
P61 (41)	32	8	16
P63* (42)	32	16	8
P64* (43)	8	4	4
P72 (48)	16	4	16
P73 (49)	16	4	16
P75 (51)	32	8	8
P94* (60)	16	8	8
P97 (165)	8	4	4
P105* (69)	32	8	16
P111 (75)	8	4	4
P119 (81)	8	4	8
P124 (86)	8	4	16
P146 (108)	16	8	8
P153 (115)	16	4	2
P157 (119)	32	4	8
P177 (138)	8	4	8
P301 (147)	8	4	8
P504 (155)	4	4	8
P510 (161)	8	4	8
P2 (2)	32	8	4
P27 (12)	8	4	4
Bold indicates broad spectrum activity; *indicates gram-positive selective

[0063]

TABLE 6
Activity against gram positive bacteria
Peptide
(SEQ ID NO:)	S. pyogenes	S. pneumoniae	S. pneumoniae	P. acne
P23 (8)	8	16	16	4
P25 (10)	8	64	8	2
P26 (11)	4	>128	16	4
P27 (12)	4	32	8	4
P34 (16)	4	8	8	8
P35 (17)	16	4		4
P37 (19)	8	64	16	4
P41 (23)	8	64	32	4
P42 (24)	4	32	8	2
P43 (25)	2	8	4	2
P44 (26)	4	8	16	4
P46 (28)	16	64	128
P49 (31)	8	64	32
P50 (32)	4	32	16	4
P54 (34)	8	64	64
P55 (35)	2	8	4	4
P59 (39)	2	16	4	2
P60 (40)	8	128	>128	4
P61 (41)	16	128	32	2
P63* (42)	8	128	16
P64* (43)	4	8	2	2
P72 (48)	16	>128	16	2
P73 (49)	16	>128	64	4
P75 (51)	4	>128	64	16
P94* (60)	8	64	128
P97 (165)	4	32	16	8
P105* (69)	16	64	32	16
P111 (75)	2	16	4	4
P119 (81)	8	128	32	8
P124 (86)	16	>128	64	8
P146 (108)	8	>128	128	16
P153 (115)	2	32	8	4
P157 (119)	8	128	16	4
P177 (138)	4	32	16	8
P301 (147)	8	>128	8	2
P504 (155)	16	64	8	4
P510 (161)	8	64	16	2
P2A* (2)	8	128	32
P97 (165)	8	32	32	16
P27 (12)	4	16	4	4
Bold indicates broad spectrum activity; *indicates gram-positive selective; S. pyogenes ATCC19615; S. pneumoniae C718; S. pneumoniae C719; P.acne ATCC 6919

[0064]

TABLE 7
Activity against gram-negative bacteria
	E.coli	S. typhimurium	P. aeruginosa
Peptide (SEQ ID NO:)	UB1005	14028S	H374
P12 (5)	1	4	8
P39 (21)	4	16	16
P41 (23)	2	4	4
P46 (28)	4	8	4
P61 (41)	2	4	4
P71 (47)	2	8	4
P100 (163)	0.5	4	8
P109 (73)	16	32	8
P110 (74)	16	32	8
P157 (119)	8	8	8
P306 (150)	4	4	8
P46 (28)	8	16	4
P29 (14)	8	8	16

[0065]

TABLE 8
Activity against gram-negative bacteria
	P. aeruginosa	C. glabrata
Peptide	H187	ATCC15126
P12 (5)	16	128
P39 (21)	32	16
P41 (23)	8	32
P46 (28)	16	32
P61 (41)	8	32
P71 (47)	8	32
P100 (163)	32	>128
P109 (73)	64	128
P110 (74)	64	128
P157 (119)	8	64
P306 (150)	16	>128
P46 (28)	8	32
P29 (14)	32	128

[0066]

TABLE 9
Activity against Pseudomonas bacterial strains
		P.	P.
Peptide	P.	aerugi-	aerugi-	P.
(SEQ ID	aeruginosa	nosa	nosa	aeruginosa
NO:)	H374	H187	Tb 105663	Tb 100609
P12 (5)	8	16	8	8
P25 (10)	8	8	8	8
P27 (12)	8	8	16	16
P35 (17)	8	8	4	4
P37 (19)	8	8	16	16
P39 (21)	16	32	32	32
P41 (23)	4	8	8	8
P42 (24)	4	8	8	8
P43 (25)	8	8	8	8
P44 (26)	8	8	16	8
P45 (27)	8	16	32	32
P46 (28)	4	16	32	16
P50 (32)	4	4	8	4
P55 (35)	8	8	16	8
P59 (39)	8	8	8	8
P61 (41)	4	8	8	16
P71 (47)	4	8	16	16
P72 (48)	4	8	8	8
P73 (49)	8	16	16	16
P97 (165)	8	16	16	16
P111 (75)	8	8	32	16
P119 (81)	8	16	16	16
P124 (86)	16	32	64	64
P146 (108)	2	4	8	8
P153 (115)	4	8	8	8
P157 (119)	8	8	16	16
P177 (138)	16	16	32	32
P301 (247)	4	8	8	8
P306 (150)	8	16	32	16
P504 (155)	8	8	16	8
P510 (161)	8	8	16	16
P2 (2)	16	16	16	32
P13 (6)	16	16	16	16
P27 (12)	8	8	8	8
P11 (4)	16	16	16	16
Bold indicates broad spectrum activity.

[0067] The following tables compare the anti-fungal and anti-bacterial properties of a representative sample of peptides.

TABLE 10
Comparison of anti-fungal and anti-bacterial
activities of selected peptides
Peptide		C. tropicalis	C. glabrata
(SEQ ID NO:)	C. albicans 105	ATCC13803	ATCC15126
P40 (22)	32	1	32
P47 (29)	32	1	64
P49 (31)	16	2	16
P74 (50)	16	1	16
P77 (53)	16	1	64
P79 (54)	32	2	128
P101 (65)	32	4	32
P103 (67)	16	2	16
P106 (70)	32	2	64
P113 (77)	32	4	32
P122 (84)	32	4	64
P154 (116)	64	8	128
P167 (128)	64	8	128
P169 (130)	64	8	128

[0068]

TABLE 11
Comparison of anti-fungal and anti-bacterial
activities of selected peptides
		S.
Peptide	E. coli	typhimurium	P. aeruginosa	S. aureus
(SEQ ID NO:)	UB1005	14028S	H187	SAP0017
P40 (22)	64	>128	>128	>128
P47 (29)	64	>128	64-128	>128
P49 (31)	32	64	16-64	64
P74 (50)	16	64	32-128	>128
P77 (53)	64	>128	64-128	>128
P79 (54)	32	>128	>128	>128
P101 (65)	32	128	32-128	128
P103 (67)	32	128	64	64
P106 (70)	64	>128	>128	>128
P113 (77)	32	44	32-128	32
P122 (84)	64	128	32-128	128
P154 (116)	64	>128	>128	>128
P167 (128)	32	64	128	128
P169 (130)	32	64	128	>128

[0069] Many of the disclosed FLAK peptides have activity against a wide array of microorganisms. The following tables illustrate these properties for a representative sample of peptides.

TABLE 12
Broad spectrum activities
		S.
Peptide	E. coli	typhimurium	P. aeruginosa	P. aeruginosa
(SEQ ID NO:)	UB1005	1402S	H374	H187
P25 (10)	8	8	8	8
P27 (12)	8	16	8	8
P35 (17)	2	4	8	8
P37 (19)	4	8	8	8
P42 (24)	4	8	4	8
P43 (25)	8	8	8	8
P44 (26)	1	4	8	8
P45 (27)	4	32	8	16
P50 (32)	2	4	4	4
P55 (35)	4	4	8	8
P59 (39)	8	8	8	8
P72 (48)	2	8	4	8
P73 (49)	8	16	8	16
P97 (165)	8	16	8	16
P111 (75)	16	16	8	8
P119 (81)	4	8	8	16
P124 (86)	16	16	16	32
P146 (108)	2	4	2	4
P153 (115)	8	8	4	8
P177 (138)	8	16	16	16
P301 (147)	8	8	4	8
P504 (155)	4	4	8	8
P510 (161)	8	16	8	8

[0070]

TABLE 13
Broad spectrum activities
Peptide	S. aureus	S. epidermis	C. albicans	C. glabrata
(SEQ ID NO:)	SAP0017	C621	105	ATCC15126
P25 (10)	16	4	32	32
P27 (12)	16	4	32	32
P35 (17)	8	4	32	16
P37 (19)	8	4	32	32
P42 (24)	16	2	32	64
P43 (25)	4	2	8	16
P44 (26)	8	4	8	16
P45 (27)	32	16	16	16
P50 (32)	4	4	16	16
P55 (35)	4	2	16	8
P59 (39)	8	8	32	16
P72 (48)	16	4	32	64
P73 (49)	16	4	32	128
P97 (165)	8	4	16	16
P111 (75)	8	4	32	32
P119 (81)	8	4	16	16
P124 (86)	8	4	16	16
P146 (108)	16	8	8	16
P153 (115)	16	4	16	16
P177 (138)	8	4	16	16
P301 (147)	8	4	32	32
P504 (155)	4	4	64	64
P510 (161)	8	4	32	64
P27 (12)	8	4	16	16

[0071] While FLAK peptides are generally active against an array of microbial targets, not all peptides are equally effective against all microorganisms. The following tables present some combinations of peptides and microorganisms in which the peptide was observed to have poor activity.

TABLE 14
Low observed anti-microbial activities
	E. coli	S. typhimurium	P. aeruginosa
Peptide (SEQ ID NO:)	UB1005	14028S	H374
P57 (37)	>128	>128	>128
P58 (38)	>128	>128	>128
P65 (44)	128	>128	64
P76 (52)	16	128	64
P93 (59)	128	>128	128
P95 (61)	>128	>128	>128
P96 (62)	>128	>128	>128
P107 (71)	>128	>128	>128
P112 (76)	>128	>128	>128
P114 (78)	32	128	>128
P120 (82)	>128	>128	128
P121 (83)	>128	>128	>128
P123 (85)	64	>128	>128
P126 (88)	>128	>128	>128
P127 (89)	128	>128	>128
P128 (90)	128	>128	>128
P129 (91)	64	>128	>128
P130 (92)	>128	>128	>128
P131 (93)	>128	>128	>128
P132 (94)	128	>128	>128
P133 (95)	>128	>128	>128
P134 (96)	128	>128	128
P136 (98)	128	>128	>128
P137 (99)	>128	>128	>128
P138 (100)	>128	>128	>128
P139 (101)	64	>128	>128
P140 (102)	>128	>128	>128
P141 (103)	>128	>128	>128
P142 (104)	64	128	>128
P143 (105)	>128	>128	>128
P145 (107)	>128	>128	>128
P147 (109)	64	128	128
P148 (110)	128	>128	>128
P149 (111)	32	>128	128
P151 (113)	>128	>128	128
P152 (114)	32	>128	>128
P155 (117)	>128	>128	>128
P166 (127)	>128	>128	>128
P168 (129)	128	>128	128
P169 (130)	64	64	128
P170 (131)	64	>128	>128
P171 (132)	32	>128	>128
P174 (135)	>128	>128	>128
P175 (136)	>128	>128	>128
P180 (141)	>128	>128	>128

[0072]

TABLE 15
Low observed anti-microbial activities
	P.		S.
Peptide	aeruginosa	S. aureus	epidermidis	C. albicans
(SEQ ID NO:)	H187	SAP0017	C621	105
P57 (37)	>128	>128	>128	128
P58 (38)	>128	>128	>128	64
P65 (44)	>128	>128	>128	64
P76 (52)	>128	>128	>128	64
P93 (59)	>128	>128	>128	64
P95 (61)	>128	>128	>128	>128
P96 (62)	>128	>128	>128	>128
P107 (71)	>128	>128	>128	>128
P112 (76)	>128	>128	64	128
P114 (78)	>128	>128	64	64
P120 (82)	>128	>128	>128	64
P121 (83)	>128	>128	>128	64
P123 (85)	>128	>128	16	64
P126 (88)	>128	>128	>128	>128
P127 (89)	>128	>128	64	32
P128 (90)	>128	>128	128	128
P129 (91)	>128	>128	32	128
P130 (92)	>128	>128	>128	>128
P131 (93)	>128	>128	>128	>128
P132 (94)	>128	>128	>128	128
P133 (95)	>128	>128	>128	>128
P134 (96)	>128	>128	128	64
P136 (98)	>128	>128	128	64
P137 (99)	>128	>128	>128	>128
P138 (100)	>128	>128	>128	>128
P139 (101)	128	>128	64	128
P140 (102)	>128	>128	>128	>128
P141 (103)	>128	>128	>128	>128
P142 (104)	>128	>128	128	64
P143 (105)	>128	>128	>128	>128
P145 (107)	>128	>128	>128	64
P147 (109)	>128	>128	64	64
P148 (110)	>128	>128	128	128
P149 (111)	>128	>128	>128	128
P151 (113)	>128	>128	>128	128
P152 (114)	>128	>128	32	128
P155 (117)	>128	>128	>128	>128
P166 (127)	>128	>128	>128	>128
P168 (129)	128	>128	128	128
P169 (130)	>128	>128	32	64
P170 (131)	>128	0.128	>128	128
P171 (132)	>128	>128	128	>128
P174 (135)	>128	>128	>128	>128
P175 (136)	>128	>128	>128	>128
P180 (141)	>128	>128	>128	>128

[0073] Cancer cell assays were performed in a manner similar to the anti-microbial assays described above, except that the assay procedure used the MTT dye protocol. Viability of cells is determined by the dye response. In the following procedure, approximately 1.510⁴ cells per well were added and viability was determined with the cells in a semi-confluent state. The assay was performed in a 96-well microtiter plate. After addition of peptide, the plate was set for 24 hours. MTT (5 mg/ml in phenol red-free RPMI-1640, 20 l) was added to each well including positive control wells untreated with peptide. The plate was incubated at 37 C. for 4 hours. The liquid contents of each well was removed, and isopropanol with 0.1 M HCl (100 l) was added to each well. The plate was sealed with parafilm to prevent evaporation of the isopropanol. The plate is allowed to rest for 5-10 minutes in order to solubilize the precipitate. Purified water (100 l) was added to each well. Absorbance was determined with an ELISA Reader instrument. Color intensity at 540 nm is proportional to viability of cells. Results for each concentration of peptide are plotted relative to untreated controls, and LD50 values are determined from the graphs.

[0074] W138 (ATCC No. CCL75) is a normal fibroblast line of lung diploid cells, MCF7 (ATCC No. HTB22) is a breast adenocarcinoma tumor cell line, SW480 (ATCC No. CCL228) is a colon adenocarcinoma tumor cell line, BMKC is a cloned melanoma line derived from Bowes melanoma line HMCB (ATCC No. CRL9607), H1299 (ATCC No. CRL5803) is a lung large cell carcinoma tumor line, HeLaS3 (ATCC No. CCL2.2) is a cervical epitheleal carcinoma tumor cell line, and PC3 (ATCC No. CRL1435) is a prostate adenocarcinoma tumor cell line. Numbers are LD₅₀ values (g/mL). Data on the six targets are presented in the following Tables 16 and 17.

TABLE 16
	SEQ ID
Name	NO:	P No.	WI38	MCF7	SW480	BMKC
HECATE AC	1	1	27	54	6	72
HECATE AM	2	2	66	23	46	128
SB37COOH	3	5	130	175	82	120
SB-37 AM	5	12	950	540	>	>
SHIVA 10 AC	6	13	57	>	ND	ND
FLAK01 AM	8	23	34	62	5	27
FLAK03 AM	9	24	55	26	38	85
FLAK04 AM	10	25	24	10	12	36
FLAK05 AM	11	26	96	74	8	94
FLAK06 AM	12	27	37	14	26	44
FLAK06 AC	13	27B	101	65	59	93
FLAK06 R-AC	14	27C	520	140	210	300
KAL V	15	30	93	72	62	140
FLAK 17AM	16	34	40	21	35	53
FLAK 26 AM	17	35	8	9	14	7
FLAK 25 AM	18	36	19	9	30	56
HECATE 2DAc	19	37	80	14	57	150
FLAK43 AM	20	38	12	17	13	21
FLAK44 AM	21	39	300	130	435	510
FLAK62 AM	22	40	>	760	>	>
FLAK 06R-AM	23	41	175	98	120	290
MSI-78 AM	24	42	67	31	34	140
FLAK50	25	43	5	9	9	7
FLAK51	26	44	36	140	32	47
FLAK57	27	45	200	260	180	160
FLAK71	28	46	200	300	160	150
FLAK77	29	47	>	575	>	700
FLAK50V	30	48	41	23	47	43
FLAK50F	31	49	135	40	100	115
FLAK26V AM	32	50	43	32	46	40
CAME-15	33	53	32	45		40
FLAK50C	34	54	97	60		90
FLAK50D	35	55	32	16	14	16
FLAK 50E	36	56	250	500	215	205
FLAK80	37	57	900	>	740	740
FLAK81	38	58	>	>	>	>
FLAK82	39	59	77	31	42	155
FLAK83M	40	60	>	>	>	>
FLAK 26 Ac	41	61	93	105	100	140
INDOLICIDIN	42	63	ND	64	345	200
FLAK 17 C	43	64	37	80		35
FLAK 50H	44	65	320	475	345	250
FLAK 50G	45	66	240	90	145	200
SHIVA DERIV	46	70	34	44	11	94
P69 KWKL
SHIVA 10 (1-18	47	71	355	190	250	445
AC
SHIVA 10	48	72	125	93	82	290
PEPTIDE
71 KWKL
CA(1-7)Shiva10(1-	49	73	160	150	70	360
16)
FLAK 54	50	74	335	465	340	460
FLAK 56	51	75	80	42	17	24
FLAK 58	52	76	445	970	400	750
FLAK 72	53	77	>	>	>	125
FLAK 75	54	79	>	540	>	830
SHIVA 10 (1-16)	55	80	28	29	35	76
Ac
CA(1-7)Shiva10(1-	56	81	8	63	13	12
16)-COOH
INDOLOCIDIN-ac	57	91	9	12	30	180
FLAK50B	58	92	43	23	51	46
FLAK50I	60	94	6	65	ND	11
FLAK50K	61	95	250	>	>	820
FLAK50L	62	96	>	>	>	>
Shiva-11	63	98	47	96	125	94
SHIVA 11 (1-	64	99	34	95	120	94
16)ME(2-9-
COOH
FLAK 50N	65	101	300	250	170	160
FLAK 50O	66	102	73	60	57	60
FLAK 50P	67	103	26	46	90	75
CA(1-	68	104	24	11	54	100
&HECATE(11/23)
PYL-ME	69	105	430	635	>	ND
FLAG26-D1	70	106	>	620	570	690
VISHNU3	71	107	>	>	>	>
MELITTIIN	72	108	16	9	23	18
FLAK26-D2	73	109	>	>	>	>
FLAG26-D3	74	110	45	180	325	400
FLAK50 Q1	75	111	24	35	27	26
FLAK50 Q2	76	112	420	500	800	445
FLAK50 Q3	77	113	170	150	180	115
FLAK50 Q4	78	114	>	730	>	>
FLAK50 Q5	79	117	>	>	>	>
FLAK50 Q6	80	118	170	70	115	135
FLAK50 Q7	81	119	45	54	46	36
FLAK50 Q8	82	120	600	730	630	660
FLAK50 Q9	83	121	625	400	800	670
FLAK50 Q10	84	122	720	360	570	700
FLAK50 T1	85	123	600	615	>	635
FLAK50 T2	86	124	21	18	9	10
FLAK50 T3	87	125	90	90	125	220
FLAK50 T4	88	126	>	>	>	>
FLAK50 T5	89	127	760	440	400	535
FLAK90	90	128	500	500	530	330
FLAK91	91	129	>	>	550	>
FLAK92	92	130	>	>	>	>
FLAK93	93	131	>	600	555	>
FLAK50 Z1	94	132	>	>	>	>
FLAK50 Z2	95	133	>	>	>	>
FLAK50 Z3	96	134	>	>	740	>
FLAK50 Z4	97	135	110	54	80	155
FLAK50 Z5	98	136	>	500	600	530
FLAK50 Z6	99	137	>	>	>	>
FLAK50 Z7	100	138	>	>	>	>
FLAK50 Z8	101	139	550	625	>	525
FLAK50 Z9	102	140	>	>	>	>
FLAK94	103	141	420	430	560	465
FLAK93B	104	142	73	44	38	38
FLAK50 Z10	105	143	>	>	>	>
FLAK96	106	144	750	150	285	250
FLAK97	107	145	>	>	>	>
FLAK98	108	146	270	110	380	185
FKRLA	109	147	83	106	185	110
FLAK91B	110	148	380	315	>	330
FLAK92B	111	149	>	>	>	>
FLAK99	112	150	125	160	235	190
FLAK50T6	113	151	>	>	>	>
FLAK50T7	114	152	620	430	740	>
FLAK95	115	153	130	64	61	165
FLAK50T8	116	154	600	315	750	330
FLAK50T9	117	155	>	>	>	>
FLAK100-CO2H	118	156	230	135	345	520
FAGVL	119	157	500	240	530	600
Modelin-5	120	159	82	61	140	140
Modelin-5-CO2H	121	160	700	320	370	220
FLAK120	126	165	470	360	240	240
FLAK121	127	166	>	>	>	>
FLAK96B	128	167	260	230	360	240
FLAK96G	129	168	>	630	>	590
FLAK96F	130	169	>	510	>	530
FLAK96C	131	170	>	940	>	>
FLAK96D	132	171	615	305	770	600
Modelin-8D	135	174	>	>	>	>
Modelin-8E	136	175	>	>	70	>
Flak 96H	137	176	>	>	>	>
Flak 96I	138	177	270	190	310	310
Flak 96J	139	178	405	770	>	640
Flak 96L	140	179	540	555	>	920
FLAK-120G	141	180	940	950	600	770
FLAK-120D	142	181	500	550	870	830
FLAK-120C	143	182	>	>	>	>
FLAK-120B	144	183	>	>	>	>
FLAK-120F	145	184	800	260	440	600
Magainin2wisc	146	300	52	22	60	130
D2A21	147	301	66	64	76	140
KSL-1	148	302	800	340	>	700
KSL-7	149	303	355	315	530	330
LSB-37	150	306	320	50	240	170
Anubis-2	151	307	75	38	73	83
FLAK 17 CV	152	501	26	23	ND	ND
FLAK50 Q1V	153	502	64	92	ND	ND
D2A21V	154	503	150	210	ND	ND
FLAK 25 AM V	155	504	110	130	ND	ND
FLAK43 AM V	156	505	85	86	ND	ND
FLAK50D V	157	506	75	45	ND	ND
HECATE AM V	158	507	285	340	ND	ND
HECATE AC V	159	508	190	160	ND	ND
FLAK04 AM V	160	509	95	84	ND	ND
03 AM V	161	510	77	62	ND	ND
D-Shiva 10 AC	162	67	4	7	ND	ND
Shiva 11 AC	163	100	95	175	82	120
Shiva 10 (1-18)AM	164	69	101	45	63	66
Note:
> indicates greater than 1000;
ND indicates not determined;
numbers are in g/mL.

[0075]

TABLE 17
	SEQ ID
Name	NO:	P No.	WI38	H1299	HeLaS3	PC3
HECATE AC	1	1	27	44	95	61
HECATE AM	2	2	66	140	50	44
SB37COOH	3	5	130	220	150	ND
SB-37 AM	5	12	950	720	>	630
SHIVA 10 AC	6	13	57	>	>	83
FLAK01 AM	8	23	34	64	82	41
FLAK03 AM	9	24	55	72	145	38
FLAK04 AM	10	25	24	37	20	12
FLAK05 AM	11	26	96	84	150	125
FLAK06 AM	12	27	37	16	25	8
FLAK06 AC	13	27B	101	54	80	16
FLAK06 AM	14	27C	520	170	260	280
KAL V	15	30	93	125	190	65
FLAK 17 AM	16	34	40	24	62	9
FLAK 26 AM	17	35	8	16	27	5
FLAK 25 AM	18	36	19	57	ND	19
HECATE 2DAc	19	37	80	150	ND	64
FLAK43 AM	20	38	12	33	35	10
FLAK44 AM	21	39	300	420	620	310
FLAK62 AM	22	40	>	>	>	435
FLAK 06R-AM	23	41	175	245	185	140
MSI-78 AM	24	42	67	150	ND	66
FLAK50	25	43	5	6	15	12
FLAK51	26	44	36	72	22	45
FLAK57	27	45	200	330	160	170
FLAK71	28	46	200	290	280	280
FLAK77	29	47	>	>	>	>
FLAK50V	30	48	41	17	44	32
FLAK50F	31	49	135	140	ND	77
FLAK26V AM	32	50	43	7	33	54
CAME-15	33	53	32	65	30	40
FLAK50C	34	54	97	80	190	90
FLAK50D	35	55	32	7	15	47
FLAK 50E	36	56	250	370	300	435
FLAK80	37	57	900	>	830	>
FLAK81	38	58	>	>	>	>
FLAK82	39	59	77	180	ND	81
FLAK83M	40	60	>	>	>	>
FLAK 26 Ac	41	61	93	127	170	66
INDOLICIDIN	42	63	ND	270	345	290
FLAK 17 C	43	64	37	30	30	46
FLAK 50H	44	65	320	450	210	470
FLAK 50G	45	66	240	130	140	170
SHIVA DERIV	46	70	34	63	28	82
P69 KWKL
SHIVA 10(1-18	47	71	355	320	570	270
AC
SHIVA 10	48	72	125	160	240	63
PEPTIDE
71 KWKL
CA(1-7)Shiva10(1-16)	49	73	160	115	270	97
FLAK 54	50	74	335	670	260	660
FLAK 56	51	75	80	80	74	54
FLAK 58	52	76	445	860	380	675
FLAK 72	53	77	>	>	>	>
FLAK 75	54	79	>	>	>	>
SHIVA 10(1-16)	55	80	28	64	97	28
Ac
CA(1-7)Shiva10(1-	56	81	8	22	19	170
16)-COOH
Indolocidin-ac	57	91	9	64	20	31
FLAK50B	58	92	43	25	670	83
FLAK50J	59	93	530	320	>	690
FLAK50I	60	94	6	ND	>	ND
FLAK50K	61	95	250	>	>	>
FLAK50L	62	96	>	>	>	>
Shiva-11	63	98	47	53	175	52
SHIVA 11(1-	64	99	34	54	180	28
16)ME(2-9
COOH
FLAK 50N	65	101	300	340	170	730
FLAK 50O	66	102	73	27	43	66
FLAK 50P	67	103	26	150	70	330
CA(1-	68	104	24	52	130	18
&HECATE(11/23)
PYL-ME	69	105	430	>	>	ND
FLAG26-D1	70	106	>	920	700	>
VISHNU3	71	107	>	>	>	>
MELITTIIN	72	108	16	25	35	13
FLAK26-D2	73	109	>	>	>	>
FLAG26-D3	74	110	45	95	540	>
FLAK50 Q1	75	111	24	8	7	11
FLAK50 Q2	76	112	420	470	660	640
FLAK50 Q3	77	113	170	50	190	240
FLAK50 Q4	78	114	>	>	>	>
FLAK50 Q5	79	117	>	>	>	>
FLAK50 Q6	80	118	170	74	87	330
FLAK50 Q7	81	119	45	33	30	140
FLAK50 Q8	82	120	600	620	810	>
FLAK50 Q9	83	121	625	460	830	>
FLAK50 Q10	84	122	720	830	780	800
FLAK50 T1	85	123	600	>	940	>
FLAK50 T2	86	124	21	30	14	10
FLAK50 T3	87	125	90	76	220	145
FLAK50 T4	88	126	>	>	>	>
FLAK50 T5	89	127	760	770	610	>
FLAK90	90	128	500	>	700	>
FLAK91	91	129	>	790	550	>
FLAK92	92	130	>	>	>	>
FLAK93	93	131	>	>	>	>
FLAK50 Z1	94	132	>	>	>	>
FLAK50 Z2	95	133	>	>	>	>
FLAK50 Z3	96	134	>	>	>	>
FLAK50 Z4	97	135	110	115	215	310
FLAK50 Z5	98	136	>	450	400	900
FLAK50 Z6	99	137	>	>	>	>
FLAK50 Z7	100	138	>	>	>	>
FLAK50 Z8	101	139	550	850	>	>
FLAK50 Z9	102	140	>	>	285	>
FLAK94	103	141	420	>	>	ND
FLAK93B	104	142	73	115	55	60
FLAK50 Z10	105	143	>	>	>	>
FLAK96	106	144	750	225	275	350
FLAK97	107	145	>	>	240	>
FLAK98	108	146	270	93	640	440
FKRLA	109	147	83	93	>	340
FLAK91B	110	148	380	660	>	>
FLAK92B	111	149	>	>	>	>
FLAK99	112	150	125	185	320	74
FLAK50T6	113	151	>	>	>	>
FLAK50T7	114	152	620	410	>	>
FLAK95	115	153	130	50	140	97
FLAK50T8	116	154	600	400	>	640
FLAK50T9	117	155	>	>	>	ND
FLAK100-CO2H	118	156	230	ND	>	260
FAGVL	119	157	500	315	>	375
Modelin-5	120	159	82	74	275	145
Modelin-5-CO2H	121	160	700	470	550	450
FLAK120	126	165	470	56	400	340
FLAK121	127	166	>	>	>	>
FLAK96B	128	167	260	300	325	320
FLAK96G	129	168	>	>	>	>
FLAK96F	130	169	>	640	>	>
FLAK96C	131	170	>	>	>	>
FLAK96D	132	171	615	540	820	600
Modelin-8D	135	174	>	>	>	>
Modelin-8E	136	175	>	>	510	>
Flak 96H	137	176	>	>	>	>
Flak 96I	138	177	270	240	380	120
Flak 96J	139	178	405	>	>	>
Flak 96L	140	179	540	>	>	>
FLAK-120G	141	180	940	>	760	>
FLAK-120D	142	181	500	>	>	>
FLAK-120C	143	182	>	>	>	>
FLAK-120B	144	183	>	>	>	>
FLAK-120F	145	184	800	370	302	570
Magainin2wisc	146	300	52	60	125	45
D2A21	147	301	66	77	170	45
KSL-1	148	302	800	720	>	>
KSL-7	149	303	355	345	>	530
LSB-37	150	306	320	120	250	370
Anubis-2	151	307	75	160	100	66
D-Shiva 10 AC	163	100	95	220	150	ND
Shiva 10 (1-18)AM	164	69	101	71	190	81
Note:
> indicates greater than 1000;
ND indicates not determined; numbers are in g/mL.