Patent Application
20160326529
The disclosure relates to anti-viral agents that either mimic or bind to packaging signals of RNA viruses that function in viral capsid formation; pharmaceutical and plant viral control compositions for use in the treatment of viral infections; methods to treat viral infections; and methods to screen for packaging signals in viral RNA genomes.
Several diseases in humans, animals and plants are caused by so called RNA viruses. Single-stranded RNA viruses are divided into three groups: Positive-sense ssRNA viruses (Group IV), negative-sense ssRNA viruses (Group V) and retroviruses (Group VI). On infection, the viral RNA enters the host cells and, dependent on the type of virus, RNA is directly translated (Group IV) into the viral proteins necessary for replication or is, prior to translation, transcribed into a more suitable form of RNA by an RNA-dependent RNA polymerase (Group V). Group VI RNA viruses utilise a virally encoded reverse transcriptase to produce DNA from the RNA genome, which is often integrated into the host genome and so replicated and transcribed by the host. Group IV viruses include the picornaviruses, such as polio, foot & mouth disease virus, human rhinovirus, Coxsackievirus B, and other enteroviruses, as well as the alpha viruses, including Chikungunya and West Nile virus and the hepatitis viruses A, C-E. Hepatitis B is a dsDNA virus but co-assembles via a pro-genomic ssRNA.
RNA viruses have a simple structure comprising RNA enclosed in a protein shell called a capsid, (i.e. they form a nucleocapsid). The formation of a protein container that encapsulates and provides protection for the viral genome is a vital step in most viral life-cycles (M. G. Rossmann and J. E. Johnson, Icosahedral RNA virus structure Annu Rev Biochem. 58, 533-73 (1989)). It is a prime example of molecular self-assembly, exemplifying the fundamental principles underlying the formation of protein nano-containers that are important both in virology (Isolation of an asymmetric RNA uncoating intermediate for a single-stranded RNA plant virus Bakker S E, Ford R J, Barker A M, Robottom J, Saunders K, Pearson A R, Ranson N A, Stockley P G. J Mol Biol. 2012 Mar. 16; 417(1-2):65-78.), and for applications in bionanotechnology (M. Wu, W. L. Brown, and P. G. Stockley, Cell-specific delivery of bacteriophage-encapsidated ricin A chain. Bioconjug Chem. 6, 587-95 (1995)) and synthetic biology (N. F. Steinmetz, V. Hong, E. D. Spoerke, P. Lu, K. Breitenkamp, M. G. Finn, and M. Manchester, Buckyballs meet viral nanoparticles: candidates for biomedicine J Am Chem Soc. 131, 17093-5 (2009)).
Methods and compositions for controlling capsid formation are disclosed in US2013156818. Similarly, US2013/0165489 discloses small molecule modulators of HIV-1 capsid stability.
While the mechanisms of (nucleo-) capsid formation and genome encapsulation vary across viral families, there are a number of common features that can be characterised collectively. For example, pro-capsid formation may occur via the self- or assisted assembly of protein subunits and be followed by the introduction of the genomic material via a packaging motor, as seen in many double-stranded DNA viruses (S. Sun, S. Gao, K. Kondabagil, Y. Xiang, M. G. Rossmann, and V. B. Rao. Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages. Proc Natl Acad Sci USA. 109, 817-22 (2012)). Alternatively, capsid assembly may follow a co-assembly process involving protein subunits and the viral genome, a phenomenon occurring in many single-stranded RNA viruses [5,6]. These latter comprise one of the largest viral families and include major human, animal and plant pathogens.
In contrast to bacterial infections, once a subject has contracted a virus there is little that can be done to cure the patient. Viruses cause debilitating diseases in humans which can ultimately result in the death of the infected subject. The detrimental effect of viruses is not just restricted to human related illnesses, viruses cause also many important animal and plant diseases, causing huge losses of animal related products such as meat or diary, or resulting in severely reduced crop yields.
Vaccination is the most effective form of disease prevention and has been successfully developed for some viral diseases such as influenza, hepatitis B, polio or measles. Vaccination is the administration of antigenic material to stimulate an individual's immune system to develop adaptive immunity to a pathogen. The active agent of a vaccine may be, for example, an inactivated form of the pathogen, or highly immunogenic components of the pathogen. Although vaccines provide effective protection against many diseases, and have almost eradicated diseases such as polio, measles and tetanus from many parts of the world, some viral infections such as HIV are less susceptible to vaccines and moreover, RNA viruses have enormously high mutation rates, making the development of vaccines difficult and reducing their effectiveness.
Additionally, there are no vaccines available for the use in plants, and control of plant viruses requires typically a great amount of effort such as the development of disease resistant plants or employing carefully controlled growth conditions to minimise infections.
We disclose that single-stranded RNA viruses assemble their capsids with great fidelity and efficiency at low concentrations using a mechanism that involves multiple coat protein (CP)-genomic RNA interactions at sites consisting of sequence-degenerate short fragments of RNA called Packaging Signals (PSs) [1-2].
This disclosure relates to an anti-viral therapy comprising: 1) the use of small organic compounds or example nucleic acid based compounds, ablating PS-CP interaction and therefore preventing or severely reducing capsid assembly; or 2) the production of decoy RNAs in plants displaying PSs on non-genomic and therefore non-pathogenic RNAs. Defective capsid assembly has several beneficial effects such as lower viral titres and therefore reducing symptoms caused by a viral infection, exposing conserved protein epitopes in animal viruses thus acting as good adjuvants for immune recognition and exposing viral genomes to RNA silencing in plants. Since PSs function collectively during assembly and are also part of the coding of viral genes, development of resistances are reduced when compared to methods that target the functions of individual viral proteins.
According to an aspect of the invention there is provided an anti-viral agent effective in controlling the formation of the viral capsid of an RNA virus wherein said agent is a nucleic acid stem-loop structure and comprises:
In a preferred embodiment of the invention said loop domain comprises at least 4 nucleotides; preferably said loop domain comprises between 4 and 8 nucleotides.
In a preferred embodiment of the invention said stem domain comprises at least 2 nucleotides wherein at least one nucleotide is base paired with a complementary base.
In a preferred embodiment of the invention said stem domain comprises between 2 and 13 nucleotides which are base paired by intramolecular complementary base paring.
In a preferred embodiment of the invention said loop domain comprises at least one uracil base; preferably at least 2, 3 or 4 uracil bases.
In a preferred embodiment of the invention said RNA virus is an animal virus.
In a preferred embodiment of the invention said animal RNA virus is a human virus.
In a preferred embodiment of the invention said human virus is a hepatitis virus; preferably hepatitis B virus [HBV] or hepatitis C virus [HCV].
In a preferred embodiment of the invention said human virus is hepatitis B virus [HBV].
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said binding motif comprises an A-G nucleotide base rich loop motif separated by 3 to 5 nucleotide base pairs from a bulge region containing A and/or G nucleotide base[s].
In a preferred embodiment of the invention said stem domain comprises between 3 and 5 nucleotide base pairs, followed by a bulge region that preferentially contains A and G nucleotide bases.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: SEQ ID NO: 142, 143 or 144.
In a preferred embodiment of the invention said human virus is hepatitis C virus [HCV]
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said binding motif comprises a G-rich nucleotide base motif; preferably GGG, and an A and/or G nucleotide base at the start and/or end of the loop portion.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: 184, 185, 186, 187, 188, 189, 190 or 191.
In a preferred embodiment of the invention said human virus is human parechovirus (HPeV).
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said binding motif comprises a poly-U nucleotide base motif with a single purine, preferably a G nucleotide base
In a preferred embodiment of the invention said stem domain comprises between 2 and 5 base pairs adjacent to a bulge region which is preferentially pyrimidine rich.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: SEQ ID NO: 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600 or 601.
In a further embodiment of the invention said human virus is human immune deficiency virus [HIV].
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said binding motif comprises a nucleic acid loop with one or two of the nucleotide base motifs selected from the group consisting of: [AAX . . . X], [X . . . XAA], [CAX . . . X], [X . . . XCA], [ACX . . . X], [X . . . XAC] wherein X is any nucleotide base and further wherein the nucleotide bases AA, CA, or AC is separated by one or more nucleotide bases, preferably separated by 1, 2 or 3 nucleotide bases.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence as set forth in the group: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53.
In a further preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence as set forth in the group: SEQ ID NO: 573, 574, 575, 576 or 577.
In an alternative preferred embodiment of the invention said RNA virus is a plant RNA virus.
In a preferred embodiment of the invention said plant virus is Turnip Crinkle Virus.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said nucleotide binding motif comprises a purine rich binding motif; preferably said motif comprises the nucleotide bases GGG or AAA.
In a preferred embodiment of the invention said stem domain comprises at least one purine rich bulge of three or more nucleotide bases.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: SEQ ID NO: 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group 472, 473, 474 or 475.
In a preferred embodiment of the invention said plant virus is Cowpea Chlorotic Mottle Virus 1, 2 or 3.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said binding motif comprises the sequence UUXX or XXUU wherein X is any nucleotide base; preferably said motif comprises the sequence UUXA.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369 or 370.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said binding motif comprises the sequence UUXX or XXUU wherein X is any nucleotide base; preferably the sequence UUXA.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, or 429.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said binding motif comprises the sequence the sequence UUXX or XXUU wherein X is any nucleotide base; preferably the sequence UUXA.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470 or 471.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: SEQ ID NO: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113.
In a preferred embodiment of the invention said plant virus is Brome Mosaic Virus 1, 2, or 3.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said binding motif comprises the sequence UUXX or XXUU wherein X is any nucleotide base; preferably the sequence UUXA or UUXC.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 or 183.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255 or 256,
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said binding motif comprises the sequence UUXX or XXUU wherein X is any nucleotide base; preferably said sequence is UUXA or UUXC.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294 or 295.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: SEQ ID NO: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises:
In a preferred embodiment of the invention said binding motif comprises the motif selected from the group consisting of: [AX . . . XA] or [XAX . . . XA] or [AX . . . XAX] wherein X is any nucleotide base and further wherein each A nucleotide base is separated by at least one nucleotide base; preferably 1, 2 or 3 nucleotide bases
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: SEQ ID NO: 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504 or 505.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: SEQ ID NO: 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536 or 537.
In a preferred embodiment of the invention said nucleic acid based anti-viral agent comprises or consists of a nucleotide sequence set forth in the group: SEQ ID NO: 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571 or 572.
In a preferred embodiment of the invention said nucleic acid based agent comprises modified nucleotides.
The term “modified” as used herein describes a nucleic acid molecule in which:
i) at least two of its nucleotides are covalently linked via a synthetic internucleotide linkage (i.e., a linkage other than a phosphodiester linkage between the 5′ end of one nucleotide and the 3′ end of another nucleotide). Alternatively or preferably said linkage may be the 5′ end of one nucleotide linked to the 5′ end of another nucleotide or the 3′ end of one nucleotide with the 3′ end of another nucleotide; and/or
ii) a chemical group, such as cholesterol, not normally associated with nucleic acids has been covalently attached to the single-stranded nucleic acid.
iii) Preferred synthetic internucleotide linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, phosphate triesters, acetamidates, peptides, and carboxymethyl esters.
The term “modified” also encompasses nucleotides with a covalently modified base and/or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3′ position and other than a phosphate group at the 5′ position. Thus modified nucleotides may also include 2′ substituted sugars such as 2′-O-methyl-; 2-O-alkyl; 2-O-allyl; 2′-S-alkyl; 2′-S-allyl; 2′-fluoro-; 2′-halo or 2; azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.
Modified nucleotides are known in the art and include alkylated purines and/or pyrimidines; acylated purines and/or pyrimidines; or other heterocycles. These classes of pyrimidines and purines are known in the art and include, pseudoisocytosine; N4, N4-ethanocytosine; 8-hydroxy-N6-methyladenine; 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil; 5-carboxymethylaminomethyl-2-thiouracil; 5 carboxymethylaminomethyl uracil; dihydrouracil; inosine; N6-isopentyl-adenine; I-methyladenine; 1-methylpseudouracil; 1-methylguanine; 2,2-dimethylguanine; 2-methyladenine; 2-methylguanine; 3-methylcytosine; 5-methylcytosine; N6-methyladenine; 7-methylguanine; 5-methylaminomethyl uracil; 5-methoxy amino methyl-2-thiouracil; □-D-mannosylqueosine; 5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2 methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl ester; psuedouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil; 4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester; uracil 5-oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil; 5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil; 5-pentyluracil; 5-pentylcytosine; and 2,6,-diaminopurine; methylpsuedouracil; 1-methylguanine; 1-methylcytosine. Modified double stranded nucleic acids also can include base analogs such as C-5 propyne modified bases (see Wagner et al., Nature Biotechnology 14:840-844, 1996). The use of modified nucleotides confers, amongst other properties, resistance to nuclease digestion and improved stability.
According to a further aspect of the invention there is provided an anti-viral agent according to the invention for use in the treatment of viral infections.
According to a further aspect of the invention there is a pharmaceutical composition comprising an anti-viral agent and a pharmaceutical excipient.
When administered the compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers and supplementary therapeutic agents.
The compositions of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, transdermal or trans-epithelial. The compositions of the invention are administered in effective amounts. An “effective amount” is that amount of a composition that alone, or together with further doses, produces the desired response. In the case of treating a particular viral disease the desired response is inhibiting or reversing the progression of the disease. This may involve only slowing the progression of the disease temporarily to enable the host's natural antiviral defences to clear the infection and ideally reversing disease phenotype. This can be monitored by routine methods.
Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of agent according to the invention for producing the desired response in a unit of weight or volume suitable for administration to a patient.
The doses of the agent according to the invention administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
In general, doses of agent of between 1 nM-1 μM generally will be formulated and administered according to standard procedures. Preferably doses can range from 1 nM-500 nM, 5 nM-200 nM, and 10 nM-100 nM. Other protocols for the administration of compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing. The administration of compositions to mammals other than humans, (e.g. for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.
When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents used in the treatment of viral disease. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
Compositions may be combined, if desired, with a pharmaceutically-acceptable carrier. The term “pharmaceutically-acceptable carrier” as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term “carrier” in this context denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application, (e.g. liposome or immuno-liposome). The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as syrup, elixir or an emulsion or as a gel. Compositions may be administered as aerosols and inhaled.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of agent, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable dilutent or solvent, for example, as a solution in 1, 3-butane diol. Among the acceptable solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectable. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.
According to a further aspect of the invention there is provided a combined pharmaceutical composition comprising an agent according to the invention and one or more additional anti-viral agents different from said agent according to the invention.
In a preferred embodiment of the invention the additional anti-viral agent is an anti-retroviral agent.
Anti-viral agents are known in the art and include by example Amantadine, deoxythymidine, zidovudine, stavudine, didanosine, zalcitabine, abacavir, lamivudine, emtricitabine, tenofovir, maraviroc, efuvirtide, nevirapine, delavirdine, efavirenz, rilpivirine, Elvitegravir, Lopinavir, Indinavir, Nelfinavir, Amprenavir, Ritonavir, Bevirimat and Vivecon or combinations thereof.
Anti-viral agents also include by example: ACH-3102, Arbidol, Boceprevir, Daclatasvir, Faldaprevir, Fluvir, Ledipasvir, Moroxydine, Pleconaril, PSI-6130, Ribavirin, Rimantadine, Setrobuvir, Simeprevir, Sofosbuvir, Taribavirin and Telaprevir.
According to a further aspect of the invention the pharmaceutical composition is adapted to be delivered as an aerosol.
According to a further aspect of the invention there is provided an inhaler comprising a pharmaceutical composition according to the invention.
According to a further aspect of the invention there is provided an anti-viral agent according to the invention for use as a plant protection product in preventing or treating plant viral infections.
In a preferred embodiment of the invention said anti-viral agent is provided in a plant expression vector adapted for expression in a plant cell.
By “promoter” is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells comprised in plants depending on design. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.
Constitutive promoters include, for example CaMV 35S promoter (Odell et al. (1985) Nature 313, 9810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christian et al. (1989) Plant Mol. Biol. 18 (675-689); pEMU (Last et al. (1991) Theor Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3. 2723-2730); ALS promoter (U.S. Application Ser. No. 08/409,297), and the like. Other constitutive promoters include those in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680, 5,268,463; and 5,608,142, each of which is incorporated by reference.
Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425, and McNellis et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156, herein incorporated by reference).
Where enhanced expression in particular tissues is desired, tissue-specific promoters can be utilised. Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascni et al. (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; Mutsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90 (20): 9586-9590; and Guevara-Garcia et al (1993) Plant J. 4(3): 495-50.
“Operably linked” means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is “under transcriptional initiation regulation” of the promoter. In a preferred aspect, the promoter is a tissue specific promoter, an inducible promoter or a developmentally regulated promoter.
Particularly of interest in the present context are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success in plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148). Suitable vectors may include plant viral-derived vectors (see e.g. EP194809). If desired, selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as resistance to herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).
According to a further aspect of the invention there is provided a transgenic plant cell transfected with an expression vector according to the invention.
According to a further aspect of the invention there is provided a plant comprising a plant cell according to the invention.
According to a further aspect of the invention there is provided a method to screen for anti-viral agents that bind to one or more packaging signals and/or one or more viral capsid proteins comprising the steps:
In a preferred method of the invention said viral packaging signal is derived from human parecho virus and comprises the nucleotide sequence selected from the group: SEQ 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.
In a preferred method of the invention said viral packaging signal is derived from human parecho virus and comprises the nucleotide sequence selected from the group: SEQ ID NO: 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600 or 601.
In a preferred method of the invention said viral capsid protein is derived from human parecho virus and comprises the capsid protein SEQ ID NO: 137.
In a preferred method of the invention said viral packaging signal is derived from HIV selected from the group consisting of: SEQ ID NO: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53.
In a further preferred method of the invention said viral packaging signal is derived from HIV selected from the group consisting of: SEQ ID NO: 573, 574, 575, 576 or 577.
In a further alternative preferred method of the invention said viral capsid protein is derived from HIV and comprises the capsid protein SEQ ID NO: 140 or 141.
In a preferred method of the invention said viral packaging signal is derived from Turnip Crinkle Virus comprises the nucleotide sequence selected from the group: SEQ ID NO: 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 or 69.
In a further preferred method of the invention said viral packaging signal is derived from Turnip Crinkle Virus comprises the nucleotide sequence selected from the group: SEQ ID NO: 472, 473, 474 or 475.
In a preferred method of the invention said viral capsid protein is derived from Turnip Crinkle Virus and comprises the capsid protein SEQ ID NO: 136.
In a preferred method of the invention said viral packaging signal is derived from Cowpea Chlorotic Mottle Virus selected from the group consisting of: SEQ ID NO: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112 or 113.
In an alternative preferred method of the invention said viral packaging signal is derived from Cowpea Chlorotic Mottle Virus selected from the group consisting of: SEQ ID NO:296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470 or 471.
In an alternative method embodiment of the invention said viral capsid protein is derived from Cowpea Chlorotic Mottle Virus and comprises the capsid protein SEQ ID NO: 138.
In a preferred method of the invention said viral packaging signal is derived from Brome Mosaic Virus selected from the group consisting of: SEQ ID NO: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135.
In a preferred method of the invention said viral packaging signal is derived from Brome Mosaic Virus selected from the group consisting of: SEQ ID NO: 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294 or 295.
In an alternative method of the invention said viral capsid protein is derived from Brome Mosaic Virus and comprises the capsid protein SEQ ID NO: 139.
In a preferred method of the invention said viral packaging signal is derived from STNV-1 selected from the group consisting of: SEQ ID NO: 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504 or 505.
In a preferred method of the invention said viral packaging signal is derived from STNV-2 selected from the group consisting of: SEQ ID NO: 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536 or 537.
In a preferred method of the invention said viral packaging signal is derived from STNV-c selected from the group consisting of: SEQ ID NO: 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, or 553.
In a preferred method of the invention said viral capsid protein is derived from STNV-1.
In a preferred method of the invention said viral capsid protein is derived from STNV-2.
In a preferred method of the invention said viral capsid protein is derived from STNV-c.
According to a further aspect of the invention there is provided a modelling method to determine the association of an anti-viral agent with a viral capsid protein or a viral packaging signal comprising the steps:
In the computational design protein ligands demand various computational analyses which are necessary to determine whether a molecule is sufficiently similar to the target moiety or structure. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., Waltham, Mass.) version 3.3, and as described in the accompanying User's Guide, Volume 3 pages. 134-135. The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures). When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure.
The person skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a target. The screening process may begin by visual inspection of the target on the computer screen, generated from a machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within that binding pocket. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER.
Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include: GRID (P. J. Goodford, “A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules”, J. Med. Chem., 28, pp. 849-857 (1985)). GRID is available from Oxford University, Oxford, UK; MCSS (A. Miranker et al., “Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method.” Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)). MCSS is available from Molecular Simulations, Burlington, Mass.; AUTODOCK (D. S. Goodsell et al., “Automated Docking of Substrates to Proteins by Simulated Annealing”, Proteins: Structure, Function, and Genetics, 8, pp. 195-202 (1990)). AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.; DOCK (I. D. Kuntz et al., “A Geometric Approach to Macromolecule-Ligand Interactions”, J. Mol. Biol., 161, pp. 269-288 (1982)). DOCK is available from University of California, San Francisco, Calif. Each of these citations is incorporated by reference.
Once suitable chemical entities have been selected, they can be assembled into a single compound or complex. This would be followed by manual model building using software such as Quanta or Sybyl. Useful programs to aid the person skilled in the art in connecting the individual chemical entities or fragments include: CAVEAT (P. A. Bartlett et al, “CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules”. In: “Molecular Recognition in Chemical and Biological Problems”, Special Pub., Royal Chem. Soc., 78, pp. 182-196 (1989)). CAVEAT is available from the University of California, Berkeley, Calif., 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.). This is reviewed in Y. C. Martin, “3D Database Searching in Drug Design”, J. Med. Chem., 35, pp. 2145-2154 (1992); and HOOK (available from Molecular Simulations, Burlington, Mass.). These citations are incorporated by reference.
As the skilled reader will already know instead of proceeding to build a ligand for the target in a step-wise fashion, target-binding compounds may be designed as a whole or de novo. These methods include: LUDI (H.-J. Bohm, “The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors”, J. Comp. Aid. Molec. Design, 6, pp. 61-78 (1992)). LUDI is available from Biosym Technologies, San Diego, Calif.; LEGEND (Y. Nishibata et al., Tetrahedron, 47, p. 8985 (1991)). LEGEND is available from Molecular Simulations, Burlington, Mass.; LeapFrog (available from Tripos Associates, St. Louis, Mo.), each of which is incorporated by reference. Other molecular modelling techniques may also be employed, see, e.g., N. C. Cohen et al., “Molecular Modeling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883-894 (1990). See also, M. A. Navia et al., “The Use of Structural Information in Drug Design”, Current Opinions in Structural Biology, 2, pp. 202-210 (1992), which are incorporated by reference.
Typically, once a compound has been designed or selected by the above methods, the efficiency with which that entity binds to a target may be tested and optimized by computational evaluation. For example, an effective ligand will preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding). Thus, the most efficient ligands should preferably be designed with deformation energy of binding of not greater than about 10 kcal/mol, preferably, not greater than 7 kcal/mol.
A ligand designed or selected as binding to a target may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the inhibitor or other ligand and the target, when the inhibitor is bound to the target, preferably make a neutral or favourable contribution to the enthalpy of binding.
Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction. Examples of programs designed for such uses include: Gaussian 92, revision C (M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa. COPYRGT. 1992); AMBER, version 4.0 (P. A. Kollman, University of California at San Francisco, .COPYRGT. 1994); QUANTA/CHARMM (Molecular Simulations, Inc., Burlington, Mass. COPYRGT. 1994); and Insight II/Discover (Biosysm Technologies Inc., San Diego, Calif. COPYRGT. 1994). These programs may be implemented, for instance, using a Silicon Graphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Other hardware systems and software packages will be known to those skilled in the art.
Once the ligand has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group.
Another approach is the computational screening of small molecule data bases for chemical entities or compounds that can bind in whole, or in part, to a target. In this screening, the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy (E. C. Meng et al., J. Comp. Chem., 13, pp. 505-524 (1992)). The computational analysis and design of molecules, as well as software and computer systems therefore are described in U.S. Pat. No. 5,978,740 which is included herein by reference.
According to an aspect of the invention there is provided a screening method for identification of nucleic acid based agents comprising one or more nucleotide sequences comprising a binding motif for one or more capsid assembly domains in a viral capsid protein comprising the steps:
iii) eluting capsid bound nucleic binding agents from said capsid protein[s];
In a preferred method of the invention the nucleic acid based agent[s] are tested for inhibition of viral capsid formation.
According to a further aspect of the invention there is provided an enriched nucleic acid based agent isolated by the method according to the invention.
According to a further aspect of the invention there is provided a method to determine one or more packaging signals in an RNA virus comprising the steps:
In a preferred method of the invention the selected genomic RNA sequence is correlated with the anti-viral capsid binding activity of the nucleic acid binding agent selected in (i) above thereby ranking the importance of the selected packaging signal for assembly.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. “Consisting essentially” means having the essential integers but including integers which do not materially affect the function of the essential integers.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
An embodiment of the invention will now be described by example only and with reference to the following figures:
n
SELEX: In Vitro Isolation of RNA Oligos with High Affinity for Viral CPs.
Initial selection libraries are described as xN, where x is the number of degenerate nucleotides (N) in a row in the library. X defines the random region and is sometimes referred to as the selected region. These libraries are prepared as dsDNA fragments synthesised by commercially. As well as the random region they encompass defined sequence regions on either side. On the 5′ side they encompass a promoter for the bacteriophage T7 RNA polymerase, allowing transcription to create the RNA library, whilst at the 3′ side they have a short fixed region to allow recovery and amplification of the aptamers that bind to the desired target.
Following completion of the SELEX process pools were amplified by a further 10 rounds of PCR to produce enough material for sequencing. The PCR product for each SELEX library was then purified using a commercial PCR DNA clean up kit to remove the excess nucleotides and enzymes. Adaptor DNA sequences needed for the Illumina MiSeq next generation sequencing machine were ligated onto the PCR products and further amplification was carried out. These libraries were then loaded on the next generation sequencing machine.
Whole virions, gifts from Prof William Gilbert at UCLA, were biotinylated using the chemical modification reagent, EZ-link biotin (Pierce) which modifies surface lysine residues. The reaction is deliberately incomplete implying that lysines are modified at random and that each protein will carry one or very few biotin labels. Modified virus particles were then dissociated by altering solution conditions, thus ensuring that only the outside of the CPs was biotinylated.
Biotinylated CPs were incubated with streptavidin beads for 1 hour and then washed with 5 mM Tris-HCl (pH 7.5) 1 M NaCl (note: all buffers contained protease inhibitor) three times (to remove excess coat protein and RNA). At this point the beads were split in half and washed three times either with RNA assembly buffer (50 mM NaCl, 10 mM KCl, 5 mM MgCl2, 1 mM DTT, 50 mM Tris-HCl pH 7.2) or virus suspension buffer (50 mM sodium acetate, 8 mM magnesium acetate pH 4.5) to create pH 7.2 and pH 4.5 positive selection beads, respectively.
An N40 2′F RNA library (modified CTP and UTP) was used (to protect against nuclease activity) for selection. Three transcriptions of the N40 library were performed, pooled together and then split evenly between the two pH selections (this ensured both pH selections had the same starting material).
Fourteen standard rounds of SELEX were performed whereby the negative beads were bare streptavidin beads, which had been washed in the same manner as the positive beads (to remove RNA sequences that bound to streptavidin). The RNA library was incubated with negative and positive beads for 5 minutes at 37° C.
The 2nd and 8th rounds of selection were done as normal but before the SELEX the RNA library was exposed to 0.1 mg/mL of biotinylated capsid (this removed RNA sequences with a greater affinity either for the outside of the capsid or for the biotin linker). The capsids were then pulled out of solution using streptavidin beads. The remaining RNA was then used as normal.
The final round of selection was a standard round of SELEX but the positive beads were exposed to 0.1 mg/mL of unbiotinylated capsid (to remove RNA sequences with a greater affinity for the outside of the capsid).
Whole virions, a gift from Drs George Lomonossoff & Keith Saunders at the John Innes Centre, Norwich, were biotinylated and then dissociated into high-salt/pH buffer. Biotinylated coat proteins were incubated with streptavidin beads for 1 hour and then washed with 50 mM PIPES (ph 6.5), 2 mM MgCl2, 50 mM NaCl (note: all buffers contained protease inhibitor) three times.
An N30 RNA library was used for selection. Selection buffer was 50 mM PIPES (pH 6.5), 2 mM MgCl2, 50 mM NaCl.
Fourteen standard rounds of SELEX were performed whereby the negative beads were bare streptavidin beads, which had been washed in the same manner as the positive beads. RNA library was incubated with negative and positive beads for 5 minutes at 37° C.
The 2nd and 8th rounds of selection were done as normal but before the SELEX the RNA library was exposed to 0.1 mg/mL of biotinylated capsid. The capsids were then pulled out of solution using streptavidin beads. The remaining RNA was then used as normal.
The final round of selection was a standard round of SELEX but the positive beads were exposed to 0.1 mg/mL of unbiotinylated capsid.
Samples of HPeV1 CP as a pentamer were supplied by our collaborator, Prof Sarah Butcher from the University of Helsinki.
The virus was buffered exchanged to PBS using a 100 kDa cutoff centricon (Millipore). It was mixed with biotin (NHS-LC-LC-biotin, Pierce) at a molar ratio of 1:20 of number of lysines on the virus capsid to the biotin and kept at room temperature for 2 h. Unreacted biotin was quenched using 1M Tris-HCl, pH 8.2 and the biotinylated virus was buffer exchanged to TNM buffer (10 mM Tris-HCl pH 7.7, 150 mM NaCl and 1 mM MgCl2) using a 100 kDa cutoff centricon (Millipore).
The biotinylated virus was heated at 56° C. for 30 min to disrupt it into pentamers and centrifuged at 92000 rpm for 10 min at room temperature in Beckman Coulter Airfuge with A-110 fixed angle rotor to pellet down undisrupted capsids. The supernatant was collected and pentamer formation was confirmed by running native 4-20% (w/v) Tris glycine gel (Biorad) with NativeMark unstained protein standards (Cat#LC0725, Life technologies). In addition, thyroglobulin (669 kDa) and β amylase (200 kDa) were used as two other reference standards. A band of the expected size for a pentamer containing all three capsid proteins was observed at ˜431 kDa.
Biotinylated coat proteins were incubated with streptavidin beads for 1 hour and then washed with 5 mM Tris-HCl (pH 7.5) 1 M NaCl (note: all buffers contained protease inhibitor) three times (to remove excess coat protein and RNA) with 10 mM Tris-HCl, pH 7.7, 150 mM NaCl. An N40 RNA library was used for selection. Selection buffer was 10 mM Tris-HCl, pH 7.7, 150 mM NaCl
Eleven standard rounds of SELEX were performed whereby the negative beads were either bare streptavidin beads or biotinylated capsid. The RNA library was incubated with negative and positive beads for 5 min at 37° C.
Negative selections were alternated at each round, i.e. round 1 used bare streptavidin beads and round 2 used biotinylated capsid.
The large numbers of putative PSs uncovered by SELEX and bioinformatics cannot be analysed by traditional approaches. We have therefore devised a protocol for high-throughput screening. Single-stranded DNA oligos encompassing all the RNA sites to be tested, designed to incorporate flanking sites for amplification and T7 RNA polymerase transcription, are purchased, used to create dsDNA templates for in vitro transcription and the transcripts aliquoted into in vitro binding/assembly assays using fluorescently-labelled viral CPs. The CP-PS affinities will be determined initially using thermophoresis (MST), which monitors the movement of dye-labelled species in differentially heated solution. MST requires only ˜10 μL of sample, is rapid (<1 h), not destructive and cheap. Binding curves are constructed via titrations of up to 16 ligand concentrations at a time and we have shown that this yields the same Kd for the MS2 CP-TR (its highest affinity PS) interaction as stopped-flow fluorescence measurements. Surface Plasmon Resonance, stopped-flow fluorescence, isothermal titration calorimetry and single molecule fluorescence spectroscopy can all then be used for assessing the effects of drugs on the CP-PS interaction. If PS-CP interaction triggers assembly, it can be detected using fluorescence anisotropy. The structures of assembled material can then be assessed by negative-stain transmission electron microscopy (TEM) and determined by cryo-EM reconstruction. Those PSs with the highest CP affinity and favourable effects on CP assembly are then subjected to more thorough analysis including making sequence variants to determine the precise sequences/motifs required for CP binding.
Methods for Identifying Small Molecular Weight Drugs that Bind PSs or their CP Binding Sites.
PSs are most likely to encompass at least one stem-loop, the lowest level of secondary structure within RNAs. These do not have unique structures in solution but exist as ensembles of differing conformations in equilibrium with each other. Traditionally this has made isolation of specific binding ligands difficult. However, a generic method for isolation of ligands with nanomolar affinities has recently been developed iDiscovery of selective bioactive small molecules by targeting an RNA dynamic ensemble. Stelzer A C, Frank A T, Kratz J D, Swanson M D, Gonzalez-Hernandez M J, Lee J, Andricioaei I, Markovitz D M, Al-Hashimi H M. Nat Chem Biol. 2011 Jun. 26; 7(8):553-9) using NMR structure determination to define the principal conformers of the RNA and de novo drug design strategies that are routine within the Pharma industry. Similar ligands that bind to the PS binding sites on viral CPs can be designed/screened for, once the structures of the PS-CP complex are known from X-ray crystallography or NMR spectroscopy.
For each virus all unique aptamer sequences from next generation sequencing results were aligned to available strains using the following in-house protocols. Comparison frames were generated by sliding of the aptamer sequence along the genome in increments of 1 nucleotide, resulting in genome fragments of the same length as the aptamers (typically 40 nt length each) that are to be compared with the aptamer sequences. In order not to miss any information at the 5′ and 3′ end, we also considered shorter frames obtained by overlaps of at least 12 nucleotide length of the 3′ end of the aptamer sequence with the 5′ end of the genomic sequence and vice versa. In particular, we start the alignment procedure by aligning the last nucleotide of the aptamer sequence with the first nucleotide at the 5′ end of the genome. The comparison frame in this case is a single nucleotide. Then the aptamer is slid one nucleotide at a time across the genome, increasing the comparison frame one nt at a time until its length is the same as that of the aptamer. This was done so as not to overlook potential stem-loop structures at the 5′ and 3′ end of the genomic sequence.
For each aptamer, we calculated the maximum Bernoulli score for its overlap with each of its comparison frames. The Bernoulli score B(L,N) is normailized so that it ranges from 0 to L, with L being the length of the aptamer. It can be converted to a probability via P(L,N)=(1/4)B(L,N) which corresponds to the probability that a random sequence of B(L,N) letters would align precisely with the genome. The procedure identifies the largest fragment of the aptamer that has the highest Bernoulli score, and therefore, the lowest probability of having aligned to the genome fragment given by the comparison frame just by chance. The Bernoulli score (and associated probability) for a sequence of L letters to have N or fewer mismatches over the length of L nucleotides is calculated using the formula (Altschul & Erickson, 1986):
Note that in most if not all cases, the fragment contributing to the score is smaller than the length of the aptamer, and contains some mismatches. For each comparison frame, the fragment of the aptamer which aligned to the genome with the maximum Bernoulli score was identified. If this maximum score was larger or equal to a threshold value corresponding to the most significant alignments, we logged it into the data file that was subsequently used to compute the histogram. The bioinformatics algorithm has been developed so that the threshold value can be adjusted depending on the needs of the user.
The histogram is then used to identify areas in the genome which are potential PSs. This is done by identifying the locations of the largest peaks in the histogram (or equivalently the genomic sequence) along with the aptamer which aligns to this area with the highest Bernoulli score. After having identified the set of aptamers which align to each peak with the highest Bernoulli score B(L,N), the corresponding areas of the genome are folded into stem-loops using Mfold (Zuker 2003). These are subsequently compared with the stem-loop structures of the most abundant aptamers obtained from next generation sequencing data. Finally, we also compute the statistical significance of the peaks (individual aptamer alignments) by comparing with the number of times that the consensus motif would occur in random sequences of the same length and letter content as the genomic sequence.
We have shown using single molecule fluorescence spectroscopy assays of in vitro virus assembly that at nanomolar concentrations, e.g. approximating the conditions in vivo, there is packaging specificity with respect to the RNA for the model viruses bacteriophage MS2 and satellite tobacco necrosis virus (STNV). Assembly of capsids is also very precise and complete under these conditions. These observations mimic what is seen in vivo.
The data from Example 1 can only be interpreted in terms of multiple interaction sites (PSs) between the cognate viral RNAs and their CPs that facilitate capsid assembly. We have worked out the molecular basis of such PS action for both MS2 and STNV [4-7].
We have used RNA SELEX to identify putative PSs for a range of additional viruses, including TCV, BMV, and CCMV from plants, and HCV, HBV, HIV and HPeV from humans. In each case NextGen sequencing of the selected RNA pools yields millions of sequence reads that have been sorted and rank ordered by numbers of precise repeats of the same sequence. These individual sequences have been scanned against the cognate viral genome sequence as a reference. This yields multiple, statistically significant matches implying that there are multiple areas of each genome that have specific affinity for their cognate CPs.
Mfold has been used to generate predicted secondary structures of the matching PSs within each genome. Moreover, aptamer Logos are generated using Clustl to identify consensus motifs. In every case so far the PSs fold into extended stem-loop regions in which the selected, previously random, regions play a significant role, often exhibiting sequence similarities/identities.
For two viruses, Human Parecho virus (HPeV) and Turnip Crinkle virus (TCV), we have explored the affinity of the predicted PSs for their CPs and for the latter their effects on assembly. Specific binding (HPeV, Kd ˜100 nM) and in vitro capsid assembly (TCV) have been demonstrated for these viruses.
Throughout the description the following terminology will be used:
aptamers for the RNA sequences identified via SELEX to bind to the coat protein target;
packaging signals (PS) for the regions in the viral genomes that the aptamers are aligning to with statistical significance.
Aptamer sequences will be represented by upper case letters and PSs by lower case letters. If a mix of upper and lower case letters occurs, this signifies that matches with the aptamer sequence have been superimposed on the genomic sequence to identify consensus motifs. Matches do not need to be contiguous in the RNA primary sequence.
As earlier work on bacteriophage MS2 demonstrates [7], the RNA sequences corresponding to PSs are only required to contain (not necessarily contiguous) motifs in order to be functional (e.g. an AxxA motif in the loop portion of a stem-loop, where x denotes any nucleotide).
Aptamer alignment to the HPeV1 Harris genome (genebank id: L02971) resulted in the histogram plot in
An alignment of the 9 stem-loops in
We performed the following statistical test: Each peak area in the black curve coincides with minima of value 0 in the red curve, i.e. an area of at least 5 perfectly aligned nucleotides across the 21 genomes. The chance of having perfect alignment (i.e. a value of 0 in the red curve) is 429/7339, i.e. 0.058%; the chance that any given nucleotide is part of a peak area is approximately 1036/7339, i.e. 14%, and significantly reduced if required to be central to the peak area. Hence, the overall chance of having an area with perfect alignment (zero value of red curve) in a peak area in the black curve is 0.8%, and the chance of finding this 26 times in the genome is 0.826%, i.e. very small. This implies that these alignments are significant.
We have established that one of these PSs binds its capsid protein specifically with an affinity in the nanomolar range.
HIV assembly takes place in two stages. First, GAG protein assembles a protein shell around the bipartite RNA genome. Then GAG cleaves into three domains: the nucleocapsid domain (NC domain) that is in complex with the genomic RNA; the middle domain (CA domain); and the out (MA) domain. At this stage, CA assembles the distinctive cone structure characteristic of mature HIV particles around the RNA-NC complex and inside the spherical shell defined by the MA domain. The assembly of HIV capsid is reviewed in Bell N M & Lever A M C, (2013), Trends in Microbiology Volume (21) (3).
It has been shown previously that there exists a packaging signal in the region towards the 5′ end (Psi) that binds the NC domain of GAG. The structural determinants of the high affinity binding site within the HIV-Psi element have been characterised with different experimental techniques (Berglund et al, 1997; Clever et al., 2000; Fisher et al., 1998). Based on these studis, a characteristic G-x-G motif, where x can be any nucleotide, has been suggested to account for affinity of Psi to NC and is present in all four stem-loops of the Psi packaging site. Further analysis (Lodwell et al., 2000; Paoletti et al., 2002; Yuan et al., 2003; Webb et al., 2013) suggests that the motif does not need to be connected, but that variants including G-x in a single-stranded bulge, followed by G in the loop of a stem-loop, and locations of the G-x-G in both loops and bulges are possible.
Given this information, we did not perform a SELEX analysis for this virus as for the others, but rather searched for the G-x-G motifs (in all its allowed variants) in the published secondary structure of the entire HIV-1 RNA genome (Watts et al, 2009) in order to identify all packaging signals that bind to the NC domain of GAG during stage 1. We performed a bioinformatics analysis similar to the one outlined above to establish that this motif occurs with statistical significance across the genome, and we identified the locations of the putative multiple degenerate packaging signals with that motif across the genome. We hypothesize that they are playing an active role as packaging signals during stage 1 of the assembly process (hence termed by us primary packaging signals). This idea of multiple degenerate packaging signals in HIV is new, as it is also for all the other viruses exemplified here.
We used these results to identify which areas of the genome are likely to be in complex with the NC domain at the onset of stage 2 of the assembly process. We then analysed the remaining regions (i.e. those not in complex with NC) for possible binding sites to the CA domain that could play the role of packaging signals during cone formation. For this we isolated all stem-loops (39, see table) in the secondary structure not in complex with NC at the onset of stage 2 and preformed a similarity analysis (see weblogo) which shows a clear bias towards a specific common motif (A-rich loop). Since CA binding can only occur during stage 2 after GAG cleavage, these are termed by us secondary packaging signals.
i) Plant Viruses:
Turnip Crinkle virus (TCV):
The analysis of the TCV genome has been performed following the same protocol as above. In this case, the histogram plot shows a number of packaging signals located in close proximity of each other that we label as Pair 1-Pair 3; in addition, there are 5 packaging signals that we term S1-S5. Our discovery of multiple packaging signals and their pairing sheds new light on the assembly mechanism. The distinctive pattern of packaging signal pairs suggests that pairs may have a specific functional role, perhaps in bracketing protein dimers and hence aiding with capsid assembly.
The analysis of the three CCMV genomes (CCMV1-CCMV3) has been performed following the same protocol as above. The histogram plot shows a number of peaks above the cut-off marking statistical-significant hits. The analysis of the peaks is still in progress, which is why we are indicating sequences containing packaging signals rather than the packaging signals themselves at this stage. However, for all peaks already analysed stem-loops with a clear consensus motif are visible. An analysis of SELEX data derived at different pH values shows their occurrence at pH4.5, but not at pH7, as expected from reassembly assays which show different assembly behaviours at these pH values. Our analysis is hence consistent with their expected function as packaging signals.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1315785.4 | Sep 2013 | GB | national |
This is the U.S. National Stage of International Application No. PCT/GB2014/052696, filed Sep. 5, 2014, which was published in English under PCT Article 21(2), which in turn claims the benefit of Great Britain Application No. 1315785.4, filed Sep. 5, 2013.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2014/052696 | 9/5/2014 | WO | 00 |
